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TABLE OF CONTENTS

FOR VOLUME XXXIV, Number 1, January, 1915

Address of the President for 1914, Charles Brookover, on Some Points

in the Development of the Nose, with 5 Text Figures..........-.-. 7

Some Fresh-water Nematodes of the Douglas Lake Region of Michigan,

U. S. A, by Margaret V. Cobb....... cece cece cent e eee e eee eeeeeetes 21

Notes and Reviews: New Life Member; Entomological Notes, by

Paul S. Welch; Notes on Microscopic Technic, by H. L. Wieman ;

Method of Preparing Fly’s Tongue as a Microscopic Object, by

Oliver Kendall, Jr.; Method of Collecting Diatoms from Surface of

Mud; New Method of Examining Stools for Eggs; A Clearing Fluid

for Celloidin; Bouin’s Fluid; Mounting Zoophytes and Polyzoa;

Henning’s Solution for Fixing Flies for Sectioning, by Vida A.

Latham; Illinois Biological Monographs; Revision of the Cestode

Family Proteocephalide; Optic Projection; Evolution of Sex in

Plants; Psychobiology; Biology and Social Problems.......-..-++:: 49

Proceedings of the Philadelphia Meeting........-.-.++eseeeeereeeeeese 63

Custodian’s Report ........cccecscceceeerceeeesesccctecssecasesecsenes 65

THEABUFENS REDOFE salts esis cine sors vs paepe yeh (Ob Vaenoreia sid aia wate ee eae 66

_—________—_—_____—__—___________

Notice TO MEMBERS

The Secretary will greatly appreciate notice from members of

any change of address or any failure to receive the Transactions.

T. W. GALLoway,

Secretary.

TRANSACTIONS

American Microscopical Society

(Published in Quarterly Installments)

Vol. XXXIV JANUARY, 1915 No, 1

ADDRESS OF THE PRESIDENT FOR 1914.

SOME POINTS IN THE DEVELOPMENT OF THE NOSE.

By Charles Brookover.

Medical Department of the University of Arkansas.

No exhaustive treatment of the subject is here attempted. For

the most part this article is a review of the literature and attempts

to set certain recently discovered facts into relation with older obser-

vations on the nose and its development. Whatever may be offered

in the way of theoretical conclusions we trust will be of value in

pointing the way to a more clear understanding of our problems

yet unsolved.

Although Max Schultze showed in 1862 that the adult olfactory

nerve arises from cells located in the nasal epithelium many anato-

mists and embryologists of later date persisted in making it homolo-

gous with a typical ganglionated sensory nerve of the body region.

Some anatomists looked upon the olfactory bulb as the ganglion.

With the advent of the Golgi method and the description of the.

relation of the mitral cells and their glomeruli to the olfactory tract

this idea had to be abandoned. Moreover it was shown embryologic-

ally that the olfactory bulbs, so often quite far removed from the

brain as in the sharks, develop from the forebrain and are not in

any way similar to dorsal root ganglia.

Among those who found a ganglion in the developing olfactory

nerve may be mentioned such men as Milnes Marshall, Balfour,

von Kolliker, Beard and Chiargui. The idea in the minds of most

of these men was that the head nerves are segmental and homologous

8 CHARLES BROOKOVER

with those of the body. These attempts to homologize the cranial

with the spinal nerves may be attributed to the trend of zoological

thought in the time in which they wrote. The recently accepted

evolution theory of Darwin led embryologists to look for evidences

of the derivation of the vertebrates from segmented forms like the

annelids. On this assumption the cranial nerves, even the olfactory,

might be found to have a ganglion in its development. That these

men found a ganglion on the olfactory nerve can no longer be

doubted.

In all chordates higher than Amphioxus the first evidence of

the olfactory organ is a thickening of the epithelium of the embryo

not far from the closure of the neuropore. Following Kupffer we

may designate this the nasal placode. Kupffer found the earliest

placode to be single in the Cyclostomes and argued for a primitive

monorhinal condition in vertebrates. I think we may agree with

Karl Peter that the monorhinal condition that seems to exist in

larval Cyclostomes is secondary, on account of their parasitic habits,

perhaps. At any rate the olfactory nerves are paired and there are

two sets of folds in the Schneiderian membrane of adult Cyclos-

tomes. In other higher vertebrates these placodes are always paired

and according to Peter, distinct from each other from the first. He

was unable to confirm the finding of an unpaired connection between

the two as cited by Gage for a human embryo of 4.3 mm. length.

I thought I found an unpaired placode in Amia, but the matter

needs further investigation to learn whether this unpaired placode

is not a cord of cells left from the closure of the neuropore.

In pig and man the first thickening of the ectoderm occurs

before the embryo attains a length of 5 mm., and later the depres-

sion that deepens into the nasal sac is soon evident. At this time

there is considerable thickness of loose connective tissue mesoderm

intervening between the nasal placodes and the neural tube. This

makes it easy to determine that the olfactory nerve grows inward

from the placode and does not originate from the neural tube. In

lower forms like some of the fishes, the olfactory placode is in con-

tact with the neural tube when it can first be detected with certainty.

The veteran embryologist, Wm. His, Jr., working on mammal-

ian embryos, was the first to cause the idea that the olfactory nerve

DEVELOPMENT OF THE NOSE 9

is derived from the ectoderm exclusively, to pass current in the

realm of embryology. Also, he found a ganglion, wandering out

from the nasal epithelium to produce neuroblasts for the formation

of the fila olfactoria as well as the formation of supporting elements.

He believed that the true nervous elements of the ganglion pass

over to the olfactory bulbs. This would leave an adult olfactory

nerve free of ganglion cells in accordance with the adult condition

described by Schultze cited above. This origin of the ganglion

from the ectoderm through the thickening of the nasal epithelium is

in direct contrast with the ideas of Beard and others who sought in

the ganglion a homologue of neural crest. Disse is the other noted

investigator of the development of the olfactory nerve whose work

seems generally accepted by embryological writers today. Working

on birds, he confirmed the work of His in finding the ganglion to

arise from the olfactory epithelium. He showed that there was

never a ganglion of considerable size. By the Golgi process he

discovered that there were only a very few true nerve cells. In

older embryos these nerve cells were found nearer and nearer to the

olfactory bulbs. The majority of the cells migrating from the nasal

epithelium were believed to become sheath cells of the fila olfactoria.

Bedford followed up the work of Disse on the birds by similar

methods both cytological and neurological applied to the pig. He

confirms the findings of His and Disse as to the origin of cells along

the fila olfactoria from the nasal epithelium. He says the majority

of these cells do not form nerve cells. He mentions the suggestion

of Disse that they form supporting cells, but closes by saying he did

not determine their fate. In a series of papers undertaken to clear

up the embryological history of the nervus terminalis, I have come

to the same conclusion as the authors cited above, viz., that a

ganglion arises in the course of the olfactory nerve whose cells come

from the nasal epithelium. No evidence of origin of any part of this

ganglion from the brain or any.other source than that named, is at

hand.

It is the presence of the nervus terminalis in all groups of verte-

brates from sharks to man as shown by a series of papers published

during the past decade by various authors that calls for a new

examination of the relation of the embryological ganglion on, or near,

10 CHARLES BROOKOVER

the olfactory nerve. The older observations of the ganglion on

the olfactory nerve did not take account of the nervus terminalis.

The embryological observations undertaken since the discovery of

the nervus terminalis have not been in complete accord in regard

to the relation of the embryological ganglion to the olfactory nerve

and the nervus terminalis. Locy, the discoverer of the nervus

terminalis in the sharks, was at first of the opinion that the nervus

terminalis has a common origin with the olfactory nerve. Later

he came to the view that there is a separate origin for the two and

OLFACTORY PLACODE

Se NEURAL TUBE

OLFACTORY PLACODE SEQ

¥,

CS ARP ASO SS I) tas

= SO 7m ae >

- — ~« =F ex Sowa o>, “ Fok 7

- a 4 es Y e

ax ED ae

[FOS

Det;

AS)

Fic. 2

Fic. 1. Section of a transverse series through the olfactory placode

of Chrysemys martinata nearly 4 mm. long. On account of head flexure

the section is approximately horizontal in this region, as are all of the others.

X 250.

Fic. 2. Similar section of 4 mm. Chrysemys embryo, showing slight

advance in development of olfactory placode which is now in contact with

the neural tube. X 250.

DEVELOPMENT OF THE NOSE 11

that the nervus terminalis is a separate segmental nerve of a more

anterior segment than the olfactory.

Later Belogolowy working on selachians, found both the olfac-

tory nerve and the nervus terminalis to be derived from what he

designates a primitive olfactory ganglion arising out of the olfactory

thickening of the ectoderm. My observations on series of graded

embryos of fresh-water fishes as previously published, are in accord

with Belogolowy except that I do not see the need or propriety of

calling the ganglion a primitive olfactory ganglion. No evidence

has presented itself to me that fila olfactoria are developed from

the ganglion. The evidence is in favor of the origin of olfactory

neuraxones from cells that never wander from the Schneiderian

membrane of the nasal mucosa.

The following account of the development of the olfactory

nerve and the nervus terminalis in relation to the ganglion in the

turtle, Chrysemys marginata, will serve to illustrate the embryolog-

ical history of the two nerves. The history is the same as that

found in the fishes. I am indebted to my former pupil, Mr. Albert

B. Myers, for the drawings and help in preparation of the sections.

When the Chrysemys embryo measures 4 mm. total length

the ectodermal thickening of the nasal placode can be recognized

over a wide area in front of the optic vesicles. The nuclei of this

epithelium occupy three or four indefinite layers (fig. 1). The

thickness is slightly greater in the posterior part of the placode.

The section is cut approximately horizontal on account of the head

flexure. At the anterior end of the neural tube the ectoderm is in

contact with the neural tube, but over the greater part of the extent

of the nasal placode scattered mesodermal cells are interposed. The

mesodermal cells increase in number and compactness farther ven-

_trally and a small capillary could be detected.

Figure 2 is of a size marked the same as the specimen from

which the preceding figure was drawn but shows a slight advance

over it. The placode has thickened and come into contact with

the neural tube. Adjacent section shows limiting membranes

broken down and a slender protoplasmic bridge extending from the

placode into the neural tube. The mesodermal elements are more

compacted than in the previous stage.

12 CHARLES BROOKOVER

When the embryo has reached a slightly larger size (fig. 3)

the nasal epithelium has increased in thickness and number of cells.

The basement membrane is wanting over much of its deeper sur-

face and a strand of fibers pierces the neural tube. Adjacent to the

strand of fibers can be seen a clump of cells just emerging from

the nasal placode. They are slightly larger than the remainder of

the placodal cells and show an irregular disturbed arrangement,

whereas the placodal cells are disposed in rows perpendicular to

NEURAL TUBE

Fic. 3

Fic. 3. Section of Chrysemys about four and one-half mm. long. The

olfactory nerve fibers are beginning to develop and the cells of the nervus

terminalis are migrating from the nasal placode. X 250.

the outer surface where the mitotic figures are always plentiful.

It is from these emergent cells that the ganglion on the olfactory

nerve arises.

In an embryo of the same length as the above, which shows

by the nasal pits a degree of advance in development, the olfactory

DEVELOPMENT OF THE NOSE 13

nerve is shown cut through its entire length (fig. 4). The cells

are seen to occupy a position in the nerve and on its lateral border.

The cells here shown nearer to the brain tube than the olfactory

epithelium, are not the only ones along the nerve, as is evident from

other sections.

Bivasa

Fic. 4. Chrysemys 5 mm. long with nasal pit forming and a number

of cells of the olfactory ganglion intimately associated with the olfactory

nerve. X 250.

When the embryo has increased to nearly six mm. length, the

size of the olfactory nerve is larger and the number of cells along

it is more numerous (fig. 5). The artist has drawn all the cells

to appear as neuroblasts with vesicular nuclei. Whether all are of

this character is uncertain. The main ganglion is located near the

nasal epithelium where the olfactory nerve breaks up into smaller

twigs. :

We have not followed the development of the ganglion in the

turtle through to the nervus terminalis of the adult as we did in the

fishes, but we have no doubt that such an adult structure exists.

Johnston has described the nervus terminalis in another turtle,

14 CHARLES BROOKOVER

practically adult. We have followed out the early developmental

history in a cyprinoid fish (probably a horned Dace, work still

unpublished) and found the evidence the same, viz., that the cells

NASAL EPITHELIUM

Fic. 5

Fic. 5. Chrysemys of nearly 6 mm. length showing the ganglion of

the nervus terminalis near the blood vessels at the base of the nasal epithe-

lium. These cells show the characters of neuroblasts. There are other

cells scattered along the olfactory nerve as far as its union with the brain.

X 250.

along the olfactory nerve arise from the nasal epithelium and

that these cells give rise to the nervus terminalis. That some of

these cells may give rise to the sheath cells of the olfactory nerve 1s

very probable. As the sheath cells are distributed as far posteriorly

as the olfactory bulbs and there is often a ganglion terminale not

far from the olfactory bulbs, we readily harmonize our findings with

the statements of His and Disse, noted above, that cells from the

embryological ganglion pass over to the olfactory bulbs.

DEVELOPMENT OF THE NOSE 15

DISCUSSION

If other embryological investigations show that the above ac-

count of the origin of the nervus terminalis from the olfactory

placode is correct, it would seem to me to prove that the nervus

terminalis is a component of the olfactory nerve. I have previously

taken this view of the matter and Johnston has emphasized it. That

the nervus terminalis is not homologous with the nerve to Jacob-

son’s organ is quite clear, The nervus terminalis is present in

forms where the vomeronasal organ is absent or rudimentary as

in man. Read has shown that Jacobson’s organ has nerve fibers

of the true osmatic type. Although the nervus terminalis is fre-

quently intimately associated with the ramus to the organon vomer-

onasale it may exist side by side with the nerve to Jacobson’s organ,

as Johnston has shown, or be distributed in part to a region anterior

to the organ of Jacobson as demonstrated by Huber and Guild.

Weare in no position to state definitely the relation existing between

the organ of Jacobson and the nervus terminalis so long as we are

uncertain of the function of either of them. With regard to Jacob-

son’s organ we may incline to the view expressed by Peter that it

serves various functions in the different vertebrate classes. It may

be glandular in part and it may serve for sex recognition, for testing

the expired air, or for recognition of harmful food-stuff in the

mouth. If it serves these various functions it may very well be

that the structure is not strictly a homologous organ in all the verte-

brates where found. Evidently it is not so essential as the nervus

terminalis which seems everywhere present in the vertebrates so

far as known.

I have been hunting for a type of vertebrate where the nervus

terminalis is hypertrophied so as to gain further evidence of its func-

tion. No such case has been found. The nerve is developed part

passu with the development of the nasal sac. This is in keeping

with the function that I have suggested previously, that it is in part

an autonomic nerve. The opinion of Huber and Guild is in accord

with this view with the reservation that its central connection does

not conform to their idea of a typical autonomic nerve. It may be

that it contains general somatic sensory fibers as suggested by John-

ston. The recent work of McKibben on the sharks by intra vitam

16 CHARLES BROOKOVER

methylene blue emphasizes two facts. The cells are not constant in

position or grouping and they are not of a type such as we should

expect in a typical somatic sensory ganglion. Instead of being

T-shaped unipolar cells, or bipolar, they are largely of multipolar

variety so far as impregnated in his various preparations. Huber

and Guild noted many multipolar cells in the rabbit and they have

been conspicuous in the fishes that have come under my observation.

In addition to the common embryological origin of the nervus

terminalis and the olfactory nerve noted above is to be mentioned

the observation of Pinkus, the discoverer of the nervus terminalis

in fishes, that it has the same peripheral distribution (nasal mucous

membrane), the same central ending (prosencephalon), and the

same nature of supporting (sheath) cells without medullation, as

has the olfactory nerve. In general these statements hold true today

in light of our extended knowledge. However, there seems to be a

central connection with parts of the brain more posterior than the

osmatic region of the forebrain. McKibben traced the central path

to the interpeduncular region of the mesencephalon, and in Amia

there is apparently a tract reaching as far as the optic chiasm or

farther.

The exclusively placodal origin of the nervus terminalis and

the olfactory nerve does not bring the nasal segment into closer

correspondence with the other cranial nerves. The homologue

of the neural crest which the early workers on the cranial nerves

sought does not seem to be present. However, it does seem to

emphasize the close relationship between the sense of smell and

that of taste. It is quite well known that there is a marked physio-

logical correspondence in the fact that we discriminate between taste

and smell with difficulty in many instances. Herrick has discussed

their probable origin from a common chemical sense, the olfactory

organ into an exteroceptor and the taste-buds into interoceptors.

Landacre has shown that epibranchial placodes occur on those nerves

only that contain gustatory fibers. Thus it might seem that the

sense of smell and the sense of taste originated from cells that were

located not in the main axial group included in the neural plate, but

from cells scattered in the skin as placodes, primitive sense organs.

When we attempt the comparison of the sense of smell and

the sense of taste on the morphological side we meet with some

DEVELOPMENT OF THE NOSE 17

difficulties. It is true there are arguments that can be adduced to

show that the nasal cavities are probably gill slits. The nose is

quite commonly intimately connected with the mouth, which is

admittedly a gill slit. I have gone so far as to figure a supply of

blood to the nose of Amia through the internal and external carotid

arteries similar to that given to the pseudobranch. The nasal folds

in the Schneiderian membrane of fishes can be compared to gill

filaments and Blaue went so far as to compare the groups of osmatic

cells in some fishes to taste-buds. I have noticed an apparently

similar grouping in the nasal cavity of young Amblystome. But

if the structures in the nose sometimes resemble taste-buds, they

are not really similar. Gustatory cells are conceded to have very

brief basal processes not reaching beyond the taste-bud, while olfac-

tory cells send their axones into the glomeruli of the olfactory bulb

and leave the olfactory nerve without a ganglion except the nervus

terminalis. This is not thought to be large enough to serve the

transmission of the sense of smell if it made the proper cranial con-

nections. Thus, the osmatic sense retains that primitive position

of its neurones in the integument like invertebrates, as was first

pointed out by Retzius, I believe. It may be that an early develop-

ment of a protective nasal capsule has lessened the necessity for

withdrawal of the neurones from the outside. It has occurred to

me that the residence of the neurone body in the epithelium where

the chemical stimuli are received may add to the delicacy of smell

over taste as shown by Parker and Stabler in experiments on each

other with dilutions of alcohol.

We may summarize the development of the olfactory nerve

by saying that the present evidence points to the embryological as

well as morphological distinctness from other nerves. It is more

closely allied in its development to taste-bud components of cranial

nerves, but its neurones remain in the ectoderm. The development

of the nervus terminalis along with the olfactory nerve from the

same placode is unique among nerves that are somatic sensory or

sympathetic.

Little Rock, Arkansas.

February 22, 1015.

18 CHARLES BROOKOVER

REFERENCES

Beprorp, E. A.

1904 The Early History of the Olfactory Nerve in Swine. Jour. Comp.

Neur., vol. 14, pp. 390-410.

BetocoLowy, G.

1912 Studien zur Morphplogie des Nervensystems der Wirbeltiere. Aus

Bull. de la Imper. des Nat. de Moscou.

BLAUE, JULIUS.

1884 Untersuchungen iiber den Bau der Nasenschleimhaut bei Fischen

und Amphibien, namentlich iber Endknospen als Endapparate des Ner-

yus olfactorius. Archiv fir Anat. und Phys. Hft. 3 und 4 p. 231.

Brookover, CHAS.

1910 The olfactory nerve, the nervus terminalis and the preoptic sym-

pathetic in Amia calva, L. Jour. Comp. Neur., vol. 20, no. 2.

1914 The Nervus Terminalis in adult Man. Jour, Comp. Neur., vol. 24,

no. 2.

1914 The Development of the olfactory nerve and its associated ganglion

in Lepidosteus. Sci. N. S. vol. 39, no. 1001, Mch. 6, ‘l4. p. 367. (Ref-

erence to n. term. cells in man).

Brookover, CHAS. AND Jackson, T. S.

1911 The olfactory nerve and the nervus terminalis of Ameiurus. Jour.

Comp. Neur., vol. 21, no. 3.

Dissz, J.

1897 Die erste Entwicklung des Reichnerven. Anat. Hefte. Bd. IX, Fest-

schrift fur F. Merkel.

Herrick, C. Jupson.

1908 On the Phylogenetic Differentiation of the Organs of Smell and

Taste. Jour. Comp. Neur., vol. XVII, no. 2.

His, W.

1889 Uber die Entwicklung des Riechlappens und des Riechganglions

und uber diejenige des verlangerten Marks. Verhadl, den Anat. Gesell.

DEVELOPMENT OF THE NOSE 19

Huser, G. Cart, aNp Guin, Stacy R.

1913 Observations on the peripheral distributions of the nervus termin-

alis in Mammalia. Anatomical Record, vol. 7, no. 8, p. 253.

Jounston, J. B.

1913 Nervus Terminalis in Reptiles and Mammals. Jour. Comp. Neur.

vol, 23, no. 2.

v. Kuprrer, C.

1891 The Development of the Cranial Nerves of Vertebrates. Jour.

Comp. Neur. vol. I, p. 315-333.

McKunren, Pavt S.

1911 The Nervus Terminalis in urodele Amphibia. Jour. Comp. Neur.,

vol, 21, no. 3.

1914 Ganglion Cells of the Nervus Terminalis in the Dogfish (Mustelus

canis). Jour. Comp. Neur., vol. 24, no. 5.

Lanpacre, F. L.

1912 The epibranchial Placodes of Lepidosteus osseus and their relation

to the cerebral Ganglia. Jour. Comp. Neur., vol. 22, no. 1.

Locy, W. A.

1899 New Facts Regarding the Development of the Olfactory Nerve.

Anat. Anz. Bd. XVI, p. 273-290.

1905 On a Newly Recognized Nerve Connected with the Fore-Brain of

Selachians. Anat. Anz. Bd. 26.

Parker, Geo. H., ann Srasiter, ELeanor M.

1913 On Certain Distinctions between Taste and Smell. Amer. Jour.

Physiol., vol. XXXII, No. 4.

Perer, Kar.

1913 Atlas der Entwicklung der Nase und des Gaumens biem Menschen.

Gustav Fischer, Jena.

20 CHARLES BROOKOVER

PINKUS, FELIX.

1895 Die Hirnnerven des Protopterus annectens. Morphologischen

Arbeiten (G. Schwalbe) p. 275-346, Bd. 4.

1905 t!ber den zwischen Olfactorius und Opticusursprung das Vorder-

hirn (Zwischenhirn) verlassenden Hirnnerven der Dipnoier und Sel-

achier. Arch. Physiol. Jahrb. Supplementb. Hefte 2S. 447-452.

Reap, E. A.

1908 A Contribution to the Knowledge of the Olfactory Apparatus in

Dog, Cat and Man. Amer. Jour. Anat. vol. VIII, no. 1.

ScHULTZE, MAX.

1862 Untersuchungen iiber den Bau der Nasenschleimhaut namentlich

die Structur und Endigungsweise der Geruchsnerven bei dem Menschen

und den Wirbelthieren. Abhabdl. d. Naturforsch. Gesell. zu Halle, Bd.

VII.

SOME FRESH-WATER NEMATODES OF THE DOUGLAS

LAKE REGION OF MICHIGAN, U. S. A.*

MarGaret V. Coss. (Descriptions by N. A. Copp).

I. Introduction.

This account of the fresh-water nematodes of the Douglas Lake

region is based on work made possible by a research fellowship from

the University of Michigan for work at the Biological Station at

Douglas Lake. The work was carried on in the summer of 1913,

under the supervision of Professor Frank Smith of the University

of Illinois. Most of the specific determinations have been made by

Dr. N. A. Cobb, Department of Agriculture, Washington, D. C., and

all have received his confirmation. I wish to acknowledge also the

hospitality of the Bureau of Plant Industry, of the Department of

Agriculture, during a part of the work.

II. The Region.

Douglas Lake lies in the sandy country of the northern end of

the lower peninsula of Michigan, about halfway between Petosky

and Mackinaw. It is about four miles long by one to two miles

wide, and is one of many small lakes in the region. Collections were

made from the waters of the lake itself; from its beach-pools of

various ages; from its inlet, Bessey Creek; its outlet, Maple River;

from a sphagnum bog cut off from the lake; and from three neigh-

boring lakes. Of these, Monroe Lake lies about a mile north of

Douglas Lake. Lancaster Lake, also to the north, is connected with

Douglas Lake by Bessey Creek, which flows from the former into

the latter. Dead Lake, close to Douglas Lake, has dwindled to a

mere series of marshy pools. Collections were made in the follow-

ing situations :

1. Maple River, entrance. Sand and mud near bank. About eighty-

five specimens.

2. Maple River, short distance from entrance. Sand and mud near

bank. About eighteen specimens.

3. Fairy Island, south shore. Detritus on sand, among rushes. Eight

specimens. 3

* Publication No. 30 from the Biological Station of the University of Michigan.

22 MARGARET V. COBB

4. Beach pool, south end of North Fish Tail Bay. Sand and mud,

near crayfish burrows. Eight hundred specimens.

5. Large beach pool, Pine Point. Among moss. Two hundred fifty

specimens.

6. Large beach pool, Pine Point, east end. Sand and mud. Thirty-

three specimens.

7. Old beach pool, Pine Point. Mud, among rushes. One hundred

specimens.

8. New beach pool, Pine Point. Sand and mud. One hundred s€v-

enty-five specimens.

9. Bessey Creek, middle bridge. Sand and mud near bank. Very

small collection. About one hundred specimens.

10. Lancaster Lake, north shore. Two specimens.

11. Inlet of Lancaster Lake. Four specimens.

12. Monroe Lake. Among smartweed growing in water along shore.

Eighteen specimens.

13. Dead Lake. Pool near roadside bridge, among moss and alge.

One hundred fifty specimens.

14. Bryant’s Bog, east end. Among sphagnum. Thirteen hundred

specimens.

15. Bryant’s Bog, west end. Among sphagnum. Six hundred _speci-

mens.

III. Preparation of Material.

Free-living nematodes are abundant in this region, as elsewhere.

They were collected by taking samples of the sand or mud and water

of the pool or stream bottom, and of the aquatic vegetation. These

were washed through a series of graded sieves, from coarse to fine,

which removed the coarser debris, until examination with a lens

showed that nematodes also were caught on the sieve. The collec-

tion was then allowed to settle for five minutes or more, and the

superfluous water poured off. The nematodes were killed and fixed

by adding to this watery mud an equal quantity of boiling hot satu-

rated solution of corrosive sublimate.

Staining and mounting these small specimens is a less summary

matter. Each sample was treated as follows. The sediment was

examined, a little at a time, in a Syracuse watch-glass under a dissect-

ing lens ; the nematodes were picked out one at a time with a bamboo

FRESH-WATER NEMATODES 23

splinter, and placed in water in the object-box of a differentiator’,

in which they were gradually passed up through the grades of alco-

hol to 80% alcohol. At this point they were treated with acid alco-

hol to dissolve out impurities (10 drops conc. HC1 to 100 cc. 80%

alc.) and overstained with Mayer’s acid carmine according to the

following formula:

APES Tock ea us LER ERE a Manvel dae oad oh 4 grams

BE Oe Ric evewbacele Wight s Sra cy ad nso ete nae e saia's aMle so Hs Lace

EE Race a rete, a ak cote eer G cos woh PEG eae pie ds 30 drops

Add 95 cc. of 85% alcohol, boil until carmine is dissolved,

neutralize with ammonia until carmine begins to precipitate,

filter through glass wool.

For differentiation of the tissues acid alcohol was used (4 drops

conc. HC1 to 100 cc. 80% alc.). The specimens, still in the object-

box, were then passed up to and through absolute alcohol and tur-

pentine to thin balsam. This was done without removing the object-

box from the differentiator, except to remove it to another type of

differentiator when the change to heavier fluids began. The object-

box was now opened in thin balsam in a Syracuse watch-glass, and

the nematodes mounted in balsam.? From ten to one hundred, ac-

cording to size, can be arranged in one drop of balsam without much

crossing of specimens. This also is best done under magnification ;

it is convenient to have two dissecting microscopes, keeping the

watch-glass of specimens under one, and the slide which is being

prepared under the other. These methods were developed by Dr.

N. A. Cobb.

IV. Results.

These collections from the Douglas Lake region have yielded

thirty-six species, two-thirds of which are new species. They

belong to twenty-seven genera; two, Dolichodorus and Tylencho-

laimellus, are new, and eight others have not previously been

1For description, see New South Wales Dep’t Agriculture Miscellaneous Publication

No. 215. ‘‘Nematode Parasites; Their Relation to Man and Domesticated Animals’. (N.

A. Cobb.)

2The mounts were made on extra thin coverglasses held in perforated aluminum

frame of regulation size, and covered by a smaller thin coverglass. The specimens may

then be examined from either side under short focus oil immersion lens. This procedure

is necessary for determining the finer histological details. Aluminum slides may be

obtained from F. Cobb, Falls Church, Virginia, -

24 MARGARET V. COBB

recorded from North America. There is little doubt that further

collections will’add to this list; for while certain ubiquitous species

were common to most of the collections, and many were taken from

more than one situation, each of the larger collections yielded some

forms found in none of the others. For some time to come each new

collection may be expected to yield new forms.

The present species are as follows:

A. Known species (see bibliography for descriptions)

1. Aphelenchus microlaimus Cobb. (1).

2. Diplogaster fictor, Bastian (3).

3. Achromadora minina, Cobb (1).

4. Cryptonchus nudus, Cobb (2).

5. Plectus tenuis, Bastian (4).

6. Chronogaster gracilis, Cobb (2).

7. Mononchus longicaudatus, Cobb (1).

8. Diploscapter coronata, Cobb (2, 1):

9. Monhystera filiformis, Bastian (4).

10. Tripyla affinis, de Man (4).

11. Trilobus longus (Leidy) Bastian (3).

12. Anonchus monhystera, Cobb (2).

B. New species, described in Trans. American Micr. Soc., for

April, 1914 (3).

Dolichodorus n. g. heterocephalus.

Microlaimus fluviatilis.

Rhabdolaimus minor.

Cephalobus sub-elongatus.

Teratocephalus cornutus.

Prismatolaimus stenurus.

Am kh WD

C. New species here described.

Tylencholaimellus n. g. diplodorus n. sp.

Actinolaimus propinquus, n. sp.

Dorylaimus sulcatus, n. sp.

Jronus minor, n. sp.

Mononchus lacustris, n. sp.

Cyatholaimus fluviatilis, n. sp.

Chromadora inornata, n. sp.

Ethmolaimus gracilicaudatus n. sp.

Aphanolaimus viviparus, n. sp.

Aphanolaimus communis, n. sp.

Prismatolaimus digitatus, n. sp.

CONTA NP WwW DW

— ps

— © oO

FRESH-WATER NEMATODES

D. Undetermined species.

1. Dorylaimus sp. 5. Plectus sp.

2. Dorylaimus sp. 6. Rhabditis sp.

3. Dorylaimus sp. 7. Monhystera sp.

4. Dorylaimus sp.

TABLE SHOWING LOCAL DISTRIBUTION OF THE SPECIES

As,

(e 3

Be ok 4

fom Zoe n 2

. S 3335 48 Be

Gr eS hy thet o Of a6 SB aA, a

Sw «v a > 5 2d Peg AL oD SPER ies Pa =

53 ea 29 US Ge ak eS S285 08 85 ‘ad 53

set Se go te em AU Ze om am Oe mm OF am

Aphelenchus micro-

laimus Xx

Tylencholaimellus dip-

lodorus ce, Sethe ATL Y AaA ON

Dolichodorus hetero-

cephalus Ry anh cla < Parkhead Ladue toto Be, aed ek aie

Dorylaimus sulcatus AP eh eee NaN May POM ain ONO Pe erat ah ad i oe a a eS

Dorylaimus spp. RE, Oe GaP. Geer see On ae fae Crue, heat ND) fo

Actinolaimus propin-

quus SAW OEM i SG WR Ae ade 7 bad aD

Ironus minor Ba eed ee UAE eh WPGAGM i chet ries cata Ma x

Mononchus longicauda-

tus SRT oh AR 8 BREESE IPRS LEE ita Md ee aac AME va

MEF C THIN? PRCHBTLTES och te Ra ER PR eke ee rare PME pag? ae

Diplogaster fictor se SOLAPUR teeta Uilce GUESS ILI TAP GaAs Sp ata gh 1 9”

Cyatholaimus fluviatilis .. .. .. .. ™

Melromatora minima 60. ee ee eae poe eames, ae

Chromadora inornata .. .. .. .. X X

Ethmolaimus gracili-

caudatus x Rr ee x?

Microlaimus Auviatilig c.hee i OK RK MX OK x

Cryptonchus nudus O68 oe MK ce ps si

Plectus tenuis Err its tO eS Oe in, ae Rane

Plectus sp. Ba Ah os See LR hy rg von Ga for ee

Chronogaster gracilis). 00.0% oleic (RK MOK Ae Xx

xm)

be ge %

ag Ae Ee view oe

y oA g 88 $e 33 Be

ee PERE BO Re tee lan oa h. Selaan) Cake

ef of uf pS af FS ys Sa 35 Ss Be ey Fy

3 —aswm a Dw a) Fars 6 Co. po

SH sn cr on is Pte 7 & lam A Ao 5 & Aan Ar om

Rhabditis sp. a soNat

Rhabdolaimus minor MK: OR es eee x

Cephalobus sub-elong-

gatus x

Teratocephalus cornut-

us x “ Ge x x

Aphanolaimus vivipar-

us ME he oom Rey i RSS PR Re ae Ee oe

Aphanolaimus com-

munis © AS Gath, Gt? SEN Ae, oi MD hes Fee

Prismatolaimus stenu-

rus x? X EP

Prismatolaimus digitat-

us Ses oe a ea COE |, es caw ta eae

Monhystera filiformis X<°.. .. .. KX X?X KX XK XK X x

Monhystera sp. r Ene inn kad A De ys ith we S.! me

Trilobus longus +S, sane n. S Ro: PR Aa 4 x

Tripyla affinis Dass et ai Oe Sie ns

Anonchus monhystera 3.5) Sss5 oe ae GOR et CN Pe Orca cie ee s cis aan

Diploscapter coronata f x

V. Key to the Genera Found in These Collections.

1. (10) Oral end armed with protrusile spear or sting............ 2

26 tS). Spear with ‘bulbous, base sci 72sec veuw cesses ahaa ee eee 3

3. (4) CEKsophagus with median bulb only......... Aphelenchus, Bastian

4, (3) (Esophagus with median bulb only....... Tylencholaimellus, Cobb

St 2) pear without 4 bulbous bases. os 7iuacwe corer a eee 6

6. (7) C&sophagus with median bulb; males with bursa...........

PE TO ab ale Woe « Uiath titelrd ware ie Seen os ocak Dolichodorus, Cobb

7. (6) (Esophagus with only an elongated posterior swelling...... 8

8. (9) Pharynx simple, male supplementary organs not in fascicles

NE AES: Avtech 4} SAE eer EME | Mi iv "ah pe Bee BA ole Dorylaimus, Dujardin

9. (8) Pharynx with complicated radiate framework, male supple-

MARGARET V. COBB

mentary organs in fascicles............... Actinolaimus, Cobb.

(1)

(28)

(13)

(12)

(15)

(14)

(27)

(18)

(17)

(20)

(19)

(26)

(25)

(24)

(23)

(22)

(21)

(16)

(11)

(42)

(33)

(32)

(31)

(30)

(37)

(36)

(35)

(34)

(39)

FRESH-WATER NEMATODES 2A

Oral end without protrusile spear or sting...........+-.-+- 11

Pharynx armed with one or more refractive chitinous

RECRTE TER so Fea FTE Liao een Redden evince on Ana eee aN Res 12

Number of teeth three; equal, small, mobile, well forward

Pegi CHG: SHOT fd Ao Leak ne wed Wine le'o a's, 5 be pidae We Ironus, Bastian

Number of teeth one; or more and unequal...............- 14

Teeth, especially dorsal tooth, usually massive; thick, more

or less prominently papillate lips closing over the pharynx

POY Wess ies a and oi Ree rene hes ENA Mononchus, Bastian

Teeth small, often only one, then dorsal; lips with incon-

spicuous papilla; pharynx moderate............+-eeeeees 16

(Esophagus usually with one or two bulbs...............-- 17

Bulbs two, spinneret absent............- Diplogaster, M. Schultze

Bulb one, then cardiac; or none. Spinneret present........ 19

Pharynx cupshaped, then conoid, longitudinally ribbed;

without cesophageal bulb.............+4-- Cyatholaimus Bastian

Pharynx less conspicuously ribbed; cesophagus with a dis-

SPIO Ca TIRE PATIO Boe Ea As CLAM RM ARCANE rere due ew tas wars 21

Dorsal tooth well developed..........cc ce eeeceereeeeerees 22

Pharynx cyathiform, then conoid, joining the cesophagus

Sather TNC eUTLELY <0 cc vos a sh sb ya e oln wy Kn eee me pea 23

Dorsal tooth opposed by one or more small teeth, amphids

eniral at sitle Of-Head va... eeccccsc veces dues Achromadora, Cobb

Dorsal tooth single, amphids invisible or inconspicuous, to-

MPA TCE LORE. OL¢ OBO viowcla ck cc dele sity eee v ins Chromadora, Bastian

Pharynx cyathiform, then prismoid, ending behind very

definitely ; amphids distinct.............. Ethmolaimus, De Man

Dorsal tooth very small, circular amphids well developed...

DE Ee, Naa eh Rye Pe «a hin sah we LAE Microlaimus, De Man

CE SODHAGB DIAN. 7k vine od oR EV Ae neh de been Cryptonchus, Cobb

Pharynx without teeth..........ccccceccsssceeeerseceeees 29

(Esophagus. with) bulbgins ccks.decica sce ek os nahin dla bs y'ends 30

Amphids circular or nearly so; pharynx compound, much

Pinna I ys oka Ora da ieee eee f keiny hog haa ee 31

Chitinous external marking of amphids circular..Plectus, Bastian

Chitinous external marking of amphids not circular......

1 aE AA Ope eee ACN RC PSR eee Ere EP es Chronogaster, Cobb

Amphids apparently absent... .....25. 00. cecnc cece eens ees 34

(Esophagus with two more or less well developed bulbs;

PUR Swat a) IIT E AT Vos wath i, oth cia'p alciare td wry wed 4 © aia Wikio kg she

With: ne. “dipgers’ ‘on head). crpawck he wee 8 os Rhabditis, Dujardin

With dorsal and ventral “diggers” on head....Diploscapter, Cobb

Esophagus with only one well developed bulb; males with-

Cer Pemee en uh ake rh Ih ce. 2 ao o 5:8 ue ain a 38

28 MARGARET V. COBB

39.2.) (38). Pharynx not long and narrow... i. cde. s ree cen ste rerswes 40

40. (41) Striz not resolvable into rows of dots....... Cephalobus, Bastian

41. (40) Striz resolvable into rows of dots, altered on lateral fields

ee, a Tt he Ter Oy per dea, PERE Teratocephalus, de Man

42. (29) (Esophagus without bulbs............. SP WA A iy Ds AM gd 43

BSCC CIO) I PHATYUE NOME. Swiss as op win Gelade eh poh Mets Ot ore ee 44

Rae CES) NII PINOS “EDILAL, 4 we Kenta areas Gita ee eee Aphanolaimus, de Man

45. (44) Amphids circular or ellipsoidal.................. Tripyla, Bastian

SGcth (45) soe NATeNE PLOSCNt 2 foasiy ous Foes bee te rie siya bens eee Rie 47

47. (48) Cavity relatively large, amphids very small................

Sunheat Os eck cS Gate eid dae dine Miers barat Prismatolaimus, de Man

48. (49) Cavity small, amphids usually well developed............. 49

49. (50) Form of cavity conoid, open in front; circular amphids

COMSIGSTADIV) OEDMIG ICs oo, clckds stole tbh Monhystera, Bastian

50. (49) Form of cavity various, closed in front; amphids nearly

OPPOSE. It. Coa. cnndy enue awe Ca ad peice x dae Send ee te 51

Bis: (52). Lateral organs inconspicuous.....:.. 005% sc25 0s Trilobus, Bastian

52. (51) Lateral organs more or less conspicuous spirals or circles..

PETC RCLET Oa ct oaes FOCACRL EME Ole E tao Kaas Anonchus, Cobb

VII. Literature Cited.

The following specific descriptions have been prepared by Dr.

N. A. Cobb, Department of Agriculture, Washington, D. C., who

examined the collection with the view of adding to the completeness

of the key he is preparing for the identification of the American

free-living freshwater nematodes.

1. Tylencholaimellus n. g. 2 6.6 12. -M- a

diplodorus, n.sp. 22 36 47 8 mm.

The thin transparent layers of the vieeayl naked ais are traversed

by exceedingly fine transverse strie visible for the most part only in the

subcuticle. Longitudinal striz exist throughout the body, probably associated

with the attachment of the muscle cells to the subcuticle. The conoid neck

becomes convex-conoid near the head which is rounded and not set off in any

way. On the outer margin of the head there are six minute flattish conoid

papillae which when seen in profile slightly interrupt the outer contour of

the head. Surrounding the mouth opening there are what appear to be similar

papillae, but they are very inconspicuous so that at first glance the head

appears to have only a single circlet of cephalic palille, namely, the

posterior. The vestibule is very narrow, the lips closing together over the

apex of the spear, which apparently is located normally a distance twice as

far from the anterior extremity as the posterior circlet of papille, The main

portion of the spear is one-fifth as wide as the corresponding portion of the

head. This is followed by a “hilt” half as long as the main shaft, and

half as wide again, ending in a short, three-bulbed base fully one-third as

FRESH-WATER NEMATODES 29

wide as the corresponding portion of the neck. The entire length of the

spear is about four times the width of the lip region measured opposite the

posterior circlet of papille. The anterior portion of the spear is more

strongly chitinized than the posterior portion. It has a dorsally oblique

opening similar to that seen in Dorylaimus, and presents the striking pecul-

iarity of having an extra piece of chitin on the dorsal side. This extra

piece is bowed outward very slightly so that there is a space between it and

what would be regarded as the normal dorsal wall of the spear,—that is,

the wall which comes next to the lumen, through which the food must

pass. It is this peculiarity which gives rise to the specific name. The

cesophagus begins at the base of the spear as a tube about one-fourth as

wide as the corresponding portion of the neck and continues to be narrow

to near the posterior portion of the neck. At the beginning of the posterior

fifth of the neck, or thereabouts, the cesophagus suddenly enlarges to produce

an elongated cardiac swelling or bulb two-thirds as wide as the base of the

neck and two and one-half times as long as wide. The lining of the cs-

ophagus is very faint throughout its length. There is no very distinct cardia.

The intestine, which is separated from the cesophagus by a distinct constric-

tion, becomes at once three-fourths as wide as the body. Its cells contain

small scattered fairly numerous yellowish granules which are not disposed

so as to give rise to any tessellation. The appearance by transmitted light

is that of a firmament in which the stars are unusually close together.

The tail of the male begins to taper from some little distance in front

of the slightly elevated anus and is conoid to the rounded terminus which has

a diameter one-third as great as that of the base of the tail. No papille or

hairs occur on the tail. The supplementary organs are two in number, One

occurs a short distance in front of the anus opposite the junction of the

distal and middle thirds of the spicula. The second is nearly twice as far in

front of the anus as the proximal ends of the spicula. The supplementary

organs are simple in character, consisting of very low flat cones which interfere

but little with the ventral contour. The posterior is perhaps double, that 1s,

consists of two papillae side by side close to the ventral line. The two equal

arcuate spicula are one and two-thirds as long as the anal body diameter.

They are simple in character and are made of a rather frail framework.

They are widest toward the proximal extremity where the greatest width

is one-fourth as great as that of the corresponding body diameter. They

taper toward the proximal end from this widest part, and the proximal end

is almost imperceptibly cephalated by expansion. No very distinct acces-

sory pieces have been seen, though the lining of the sheath in which the

spicula glide is a little more pronounced toward the anus. The faintest

possible traces of very oblique copulatory muscles have been seen. There

are two small testicles outstretched in opposite directions after the manner

of Dorylaimus. The blind end of the anterior one is one and one-half times

as far behind the base of the neck as this latter is behind the anterior

extremity. Nothing is known concerning the renette.

Locality: Maple River, Mich.

30 MARGARET V. COBB

6 6.7 24 an 91

BLS ALOAe 3) 12 eee

The thin layers of the transparent, colorless, naked cuticle appear to be

destitute of transverse striations. If any occur they must be exceedingly

minute. Faint longitudinal striations exist throughout the length of the

body, and are most readily visible outside the borders of the lateral fields.

The posterior portion of the neck is cylindroid; toward the head it becomes

conoid. The subtruncate head is set off by a very broad, almost impercep-

tible constriction. The contour of the front of the head is approximately

flat. This flat portion occupies about two-thirds of the diameter of the front

of the head, and from its margin the head slopes at an angle of forty-five

degrees to join the short subcylindrical portion of the head opposite the

anterior part of the pharyngeal cavity. The lips are thoroughly amalgamated,

and the two blunt angles caused by the oblique marginal surface are the

locus of the two circlets of labial papillae which do not extend out so as to

materially break the contour, but are seen in optical section as refractions

rather than as elevations. The innervations are obscure. Amphids are

present in the form of somewhat elliptical markings, the plainest of which is

a refractive chitinous curve on the surface of the head opposite the anterior

portion of the collar of the spear. The width of these markings is about

one-fourth to one-third as great as that corresponding portion of the head.

When the amphids are seen dorso-ventrally they have the form of narrow

slits leading inward and backward from a distinct narrow groove opposite

the base of the lips, that is to say, opposite or slightly behind the bottom of

the anterior portion of the pharyngeal cavity. The mouth opening is circular

and fits the spear. Surrounding the mouth there is a somewhat discoid

structure which is separated from the outer portion of the wall of the head

by a circular groove as in Actinolaimus radiatus. The outer wall of this

shallow groove is very finely longitudinally striated. The disk-like structure

represents the inner portion of the lip region and is arched over the anterior

part of the pharynx which is more or less globoid in form, and one-half as

wide as the corresponding portion of the head. At its base there are six

chitinous processes which surround the spear and serve to guide it in action.

These project into the spheroidal chamber so that its base, instead of being

flat or concave, is really convex or conoid toward the lips. The optical section

of this portion of the cavity is therefore that of a somewhat broad crescent

rather than circular. The lateral walls of this cavity when seen in optical sec-

tion sometimes present appearances leading to the conclusion that it is minutely

dentate in some such way as is the wall of the pharynx of some species of

Mononchus. The observations on this point, however, are not yet sufficiently

decisive. At a distance from the anterior extremity equal to about two-

thirds of the radius of the front of the head is found the beginning of the

2. Actinolaimus propinquus, n.sp.

ae es al apis derived from a single specimen which apparently had lost a short portion

of the tail.

FRESH-WATER NEMATODES 31

guiding collar for the spear, which is compound, wider posteriorly than

it is anteriorly. Anteriorly it is but slightly wider than the spear, and

there is a narrow, refractive chitinous element or ring clearly indicating the

anterior border of the collar. The substance of the collar behind this border

is practically cylindrical, although it diminishes slightly in diameter to a

little in front of the middle of the collar, where it is flared and receives a

second element inside the space formed by the flare. This second element

is itself flared, though it is smaller than the first, and is continued backward

and ends rather indefinitely. Both these expansions, or “capes,” are margined

with refractive chitinous rings. Measuring to the limit of the second flare,

which is indicated by a chitinous ring similar to that which indicates the

anterior limits of the collar, it may be said that the collar is about as long

as it is wide in its widest part, namely, about twice as wide as the spear.

This latter is fully one and one-half times as long as the head is wide. It

is slightly swollen just behind the oblique dorsal aperture. In its posterior

three-fifths the spear is cylindrical, while in the anterior two-fifths it tapers

to an acute point, the ventral contour being slightly arcuate, while the dorsal

contour is nearly straight. The diameter of the shaft of the spear is about

one-fifth as great as that of the corresponding portion of the head, perhaps

a little less. The walls of the spear are well chitinized. Following the spear

is a tubular, rather indefinite portion, about three-fourths as long as the

spear, which joins a fusiform swelling about one-half as wide as the cor-

responding portion of the neck and about as long as the spear, through

which the lumen continues to be only slightly less narrow than in the spear

itself. The fibers in this fusiform portion extend forward as well as out-

ward. Behind this fusiform part the cesophagus is a tube nearly one-third

as wide as the corresponding portion of the neck. It continues to have this

diameter, although it varies a little, to a short distance in front of the nerve

ring, where there is an obscure swelling of very faint character. Behind

this swelling, the cesophagus for a short distance, where it passes through

the nerve ring, is about one-fourth as wide as the corresponding portion of

the neck, or a little less. Behind the nerve ring it expands gradually to a

point where there is a break in the musculature. At this point the cesophagus

is nearly two-fifths as wide as the corresponding portion of the neck. It

continues to have this diameter for a distance nearly equal to two of the

corresponding body diameters and then rather suddenly enlarges at a point

where there is a second break in the musculature. Thence onward the

cesophagus is cylindrical and nearly two-thirds as wide as the corresponding

portion of the neck. For a short distance near the base of the cesophagus

the internal tissues stain differently from those farther forward. They take

the carmine more strongly. This portion of the cesophagus is one and one-

half times as long as it is wide. It is not set off from the rest of the

cesophagus by any striking difference in the internal structure. The lining

of the cesophagus is a distinct feature, especially in the enlarged cylindrical

portion, where its optical expression consists of several marked refractive,

32 MARGARET V. COBB

parallel, longitudinal lines occupying fully one-fourth of the optical longi-

tudinal section. The rather broad conoid cardia is two-thirds as long as

the body is wide. The cardiac collum is about one-third as wide as the body

and presents the peculiarity that the lining of the cesophagus extends into

it a short distance. The constriction between the cesophagus and the intestine

is not only deep but rather broad, and one or two special spherical cells are

to be seen in this region. The thick-walled intestine becomes at once about

three-fifths as wide as the body, and is composed of cells of such size

that a considerable number are required to build a circumference, probably

about eight. These cells contain numerous brownish-yellow granules of

variable size, the largest having a diameter about one-half as great as

the internal diameter of the spear. The tail begins to taper from some little

distance in front of the anus and tapers at the same rate behind the anus. It

continues so to taper until near the extremity, which is sometimes blunt, some-

times conical, and sometimes subacute. For some little distance in front

of the terminus, namely, a distance fully equal to the length of the anal body

diameter, the tail is cylindrical and not more than one-twelfth to one-

twentieth as wide as at the anus. This latter is very slightly raised in that

the posterior lip is slightly elevated. From it the conspicuous rectum, which

is lined with refractive chitin, and is fully twice as long as the anal body

diameter, extends inward and forward. Anal muscles are present and rather

conspicuous, as are also rather numerous glandular cells. The pre-rectum

is about five times as long as the corresponding body diameter and about

three to four times as long as the rectum. The pre-rectum differs from the

rectum in being smaller in diameter, and its cells do not contain the brown-

ish-yellow granules seen in those of the intestine. The lateral fields are dis-

tinct and nearly one-third as wide as the body. Nothing is known concerning

the renette. The nerve ring surrounds the cesophagus somewhat squarely.

From the rather small vulva the vagina leads inward at right angles to the

ventral surface about half way across the body, where it joins the two

symmetrically placed uteri. The reflexed ovaries, which taper toward the

blind end, reach three-fourths of the way back to the vulva and contain

about two dozen developing ova, most of which are arranged in single

file and are more or less compressed into disk-like form. The eggs are

ellipsoidal and somewhat longer than the body is wide and a little less than

half as wide as long. The eggs are apparently deposited before segmenta-

tion.

Locality: Maple River and Beach Pools, Douglas Lake, Mich.

3.. Dorylaimus sulcatus, n.sp. 3 1.6 mm.

The very thick layers of the transparent, colorless, naked cuticle appear

to be destitute of transverse striations. Prominent longitudinal striz to

the number of about 32 are found throughout the length of the body except on

the lateral fields where for a distance equal to about one-fourth of the body-

width, they are absent. The cuticle becomes thinner near the lips. The

FRESH-WATER NEMATODES | 33

neck is conoid, tapering more rapidly toward the truncate head which bears a

lip region set off by an almost imperceptible, broad constriction. The lips are

thoroughly amalgamated, and each is armed with two plainly innervated

papillz, one on the outer surface and outward pointing, and one on the outer

margin of the front surface and forward pointing. These papille do not

interfere with the contour of the lips. The front of the head is very slightly

depressed in the middle. The pharynx is beaker-shaped, half as wide as the

front of the head, and a little deeper than it is wide. The apex of the spear

appears on a level with the front surface of the lips. Under these circum-

stances this beaker-shaped portion of the pharynx is continued as a some-

what narrower cylindrical part which is nearly filled by the spear for a

distance almost equal to the depth of the beaker-shaped portion. At the

base of this second narrow portion of the pharynx occurs the guiding collar

of the spear which closely surrounds the spear for a distance about

equal to its own width. The limits of the collar are indicated by two chitin-

ous rings, the anterior of which rather closely surrounds the spear, the pos-

terior and stronger of which is somewhat wider. The relatively small, some-

what stirrup-shaped amphids are just behind the outer circlet of papille.

They are about one-third as wide as the corresponding portion of the head.

Their anterior contour is a nearly straight, transverse element, from the ends

of which there extend backward and inward convex contours which become

indefinite opposite the base of the beaker-shaped portion of the pharynx.

There are no eye-spots. The massive spear is three times as long as the front

of the head is wide and tapers from base to apex. Seen in profile, the taper

of the posterior three-fifths is only slight, whereas that of the anterior two-

fifths is more pronounced; the contour is straight on the dorsal side,

where the opening is, and slightly convex on the ventral side. The apex

is acute. When seen in optical longitudinal section, the chitin forming the

spear is seen to be bifurcated at the posterior margin, more particularly on

ventral side. At its maximum width, namely, at the base, the spear is nearly

half as wide as the front.of the head and about one-fifth as wide as the cor-

responding portion of the neck. There is no distinct fusiform swelling at

some little distance behind the spear as is often the case in Dorylaimus. The

lumen of the spear is continued by that of the cesophagus for a distance

about equal to the length of the spear; at the posterior extremity of this

portion there is an obscure joint in the lining of the tube. Otherwise, there

is no indication of the secondary extending element of the spear. The

cesophagus begins behind the secondary element as a tube fully one-third

as wide as the corresponding portion of the neck, and continues to have

this diameter until it passes through the nerve ring. At this point it begins

to enlarge almost imperceptibly, and finally at the beginning of the pos-

terior third, it enlarges rather rapidly so that in this portion of the neck it

occupies one-half of the diameter. At the extremity, finally, it is really a

little more than one-half as wide as the base of the neck. There is a some-

what spheroidal cardia, one-fourth as wide as the base of the neck. The

34 MARGARET V. COBB

lining of the cesophagus is a very distinct feature throughout its length. In

the posterior enlarged portion the lining consists of several parallel longi-

tudinal elements occupying one-fourth of the optical longitudinal section ;

in the narrow tubular part the lining occupies one-third of the diameter.

The rather thick-walled intestine, which is separated from the cesophagus

by a fairly distinct constriction, becomes at once two-thirds as wide as the —

body. Its walls are composed of cells of such size that probably a consid-

erable number are required to build a circumference, perhaps six to eight.

These cells contain rather numerous brownish-yellowish granules of variable

size, the largest of which have a diameter about one-third as great as the

thickness of the cuticle, the smallest of which are much smaller. The

granules are so arranged as to suggest a faint tessellation. Of the large

granules, two to three would go side by side into the base of the spear.

From the slightly depressed anus the conspicuous, chitinized rectum, which

is one and one-half times as long as the anal body diameter, extends inward

and forward. The pre-rectum is about three times as long as the rectum

and is distinctly separated from the intestine by difference in size as well

as difference in structure; it is considerably narrower. Only a_ short

distance in front of the constriction separating the pre-rectum from the

intestine, the intestine is nearly fifty per cent wider than the pre-rectum. The

brownish-yellow granules characteristic of the cells of the intestine are

absent from those of the pre-rectum: The tail begins to taper from some

distance in front of the anus; near the anus it tapers more rapidly, and

again more rapidly still behind the anus, in such fashion that the anterior

three-fourths are concave-conoid; the reduction in diameter is such that at

the beginning of the final fourth, the tail measures only about one-tenth

as wide as at the base. Thence onward the tail tapers but very slightly to

the rather blunt, colorless terminus. The posterior fourth is destitute of axial

matter. The cuticle of the posterior portion of the body, though not striated

in the usual way, is radially striated. These radial markings come out most

clearly in optical longitudinal section, and are visible on the surface, inasmuch

as the surface appears to be finely granular. These striations are so minute

that they would escape observation except with high powers and under

favorable conditions. Once having seen this striation in the posterior part

of the cuticle, one remarks it as less plainly visible throughout the body.

Nothing is known concerning the renette. The nerve ring is rather massive

and surrounds the cesophagus rather squarely. Connected with it, and

near by, are numerous ganglion cells, somewhat more conspicuous than is

usual in this genus. From the depressed vulva, the strongly chitinized, mas-

sive vagina leads inward at right angles to the ventral surface fully half

way across the body. The vagina is rather complicated in structure, con-

sisting of central masses of structureless chitin forming a somewhat bell-:

shaped contour when seen in optical longitudinal section. These elements

are surrounded by granular and fibrous muscular structures, and into the

internal angle which the chitinous elements form near the axis there extend

FRESH-WATER NEMATODES 35

other refractive, more or less chininous elements. Small unicellular glands

are to be seen on both sides of the vagina,—anterior and posterior. The uteri

are two in number and symmetrically disposed. The reflexed ovaries reach

half way back to the vulva, at least in immature specimens such as that from

which the description is derived. At a distance in front of and behind the

vulva about equal to the length of the corresponding body diameter, there

are slightly depressed ventral areas in which the radial striation of the chitin

is materially altered and into which there appear to pass fibrous elements

from the sub-cuticle, connected with special groups of small cells, presumably

nerve cells, located in the body wall.

Locality: Maple River, Michigan.

35.9

PEAT 21 22 81 219 518 902 |,

F 01 Sy, ———__—_——__—__—_———— _ 16 mm.

y Pee sie Hon Et

The moderately thick layers of the transparent, colorless, naked cuticle

appear to be traversed by exceedingly fine transverse striae. The conoid neck

ends in a slightly expanded, rounded head whose chitinous elements are

unusually refractive. On the outer surface of the head at a distance from

the anterior extremity about equal to the radius of the head there is a circlet

of four, equal, arcuate, spreading cephalic setae, one-third as long as the

corresponding portion of the head is wide. There are three lips each armed

with a highly refractive, arcuate-conoid, acute, inward-pointing tooth with

a somewhat forward pointing apex. These teeth, which are nearly half as

long as the head is wide, are arranged opposite each other near the mouth

opening and are doubtless capable of being everted, though they have not

been seen in the everted position. In growing specimens additional sets of

these teeth are often seen at a distance from the anterior extremity about

equal to two to three head widths. Immediately behind the vestibule, which

is the narrow passage leading between the teeth, the pharynx is wider than

it is farther back. At this point, immediately behind the base of the lips the

pharynx is one-half as wide as the corresponding portion of the head. It

soon narrows, so that at a distance from the anterior extremity equal

to the diameter of the head, it is less than one-third as wide as the corre-

sponding portion of the head. Thence on, it is of uniform diameter, tubular

and slightly sinuous to the truncate end. The walls of the pharynx are

distinctly chitinized and are surrounded with little tissue. The cesophagus

begins at the base of the pharynx as a tube two-thirds as wide as the corre-

sponding portion of the neck and very gradually expands as it passes back-

ward, so that finally it is two-thirds as wide as the base of the neck. The

lining of the cesophagus is a distinct feature throughout its length and

generally appears in optical, longitudinal section as a series of two or three

parallel longitudinal lines, occupying about one-eighth of the optical sec-

tion. There is a flattish-hemispherical cardia about half as wide as the base of

the neck. The vaguely-tesselated intestine, which becomes at once about two-

36 MARGARET V. COBB

thirds as wide as the body, is separated from the cesophagus by a distinct

constriction. It is thick-walled and is composed of cells of such size that

probably but few are required to build a circumference. These cells con-

tain scattered, yellowish-brown granules of variable size. From the incon-

spicuous but slightly elevated anus, the rectum, which is one and one-half

to two times as long as the anal body diameter, extends inward and forward.

The rectum is separated from the intestine by a fairly definite pyloric con-

striction. The tail tapers from some distance in front of the anus and con-

tinues to taper behind the anus at about the same rate to the nearly hair-

fine terminus, which is destitute of a spinneret. The longitudinal fields

appear to be one-third as wide as the body. The nerve ring surrounds the

cesophagus somewhat obliquely. Nothing is known concerning the renette.

From the elevated vulva, the well developed vagina leads inward at right

angles to the ventral surface half-way across the body, where it joins the two

symmetrically placed uteri. The reflexed, tapering ovaries reach about half-

way back to the vulva and contain about eight developing ova. The moder-

ately thin-shelled, smooth eggs are much elongated and about five times

as long as the body is wide, and about one-sixth as wide as long. They

appear to be deposited before segmentation begins.

Locality: Beach pools and bogs, Douglas Lake, Michigan.

19,

A Wis ei ale YN 5 ROR

5. Mononchus lacustris, n.sp. |g 2] 24 27 18 1.7 mm.

The thin layers of the transparent, colorless, naked cuticle appear to be

destitute of transverse striations. If any are present they must be exceed-

ingly minute. The cylindroid neck tapers slightly toward the head, which

bears a very slightly expanded lip region composed of six confluent lips, each

bearing two papillz, one on the outer surface and outward pointing, and

one on the front surface halfway between the margin and the body axis, and

forward pointing. This inner circlet of papille is composed of members of

somewhat larger size than those of the outer circlet. Both are flattish conoid

in shape, and impart to the contour of the lips a somewhat angular appear-

ance. The thick lips are arched together over the goblet-shaped pharynx,

which has a depth equal to the diameter of the head, measured opposite the

base of the pharynx. At its widest part, a short distance behind the lips,

opposite the apex of the dorsal tooth, the pharynx is three-fifths as wide

as the corresponding portion of the head. Thence backward it tapers rather

regularly to near the base, where it tapers a little more rapidly in that part

which extends into the anterior end of the cesophagus. The walls of the

cesophagus are well chitinized. The dorsal tooth springs from near the

middle of the dorsal side of the pharynx, and extends inward and forward.

The apex is forward pointing. At its greatest width the tooth is about

one-third as wide as the corresponding portion of the pharynx, though it

projects forward so that its apex lies opposite the axis of the head. The

FRESH-WATER NEMATODES 37

axis of the dorsal tooth is not straight, but slightly curved, that is to say,

has the form of a line of beauty. Surrounding the cesophagus opposite the

dorsal tooth there is a band of minute chitinous teeth on the wall of the

pharynx arranged in transverse series. Where these teeth are most numer-

ous there are at least six transverse rows. They begin near one lateral line

and extend around the ventral side of the pharynx to the other lateral line.

Viewed at a certain angle, which is very near the lateral view, these teeth

appear, on a surface inspection, to occupy an elongated elliptical area, and

with low powers one might easily mistake one of these areas for the outer

expression of one of the amphids. Behind this area of minute rasp-like teeth

there are two small projections from the inner surface of the pharynx,

which are undoubtedly rudimentary teeth of the same character as the large

dorsal teeth. These are several times larger than the minute teeth just men-

tioned, and are located opposite the base of the large dorsal tooth just behind

the rasp-like area. They are practically submedian in position, one near each

ventrally submedian line. The inconspicuous amphids are on the lips just

behind the outer papille. They are somewhat circular in contour, one-fifth as

wide as the lip-region, and only their anterior contours are definite. There are

no eye-spots. The cesophagus receives the base of the pharynx, and is at

that point almost imperceptibly enlarged. A.short distance behind the

pharynx the cesophagus is nearly three-fifths as wide as the corresponding .

portion of the neck. Thereafter it diminishes very slightly, until after it

passes through the nerve-ring, when it begins to increase, and continues

gradually to increase throughout the remainder of its length. It finally

becomes two-thirds as wide as the base of the neck. The lining of the

cesophagus is a very distinct feature throughout its length, more particularly

in the anterior part, where it occupies about one-fourth of the longitudinal

optical section, and finds optical expression in three refractive parallel lines.

There is no very distinct cardia. The intestine, which is set off from

the cesophagus by a distinct constriction, becomes at once about two-thirds

as wide as the body. It is composed of cells of such a size that six

to eight posteriorly, and ten to twelve anteriorly, are required to build

a circumference. These cells are packed with minute granules of rather

uniform size, which, together with refractive cell walls, give rise to an

indistinct tessellation. From the raised anus the conspicuous rectum,

which is about as long as the anal body diameter, extends inward and

forward. The conoid tail tapers from the anus and ends in a subtruncated

terminus, containing a single somewhat depressed axial pore, which forms

the outlet for the secretions of the three caudal glands. These latter lie in

a tandem series, one opposite the rectum, and the other two immediately

behind, the posterior cell being slightly the smallest, and having its limits

near the middle of the tail. The ducts form a conspicuous feature, and to-

gether occupy about one-third the diameter of the middle of the tail. They

are slightly expanded posteriorly, so that one may with correctness speak

38 MARGARET V. COBB

of the presence of ampulla. The lateral fields are about one-third as wide

as the body. From the slightly elevated vulva the vagina extends forward

at right angles to the ventral surface nearly halfway across the body, where it

joins the two symmetrically placed uteri. The reflexed ovaries reach

nearly three-fourths the distance back to the vulva, and contain about a

dozen developing ova, arranged for the most part somewhat irregularly.

The distal end of the ovary tapers rather rapidly. Judging from the

size of the ripe ova the eggs are nearly twice as long as the body is wide,

and not much more than one-third as wide as long. Nothing is known

concerning the renette. The nerve-ring surrounds the cesophagus somewhat

squarely. The single specimen examined contains the remains of an organism

that has been swallowed. This may be the skin of another nematode.

Locality: Beach Pools, Douglas Lake, Michigan.

; Sade 17 92 16).~49, So,

6. Cyatholaimus fluviatilis n.sp. A 2627 39 23 9 mm.

The thin layers of the transparent, colorless, naked cuticle are traversed

by exceedingly fine transverse strie, resolvable with the very greatest diff-

culty into rows of dots which are not modified on the lateral fields. The

conoid neck ends in a rounded head which is not set off in any way. The

lip region is divided internally into about twelve parts, whose refractions

give rise to a striated appearance of the vestibule extending backward from

the front of the head to near the apex of the dorsal tooth. When the

mouth is partially open each of these elements has a triangular contour, the

bases being adjacent to a sinuous, refractive, chitinous line encircling the

base of the lips on the inside of the vestibule. The pharynx is open and

somewhat elongated. Near the base it is about one-fourth as wide as the

base of the head. A little farther forward it is 50% wider; in this part the

thumb-shaped dorsal tooth is found. This tooth springs from the dorsal

side of the pharynx and extends inward at an angle of approximately 30°

with the axis of the head. It is slightly arcuate, so that its apex is directed

nearly forward. The wall of the pharynx is fairly well chitinized. There

are ten cephalic sete on the outer margin of the head opposite the apex

of the dorsal tooth, two on each submedian line, and one on each lateral line.

These sete are about equal in length throughout the series,—about one-third

as long as the corresponding diameter of the head. The amphids are so

located that their posterior margins are a little in front of the base of the

pharynx; they consist of spirals of a little more than two winds, and are

about one-third as wide as the corresponding part of the head. The cesoph-

agus surrounds the pharynx without being enlarged. It begins at the base

of the pharynx as a tube fully two-thirds as wide as the corresponding portion

of the neck, and retains this diameter throughout most of its length, but

finally expands to form an obscure, ellipsoidal bulb or pseudo-bulb, five-

sixths as wide as the base of the neck. The lining of the cesophagus is a

FRESH-WATER NEMATODES 39

fairly distinct feature throughout its length. There is no distinct valve in

the pseudo-bulb. The moderately thick-walled intestine is separated from

the cesophagus by a very deep and distinct constriction and becomes at once

two-thirds as wide as the body. The cells composing the intestine are of

such size that about eight to ten are required to build a circumference. They

contain scattered granules of variable size, the largest of which have a diam-

eter such that they would slip inside the dorsal tooth. From the inconspicu-

ous anus the very unusually long rectum extends inward and forward. The

rectum is separated from the intestine by a distinct pyloric constriction. The

entire length of the rectum is three to four times as great as the anal body

diameter. The tail is conoid from the anus to the terminus, whose diameter

is about one-fifth as great as that of the base of the tail and bears a plain,

conoid, apparently unarmed spinneret. There do not appear to be any sete

on the tail. The lateral fields are about one-half as wide as the body and

are composed of cells of large size arranged somewhat irregularly in two

rows. From the slightly elevated vulva the chitinized vagina leads inward

at right angles to the ventral surface nearly half-way across the body, where

it joins the two symmetrically placed uteri. The reflexed, tapering ovaries

reach half way back to the vulva and contain comparatively few developing

ova. The thin-shelled eggs have been seen in the uterus one at a time and

are three times as long as the body is wide, and somewhat less than one-

third as wide as long. They are apparently deposited before segmentation

begins.

Locality: Maple River, Michigan.

3.2 10.8 19. 49. 90.

7. Chromadora inornata, n.sp. AO. Ole au Rk TOA 5 mm,

The thin layers of the transparent, colorless, naked cuticle are traversed

by very fine striz which do not appear to be further resolvable. It should

be noted, however, that the examination was confined to a single balsam

specimen, and that a more complete observation may perhaps show the

striz to be further resolvable. Along the lateral field are faint longitudinal

striz, beginning near the middle of the neck and extending on to the tail.

Of these there are two double lines quite near together, occupying a space

probably not greater than one-twelfth of the diameter; outside these, other

fainter lines. No traces of cephalic sete have been seen, but, as the descrip-

tion is derived from a single specimen, it is barely possible that they are

present and have been broken off. Other specimens in the same lot being

in fairly good preservation, it seems likely that there are no cephalic sete.

Unfortunately this specimen is dirty at the head end, so these matters have

to be left in uncertainty. The conoid neck ends in a somewhat rounded

head which is not set off in any way. In the specimen examined the lips

are drawn together and the dorsal tooth is located at a distance from the

anterior extremity nearly equal to the radius of the front of the head.

Under these circumstances the lips are closely surrounded by a narrow vesti-

40 MARGARET V. COBB

bule. The number of lips remains uncertain, but there are at least six, and

probably may be twelve. The vestibule, however, does not appear to be

striated. That portion of the pharynx behind the lips and in front of the

dorsal tooth is small, not more than one-fourth as wide as the corresponding

portion of the head. The narrow portion behind the dorsal tooth is tubular

or prismoid and extends backward to a point removed from the anterior

extremity a distance slightly greater than the diameter of the head. This

portion of the pharynx is only about one-eighth as wide as the corresponding

portion of the head. The dorsal tooth is small, rather narrowly conoid, acute,

and forward pointing, and is located at the body axis. Its altitude is about

equal to the width of the posterior portion of the pharynx. There are no

eye-spots. No amphids have been seen. There is a fairly distinct pharyngeal

swelling, whose presence is indicated not so muclhr by its size as by the fact

that it is separated from the cesophagus by a difference in internal structure;

the fibers composing it are arranged in longitudinal manner. Beginning at

the base of the pharynx the cesophagus is about three-fifths as wide as the

corresponding portion of the neck. It continues to have this diameter

until the middle of the neck, where it diminishes very slightly in diameter

and then passes through the nerve ring. Just in front of the cardiac bulb,

its width is about two-fifths that of the corresponding portion of the neck,

The cardiac bulb is prolate and five-sixths as wide as the base of the neck.

It does not contain any distinct valve, or if it does the valve is hidden

by the radiating muscles. The bulb is divided into subequal parts by a break

in the musculature slightly behind the middle. There is no distinct cardia.

The lining of the cesophagus is a fairly distinct feature throughout its

length. The intestine, which is separated from the cesophagus by a distinct

constriction, becomes at once three-fifths as wide as the body, and the width

of the cardiac collum is about one-half as great as that of the base of the

neck. The intestine is composed of cells of such size that probably two to

three are required to build a circumference. These contain rather numerous

small granules of uniform size. The anus is nearly continuous, but the

posterior lip is very slightly elevated. The rather refractive rectum which is

somewhat longer than the anal body diameter extends inward and forward.

There is a fairly distinct pyloric collum. The tail tapers from some little

distance in front of the anus and continues to taper behind the anus at the

same rate to the terminus which bears an elongated, conical, truncate, unarmed

spinneret, whose length is considerably greater than that of the diameter of

its base. This latter is about one-eighth as wide as the base of the tail.

The three caudal glands are located in tandem series in the anterior two-

fifths of the tail. Their nuclei are placed toward the dorsal side. The cells

themselves are contiguous and are rather broadly saccate. The lateral

fields are one-third as wide as the body. The renette is located a short dis-

tance behind the base of the neck and presses the intestine rather sharply to

one side. It is about one-half as long as the body is wide and one-half as

wide as long. The location of the excretory pore remains uncertain, but it

FRESH-WATER NEMATODES eee |

seems likely that it is near the lips. The nerve ring surrounds the esophagus

somewhat squarely. The female organs are double and symmetrically

reflexed. Nothing is known concerning the size, number and form of the

eggs, as the description is derived from a single, immature female.

Locality: Maple River and Bessey Creek, Douglas Lake, Michigan.

NOTE—This species bears a general resemblance to Chromadora Orleyi, de Man,

but drleyi has a striated vestibule and cephalic sete, and also appears to have a somewhat

more elongated pharynx. The discovery of the male might show other differences.

19,

A EN Yop a

ETC ROR TB GS &

The thin layers of the transparent, colorless cuticle are traversed by

fine striz, resolvable with difficulty into rows of minute dots which are not

modified on the lateral fields. Short, scattered and very inconspicuous hairs

are found on the body. These are usually of a length not much greater

than the thickness of the cuticle. The conoid neck ends in a rounded head

which is not set off in any way. On the outer surface, removed from the

anterior extremity a distance nearly equal to the length of the radius of the

head, there are four very slender, spreading cephalic sete, each half as long

as the head is wide. There are scattered hairs to be found on the neck,

seldom more than one-fourth as long as the corresponding body diameter.

The amphids consist of spirals of a little more than one wind, placed opposite

the middle of the posterior half of the pharynx and each about one-third as

wide as the corresponding portion of the head. The anterior portion of the

pharynx is broadly conoid or cup-shaped, and at the base of this part the

forward pointing dorsal tooth is seen lying in the axis of the head. This

anterior portion of the pharynx at its widest part is half as wide as the head,

and is about two-thirds as deep as wide. Extending backward from this

is a comparatively definite rather uniform, narrow tubular portion of the

pharynx whose walls are well chitinized and which ends suddenly at the

point where the cesophagus proper begins. The pharynx is surrounded by a

distinct pharyngeal bulb which is set off from the cesophagus by a distinct

constriction. The pharyngeal bulb is three-fourths as wide as the head. The

anterior portion of the pharynx is surrounded by minute more or less con-

fluent lips, too small to be accurately enumerated. It is not unlikely that

there are twelve of them. They do not materially break the comparatively

rounded contour of the front of the head. The dorsal tooth has a length

about equal to one-sixth the diameter of the head. It is somewhat blunt and

the width of its base is nearly as great as its altitude. The cesophagus begins

as a tube two-thirds as wide as the corresponding portion of the neck and

continues to have this diameter until it expands to form the prominent ellip-

soidal cardiac bulb which is five-sixths as wide as the base of the neck.

The bulb contains a rather obscure, apparently rather simple, fusiform valvular

apparatus about one-fourth as wide as itself. The lining of the cesophagus

8. Ethmolaimus gracilicaudatus, n.sp.

bo 5 nth MARGARET V. COBB

is not a very distinct feature. The anterior portion of the intestine, which is

separated from the cesophagus by a very distinct constriction, is slightly

different in histological structure from that portion which immediately fol-

lows. The portion thus characterized is only about half as long as it is

wide. The intestine joins the middle of the posterior surface of the cardiac

bulb, and the cardiac collum is one-third as wide as the base of the neck.

The intestine becomes at once about two-thirds as wide as the body, but in

the anterior part is pushed to one side by the renette cell, so that immediately

behind its beginning it is only about one-third as wide as the body. Its cells

contain minute granules of rather uniform size, not distributed in such a way

as to give rise to any tessellation. The number of cells required to build

a circumference is few, perhaps only two. The lining of the intestine, at any

rate in the posterior part, is refractive, so that the narrow, sinuous lumen is

rather readily seen. The tail tapers from in front of the anus, but only slightly.

Behind the anus it tapers more rapidly, and rather uniformly in the anterior

three-fourths. The posterior fourth is rather slender and tapers but little.

The spinneret is very narrow and elongated-conoid, slightly blunt at the end,

and is armed with exceedingly slender, rather short sete. The caudal glands

are arranged in a close tandem series in the anterior fourth of the tail.

From the anus, only the posterior lip of which is raised, the slightly

chitinized rectum, which is about as long as the anal body diameter,

extends inward and forward. The lateral fields are one-third as wide as

the body. The nerve ring surrounds the cesophagus somewhat obliquely. The

renette is a cell half as long as the body is wide and apparently two-thirds

as wide as long, located a short distance behind the base of the neck. The

position of the excretory pore remains to be discovered. From the rather

inconspicuous vulva, the chitinized vagina leads inwards at right angles to

the ventral surface half way across the body, where it joins the two sym-

metrically placed uteri. The reflexed ovaries reach well back toward the

vulva. This description is derived from a female specimen in an unsatis-

factory state of preservation, and these details are subject to revision. Noth-

ing is known concerning the size, number and form of the eggs.

Locality: Maple River, Michigan.

The female of this species at any rate presents many of the characters be-

longing to Dr. de Man’s genus Ethmolaimus, but possesses distinct spiral am-

phids which could hardly have been overlooked in the type species of Ethmo-

laimus.

9. Aphanolaimus viviparus, n.sp.— 9. 16. 148." 87.

ie Perle ane am 85 01

The moderately thick layers of the transparent, colorless, naked cuticle

are traversed by 700 plain transverse strize which are not further resolvable

and are not modified on the lateral fields, but which are interrupted there by a

single, distinct, refractive wing. The contour throughout is somewhat crenate.

1.2 mm.

FRESH-WATER NEMATODES 43

The conoid neck ends in a rounded head whose lip region is set off by an

almost imperceptible expansion. At the base of the lip region, at a distance

from the anterior extremity nearly equal to the radius of the lip region,

there are four well-developed, submedian, spreading, rather flexible, slightly-

tapering, cephalic sete, each about one and one-half times as long as the

head is wide. The three exceedingly small lips project a little from the

middle of the front of the head, and lead directly to the cesophageal passage-

There does not seem to be any distinct pharynx. The amphids are so located

that their anterior borders are opposite the bases of the cephalic sete. They

sometimes appear to be subcircular, a little longer than they are wide, and

appear to be about two-thirds as wide as the corresponding portion of the

head. In some specimens they may be clearly seen to be open spirals of

about one and one-half winds. Their contours are distinctly refractive, and

the tissues just outside them stain a little more strongly. The strize, which

are sO prominent on the neck as to cause a distinct crenate contour, become

less distinct opposite the posterior borders of the amphids. There seem,

nevertheless, to be about three striz opposite the amphids. There are no

eye-spots. The cesophagus begins as a tube about one-half as wide as the

base of the head and expands but little and very gradually. Toward the end

it becomes half as wide as the corresponding portion of the neck and then

diminishes so as to be only one-fourth as wide as the base of the neck ;

for a short distance before joining the intestine it tapers so as to be one-half

as wide as it is a short distance farther forward. The constriction between

the cesophagus and the intestine is very broad and shallow. The lining of

the cesophagus is a distinct feature throughout its length. There is no dis-

tinct cardia, but for a short distance the cells composing the intestine are

smaller and stain more strongly than those farther back. The intestine

gradually becomes one-half as wide as the body and is composed of cells

of such size that only a few, probably three to four, are required to build

a circumference. The intestine is moderately thick-walled. It is separated

from the cesophagus by a distinct cardiac constriction. From the slightly

raised anus the chitinized rectum, which is one and one-half times as long

as the anal body diameter, extends more directly forward than is usually

the case. The tail tapers from a little distance in front of the anus and

continues to taper at the same rate behind it until near the end, where it is

cylindrical for a short distance. The terminus is not expanded and ends

in a concave-conoid, truncated, unarmed spinneret. It constitutes a short,

broad apiculum. The. caudal glands constitute a rather open tandem series

in front of, opposite to, and behind the anus,—occupying the anterior fourth

or fifth part of the tail. The lateral fields are about one-third as wide as

the body. The renette cell is a much elongated cell lying opposite the cardiac

constriction. It is fully twice as long as the body is wide and not much

wider than the cesophagus. It possesses a distinct nucleus near its middle.

The duct leading forward from it is narrow and apparently empties through

the excretory pore located opposite the nerve ring. The nerve ring sur-

44 MARGARET V. COBB

rounds the cesophagus obliquely. From the almost imperceptibly elevated

vulva the chitinized vagina leads inwards at right angles to the ventral sur-

face one-third the distance across the body, where it joins the two symmet-

rically placed uteri. The small reflexed ovaries reach about one-fourth the

distance back to the vulva and contain about a dozen developing ova arranged

for the most part single file. Each of the uteri contains a half-dozen thin-

shelled smooth eggs in various stages of development, the most advanced

containing fully-formed embryos. It is not unlikely that the species is vivi-

parous. Male supplementary organs 5 to 8.

Locality: Beach pools, Douglas Lake, Michigan.

23.

; ms ete ke oe By Wp | SA

10. Aphanolaimus communis. “6 LS 25 27 12 1. mm.

The rather thin layers of the transparent, colorless, naked cuticle are

transversed by about 600 transverse striz, which exist in the outer cuticle, and

are of such a nature that they cause the contour to be minutely crenate

throughout the length. These strie are not further resolvable. They are

interrupted on the lateral fields by a single highly refractive wing. It is

probable that there are four minute, somewhat forward pointing submedian

cephalic setz, each about one-third as long as the head is wide. The conoid

neck ends in a rounded head, which is not set off in any way. The mouth

pore in the front of the head is exceedingly minute; it is very slightly

depressed. The circular amphids are so located that their anterior borders

are removed from the anterior extremity a distance about equal to the

radius of the head, or a distance about equal to their own radius. They

appear to be about half as wide as the corresponding portion of the head.

Although they appear circular at first sight, they are probably really spiral

in form, and unclosed in the anterior border. There is a very minute

pharynx about one-third as wide as the front of the head, one-half as wide

as the amphids. Possibly there is a chamber extending backward from

this, having a length such that it extends a little farther back than the amphids,

but this is somewhat doubtful. The cesophagus begins opposite the amphids as

a tube nearly half as wide as the corresponding portion of the head, and ex-

pands very slightly in diameter as it passes backward, until it reaches the nerve-

ring. Thereafter it is uniform in diameter. There is no cardiac swelling. The

intestine is set off from the cesophagus by a distinct constriction, and in the an-

terior portion is not much wider than the base of the cesophagus. Gradually,

however, it widens until it becomes two-thirds as wide as the body. It is rela-

tively thin walled, and is composed of elongated cells of such a size that

probably three to four are required to build a circumference. From the

rather inconspicuous anus the chitinized rectum extends inward and forward.

There is a distinct pyloric collum. The tail tapers from some distance in

front of the rectum, and continues to taper through its anterior four-fifths.

Thence onward the tail is cylindroid to the convex-conoid terminus, which

FRESH-WATER NEMATODES 45

bears a truncate conoid spinneret. The wall of the tail is relatively thick,

and the caudal glands are rather small sized, and are located in a tandem

series in the anterior third of the tail. The lateral fields appear to be about

one-third as wide as the body. The renette is of relatively large size, and

is located opposite the posterior portion of the cesophagus. It is fully half

as wide as the base of the neck, and is about one-third as wide as long.

Anteriorly it tapers to join the duct, which leads forward and perhaps empties

near the base of the lips. The nerve-ring surrounds the cesophagus some-

what obliquely. From the slightly depressed vulva the chitinized vagina leads

inward at right angles to the ventral surface fully half-way across the body,

where it joins the two symmetrically placed uteri. The broad reflexed ovaries

contain a dozen or more developing ova arranged somewhat irregularly. Only

the distal third of the ovary is tapering. The somewhat thin-shelled eggs

are fully one and one-half times as long as the body is wide, and less than

half as wide as long. They have been seen in the uteri one at a time, and

apparently are deposited before segmentation begins.

oe Ui peas tS) gaa 74

6 22 24 — 18

The tail of the male is similar in size and form to that of the female.

The two equal, arcuate spicula are one and ‘one-half times as long as the

anal body diameter. They are broadest in the middle, and of uniform

width for some distance in the middle part. Near the distal extremity they di-

minish rather suddenly in size, and thence outward they are of uniform diam-

eter to the end. The formation of the proximal end is somewhat the same,

but this portion is longer and slightly arcuate. There is no distinct cephalum.

At their widest point they are one-fifth as wide as the corresponding body

diameter. In front of the anus there are ten tubular supplementary organs

arranged at an angle of about forty-five degrees with the body axis. These

organs are about half as long as the body is wide, and are themselves

somewhat arcuate. The proximal end of each is slightly cephalated. The

diameter of the main portion is about the same as the diameter of the distal

portion of the spicula. Each tubule ends in a slight depression on the ventral

line, and is probably protrusile. The posterior member of the series is

opposite the proximal ends of the spicula, and the anterior member nearly

as far in front of the anus as the terminus is behind it. The ejaculatory

duct is about one-third as wide as the body. The vas deferens is about

half as wide.

9 mm.

Locality: Beach Pools, Douglas Lake, Michigan.

11. Prismatolaimus digitatus, n.sp. 11 12. 26. ‘63’ __81._ .6 mm.

oman, Gat. 2c ae

The rather thin layers of the transparent, colorless cuticle are trav-

ersed by 380 transverse strie, resolvable with high powers into rows of

minute elements, at any rate toward the head end. Scattered sete are

found here and there on the surface of the body. These have a length

46 MARGARET V. COBB

equal to the width of two to three annules. The somewhat cylindroid neck

tapers but little, and ends in a rounded head, which is not set off in any way.

On the outer surface of the head, opposite a point between the anterior and

middle thirds, there is a circlet of six wide-spreading, finger-shaped sete,

each bearing a minute bristle at its end. These sete are nearly half as long

as the head is wide. The thin lips are arched together over the pharynx,

and each bears a minute, forward-pointing and slightly outward-pointing pa-

pilla, which may be seen quite distinctly with high powers when the lips are

which may be seen quite distinctly with high powers when the lips are

brought into exact profile. The unarmed prismoid pharynx begins immedi-

ately behind the base of the thin lips and ends suddenly at a distance

from the anterior extremity equal to the width of the head. The width

of the pharynx is about as great as the width of the head, and it is half as

long as wide. There are no eye-spots. The amphids are ellipsoidal markings

about one-third as wide as the corresponding body diameter. They have

their long axis located transversely, and are situated about twice as far

behind the base of the pharynx as this latter is behind the anterior extremity.

The csophagus receives the base of the pharynx, and is at first two-thirds

as wide as the base of the head. It continues to have about the same diam-

eter throughout its length, but it expands somewhat posteriorly so that it

becomes about two-thirds as wide as the neck. Between the cesophagus and

the intestine there is a spheroidal element nearly as wide as the cesophagus.

This perhaps may be regarded as a strongly developed cardia. It is set off

by a constriction on both sides, particularly on the side toward the cesophagus.

The lining of the cesophagus is a fairly distinct feature throughout its

length. The thick-walled intestine, which is set off from the cesophagus by

a distinct constriction, becomes at once about three-fourths as wide as the

body. It is composed of cells of such a size that three or four are required

to build a circumference. The anus is slightly raised, and the rectum,

which is as long as the anal body diameter, extends inward and forward.

The tail end begins to taper a short distance in front of the anus, and tapers

with considerable regularity to the terminus. This latter has a width about

one-eighth as great as that of the base of the tail, and appears to bear a minute

apiculum, but has no spinneret. The lateral fields appear to be about one-third

as wide as the body. The nerve-ring surrounds the cesophagus somewhat

squarely. Nothing is known concerning the renette. From the elevated vulva

the vagina leads inward and forward. The single uterus extends forward.

The ovary is reflexed, and has its blind end located in the vicinity of the vulva,

at least when the uterus is not occupied by an egg. Judging from the size of

what appears to be a mature ovum, the eggs are considerably elongated, prob-

ably three to four times as long as the body is wide, and about one-fourth

as wide as long. The ovary may contain fifteen to twenty ova, arranged

single file except toward the tapering blind end.

Locality: Beach Pools and Bogs, Douglas Lake, Michigan.

FRESH-WATER NEMATODES 47

VII. Literature Cited.

Coss, N. A.

1. ’93. Nematodes, Mostly Australian and Fijian. Dep’t Agric. New

South Wales, Misc. Publ. No. 13.

2. '13. New Nematode Genera Found -Inhabiting Fresh Water and

Non-brackish Soils. Journ. Wash. Acad. Sci. 3, No. 16.

3. 14. North American Free-living Fresh-water Nematodes. Trans.

Am. Micr. Soc. 33: 69-119, 7 Pi.

DE MAN, J. G.

4. ’84. Die frei in der reinen Erde und im stissen Wasser lebenden

atoden der Niederlandischen Fauna. Eine systematische-faunis-

tische Monographie. 206 pp., 34 PI.

DEPARTMENT OF NOTES, REVIEWS, ETC.

It is the purpose, in this department, to present from time to time brief original

notes, both of methods of work and of results, by members of the Society. All members

are invited to submit such items. In the absence of these there will be given a few brief

abstracts of recent work of more general interest to students and teachers. There will be

no attempt to make these abstracts exhaustive. They will illustrate progress without at-

tempting to define it, and will thus give to the teacher current illustrations, and to the

isolated student suggestions of suitable fields of investigation.—[Editor.]

NEW LIFE MEMBER.

The Secretary is pleased to announce that Mr. Seth Bunker

Capp of Philadelphia has been elected to Life Membership in the

American Microscopical Society. The Constitution provides that

the 50 dollars dues of life members shall become a permanent part

of the Spencer-Tolles Fund for the encouragement of microscopic

research. Members can render a real service to the Society by

bringing to the attention of people of means, who are interested in

encouraging scientific work, this provision of the American Micros-

copical Society.

ENTOMOLOGICAL NOTES.

Hearing in Saturnitide.—Turner (14, Biological Bulletin, 27:

325-332) reports results of a study of the auditory powers of Sat-

urnude. From experiments on four species: Telea polyphemus,

Samia cecropia, Philosamia cynthia and Callosamia promethia, the

author finds evidence which seems to prove that all of these species

hear. The last three species respond readily to a large range of

sounds, but Telea polyphemus normally does not respond perceptibly

to sounds. However if experimentally the moth is caused to asso-

ciate some disagreeable experience with some sounds, it can be

induced to respond to the latter. Responses are looked upon as

expressions of emotion.

Poison Glands.—Kephart (714, Journ. Parasitology, 1: 95-103)

finds that the poisonous element of the hairs of the larve of the

Brown-tail Moth which produces serious irritation to human skin

is contained in the short barbed hairs. Specialized hypodermal

cells secrete this substance, the latter gaining entrance to the blood

50 NOTES, REVIEWS, ETC.

through the point of the hair when it comes in contact with the skin.

There is a poison gland for each papilla on a tubercle but not one

for each hair.

Behavior of Insects and Spiders—Turner, (’14, Journ. Animal

Behavior, 4: 394-413) gives a summary of “The Literature for

1913 on the Behavior of Spiders and Insects other than Ants.”

The essential results of each paper are briefly discussed. At the -

end of the paper the literature for the year 1913, consisting of one

hundred and twenty-six titles, is listed.

Catocala Moths.—Turner and Schwarz (Biological Bulletin, 27:

275-293) presents a paper on the “Auditory Powers of the Catocala

Moths.” An experimental field study on several species of the

genus Catocala has shown that several of the species observed

respond definitely to certain high pitched notes of the Galton whistle

but usually fail to respond to sounds of low pitch. Degree of

responsiveness differs in different species. Lack of response to

sounds of low pitch is not attributed to failure to hear such sounds,

but it is the opinion of the authors that these moths respond “only

to such sounds as have a life significance.”

Olfactory Sense of Insects—McIndoo (14, Smithsonian Misc.

Coll., 63: No. 9) presents evidence against the old view that the

olfactory sense of insects is located in the antenne and finds that in

Hymenoptera the organs of olfaction have their seat in certain defin-

ite regions, such as the bases of the wings, the bases of the legs, the

sting of the worker and queen honey bee, and the mouth parts,

where aggregations of “olfactory pores” occur. Evidence is also

presented in favor of the view that possibly these olfactory pores

are the true olfactory organs in all irisects. No evidence is found

of any organs of olfaction on the antenne.

PauL S. WELCH.

NOTES ON MICROSCOPIC TECHNIQUE.

Euparal.—Euparal is a mounting medium composed of a mix-

ture of camsal, sandarac, eucalpytol and paraldehyde, having a re-

fractive index of 1.483. It is put up in two forms, the colorless and

the green, the latter containing a copper salt which intensifies hema-

en a a Re by Lane

~~ a

ae es

AMERICAN MICROSCOPICAL SOCIETY 51

toxylin stains. The colorless is preferable when stains other than

hematoxylin are used. Either may be obtained from G. Griibler &

Co., Leipzig.

The primary advantage of this medium is that it spares deli-

cate objects the usual treatment with absolute alcohol, since objects

may be mounted in it directly from 95% alcohol. It dries rapidly

so that preparations may be studied with safety at the end of 24

hours. These two points should be of interest not only to investi-

gators, but to those who conduct classes in embryology in which

students prepare their own serial sections for study. Further,

cover-slips may be readily removed from old preparations by immer-

sion in 95% alcohol for several hours.

Another useful property of euparal is its low index of refrac-

tion which is well adapted to cytological study, giving a much desired

increase of visibility to delicate elements. It may also be noted

that it does not bleach the stain.

During the past year I have used euparal with a variety of

material and have found it uniformly satisfactory. It is superior to

Canada balsam in every way.

Safranin O and Lichtgriin—This is a staining combination es-

pecially well adapted for the study of chromosomes, and is one that

should be more widely used than is the case at the present time. In

many ways it is superior to Iron-Alum-Hematoxylin, although it

may be used to good advantage in conjunction with the latter.

The Safranin should be of good quality preferably Gribler’s

Safranin O. The usual solvent is anilin water which may be made

by shaking up 4 or 5 cc. of either anilin oil, or old discolored anilin,

in 90 cc. of distilled water and filtering. Enough 95% alcohol is

added to the filtrate to make a 10% alcoholic solution, in which 1

gram of safranin is then dissolved.

Lichtgriin (Griibler’s Lichtgriin L. S.), the acid counterstain,

is made up by adding .5 gram of the stain to 100 cc. of 95% alcohol.

The staining procedure is as follows: Sections on the slide

are stained in safranin for from 4 to 6 hours. Excess of the stain

is removed by washing in 35% alcohol for a few minutes. The sec-

tions are then rapidly passed up through the graded alcohols to 95%

from which they are transferred to Lichtgriin in which differentia-

52 NOTES, REVIEWS, ETC.

tion is done. The secret of good preparations is to let the lichtgriin

act just long enough to extract the excess of safranin. A little prac-

tice will enable one to tell from the general appearance of the slide

held up to the light when to stop. Usually for thin sections 30

seconds is enough. It is well to move the slide about all the time

it is in the lichtgrtn.

The excess of lichtgriin is hastily washed off in 95% alcohol

after which the slide is immersed for a second in absolute alcohol

and then transferred to clove oil for one or two minutes. Before

mounting the excess of clove oil may be removed by immersion in

xylol. Euparal may be used as the mounting medium after xylol.

Safranin stains chromosomes and chromatin elements a deep

red for which the light green background gives an excellent contrast.

Many object to lichtgriin because it is not permanent. However, I

have had preparations retain their brilliancy for two years or more

and that certainly is long enough for most lines of work.

Hematoxylin-Orange G Stain for Embryos—To those inter-

ested in a satisfactory double stain for vertebrate embryos, atten-

tion is called to the method published by J. T. Morris in the

Anatomical Record, Vol. 3, 1909, under the title “A Note on

Orange G counterstaining suggesting a useful method in the hand-

ling of embryonic tissue.” I have found the method extremely sat-

isfactory in staining serial sections of human embryos. It was sur-

prising to find that it stained chromosomes in dividing cells with all

the clear-cut detail of iron-alumhzmatoxylin.

H. L. WreMan.

University of Cincinnati, Dept. of Zoology.

METHOD OF PREPARING FLY’S TONGUE AS MICROSCOPIC OBJECT.*

A fly’s tongue when properly prepared and mounted makes a

very beautiful object for the microscope, but to the novice it has

been more or less a difficult task. With the following suggestions

the worst part of the preparation is overcome with much ease, that

is, making the lobes of the tongue lie flat and evenly spread out.

We have all noticed that when the fly places his tongue on

a flat surface it spreads out the lobes in a way we would best

* Presented before State Micr. Soc. of Ill., Feb. 11, rors.

AMERICAN MICROSCOPICAL SOCIETY 53

have it on our finished mount, so we will proceed to catch it in

this position.

Have ready two glass slips and place one of them at the edge

of a table where the operation is to take place. Take a fly between

the thumb and finger of the left hand and the other glass slip in

the right hand, hold the fly’s head at the edge of the glass slip on

the table and he will lay his tongue out flat on the glass. When

all is right, place the slip that is in the hand on top of the tongue

with enough pressure to hold it and cut off the tongue close to the

head. A patent clothes-pin or elastic band may be used to hold

the two glass slips together till the specimen is further treated.

The slips with the tongue clamped between them at one end are

next put in a small dish in which some turpentine has been placed

and is allowed to remain in the turpentine bath for five hours. It

may then be taken out and will be found to be quite hard and flat,

with all the beautiful colorings retained. The tongue may now be

mounted with great ease in turpentine balsam or benzole balsam.

Fly’s feet may be prepared in the same manner.

Students who have tried in vain to make a good mount of this

common object will smile when they find with what ease this seem-

ingly difficult feat may be accomplished.

OLIVER KENDALL, JR.

METHOD OF COLLECTING DIATOMS FROM SURFACE OF MUD.

To the amateur collector of diatoms, the greatest difficulty has

been to gather them free from excess of sand and foreign particles.

The following method will be found to be of great help in this

respect, especially on the shores of tide water, and the method

requires that the surface of the mud be uncovered by the tide. The

spot for working is found by the presence of a brownish colored

film generally in streaks or patches on the mud surface.

It has been found that by removing the film of diatoms with

a spoon large quantities of sand and mud are taken up at the same

time making its removal difficult in the cleaning process.

The collector is to provide himself with several squares of

well washed cotton cloth, about the size of a handkerchief, and be

at the ground at low tide. Take a square of cloth and carefully

54 NOTES, REVIEWS, ETC.

lay it down on the mud surface in a way not to include air bells,

The cloth will in a few moments become wet and may then be

raised by one corner first and folded up with the side that was

next to the mud on the inside. After folding wrap in waxed paper

and label for future reference. When ready to clean, place the

cloth in a porcelain evaporating dish and cover with strong sulphuric

acid and enough bi-chromate of soda to make the mass a deep reddish

color. Place the dish in a sand bath over a gas stove or other source

of heat, boil the mass till crystal of chromic acid appear as a scum

on the surface of liquid. Remove and let cool and pour into a

preserve jar partly filled with water. Let settle for at least one-half

hour undisturbed, then siphon off water with a rubber tube to within

one inch of the bottom of the jar, being careful not to disturb the

sediment. Repeat the washing till clear from all color. The sedi-

ment may now be removed to a small bottle and examined and if a

small quantity of sand is present it may be removed by whirling it

with some water in the evaporating dish by means of a glass rod,

and the sand will be found to pile up in the center as a dark spot.

Carefully pour off the water with the diatoms suspended in it, leaving

the sand in the dish.

One will be surprised how the diatoms will stick to the cloth

and how little foreign matter will be collected by this method. The

above method may be used in fresh water streams provided the spot

is first drained.

Providence, R. I. OLIvER KENDALL, JR.

NEW METHOD OF EXAMINING STOOLS FOR EGGS.

C. M. Fauntleroy and R. Hayden (U. S. Naval Med. Bul. Jan.

1915) suggest the following method:

1. Mix thoroughly about 2 grams of fecal matter with 5 cc.

of a 2% aqueous solution of lysol in a centrifuge tube.

2. Centrifugalize at high speed for one minute, decant the su-

pernatant liquid, and mix a fresh quantity of the lysol solution with

the sediment in the tubes. Repeat this step three times.

3. Remove small portions of centrifugalized sediment with

pipette; place on slide; mix a small drop of anilin gentian violet

with the sediment; cover and examine.

AMERICAN MICROSCOPICAL SOCIETY 55

The authors have used the method successfully in more than a

thousand cases. All eggs, hookworms and others, stand out very

clearly. Everything is stained except the eggs. These appear in

the natural state because their membranes resist the stain, as they

would not in an alcoholic solution, which enables one to run a slide

through quickly with the certainty that no eggs are overlooked. -

The lysol is not essential but is used as a precaution for disin-

fection. The method facilitates this disagreeable work, and is so

sure that much of it can be turned over to less expert assistants.

The clearness of differentiation saves much time.

Abstracted by V. A. LATHAM.

A CLEARING FLUID FOR CELLOIDIN.

Dissolve 1 oz. absolute phenol-crystals in a mixture of equal

parts of oils of cedar and bergamot, using gentle heat to hasten the

solution. Sections may be cleared in this direct from ordinary

methylated spirit. It can be used several times.

Near keiice

BOUIN’ S FLUID: A GOOD ALL-ROUND FIXING SOLUTION

Corrosive sublimate 40 grains

Water 4 ounces

Picric acid 60 grains

Formalin 8 ounces

Dissolve corrosive sublimate in hot water. When cold add

picric acid, and then the formalin. Wash with water.

3 VECAPEE:

MOUNTING ZOOPHYTES AND POLYZOA.

Fix and stain in any suitable way. Select suitable branches,

place in a little vessel of oil of cloves or other clearing agent, but

never let dry. Mount in sunken cell-slip without pressure or dis-

tortion. To do so overfill the cell, slowly slide the cover over the

fluid, and put a clip on each edge of slide. If the clip bears on the

center it may crack the cover, or when it is removed the cover may

reassert its level and draw in a bubble of air. Larval stages of

56 NOTES, REVIEWS, ETC.

crustaceans, young fishes, annelids, etc., may be put up in the sunken _

cells. Be sure to use colorless Canada Balsam in all cases, or the

deeper parts will show yellow.

Veo An Ls

HENNING'S SOLUTION FOR FIXING FLIES FOR SECTIONING.

This solution fixes, and softens the chitin. Imbedding should

be rapid and brief. The washing should be done with iodised alco-

hol. Henning’s solution:

Nitric Acid (c. p.) 16 parts

Chromic (.5% sol.) 16 parts

Corrosive Sublimate (Sat. sol. in 60%

alc. ) 24 parts

Abs. Alcohol 42 parts

Picric Acid (saturated watery sol.) 12 parts

Vi Aas

ILLINOIS BIOLOGICAL MONOGRAPHS

Under the general series of University Studies the Biological

Department of the University of Illinois issues a double number of

Volume I of the Illinois Biological Monographs. This number is

entitled “A Revision of the Cestode Family Proteocephalidz,” by

Dr. George R. LeRue. There will be four numbers per volume.

The third number of the first volume will be “Studies on the Com-

parative Anatomy and Phylogeny of the Cestode Family Anoplo-

cephalide,”’ by H. Douthitt. Subscription price is $3.00 yearly.

The price of Dr. LaRue’s paper is $2.00. Address Manager Uni-

versity Studies, 321 Natural History Hall, Urbana, Illinois.

REVISION OF THE CESTODE FAMILY PROTEOCEPHALID.

LaRue (Ill. Biol. Monog. Vol. 1, Nos. 1 and 2, 1914) presents a

discussion of the Cestodes of the Family Proteocephalide, including

the genera Proteocephalus (29 species), Choanoscolex (1 species),

Corallobothrium, Crepidobothrium, (1 species), Acanthotenia, and

Ophiotenia (1 species). These infest the cold-blooded Vertebrates.

They were formerly included in the old genus Tenia. The chief

AMERICAN MICROSCOPICAL SOCIETY Ve

genera are Proteocephalus, from fishes, and Ophiotenia from Am-

phibians and Reptiles.

Proteocephalids have been found in fish in Europe, North and

South America, and Africa. The species of each region are peculiar,

except one species common to Europe and N. America. Those

of Amphibians and Reptiles have been found in North and South

America, Australia, Europe, Asia, and Africa. The distribution

of each of these seems narrow. None is known to occur on more

than one continent. .

Five species of Proteocephalus occur in different host species

of fishes of the same genus. Four cases are known where a species

may occur in members of closely allied genera. There is at least

one species that may infest fish belonging to widely different fam-

ilies. When species have multiple hosts the host species have a

continuous distribution.

The parasitic infestation of the host is determined by the food

eaten. The hosts of those cestodes that have multiple hosts are

alike in some of the elements of their food supply. This allows

a common intermediate host.

The author believes that this family is related to and derived

from marine forms of the order Tetraphyllidea. This probability

coupled with the fact that this family of parasites is so well repre-

sented in fresh water fishes raises an interesting question as to how

parasites, demanding as these do an intermediate host, may have

evolved from Cestode parasites of marine adaptations. Reference

is made to the salmons, eels, and other forms that pass back and

forth from salt to fresh water as possible agents. Yet one of the

salmon species that has become permanently fresh water has para-

sites exclusively characteristic of fresh water. This form was

clearly unable to establish its marine parasites, if it had any, in the

fresh water conditions, and became the host of fresh water parasites.

There is no evidence that these types are succeeding in introducing

marine parasites into fresh water.

Another possibility is that the intermediate host of some kind,

fish or invertebrate, may have made its transition from marine to

fresh water. Then some carnivorous fresh water fish with internal

conditions sufficiently like those of the original species, host to the

58 NOTES, REVIEWS, ETC.

mature Cestode, may have become infested by adopting the new

arrivals as food. This alternative seems the more plausible.

The author believes that Amphibians and Reptiles may have

become infected by eating fish or invertebrate intermediate hosts

containing larve Proteocephalids.

OPTIC PROJECTION.

Dr. Simon Henry Gage who for many years has been an active

member of this Society and has twice been its President, and his

son, Dr. Henry Phelps Gage, are the joint authors of a book, just

issued, with the above title. The expressed object of the book is

so to place before the intelligent reader the principles of optic pro-

jection, accompanied by such simple and careful directions and

illustrations, that any one may become sufficiently proficient to get

real satisfaction from the practise of the art.

The tremendous growth in the realization of the value of pro-

jection in all kinds of educational work and the great advances

in the making of projection apparatus make the book most valuable

not alone to users of such instruments, but to manufacturers and

to general students of optical processes as well. The work has

been done with characteristic painstaking and attention to detail.

It is a record of a tremendous amount of practical manipulation and

experiment with all manner of projection devices. It is safe to

predict that it will become a standard reference work and handbook

for the people for whom it is intended.

One of the outstanding features of the treatise is its suggestions

for adapting and combining parts of standard apparatus for special

uses, and thus for making economical combinations and elaborations

at home.

This book ought to do much to bring closer together the manu-

facturer and the user of projection apparatus. The manufacturer

must make allowance for the lack of optical and mechanical knowl-

edge and skill on the part of the users, and the user must under-

stand that the best results can only be had by understanding the

principles upon which the apparatus is built.

There are fifteen chapters, with appendices, bibliography, etc.

The illustrations are abundant. These consist not merely of photo- _

AMERICAN MICROSCOPICAL SOCIETY 59

graphs of apparatus, but of adequate sectional drawings of details.

The principal chapter headings are as follows: Magic Lan-

tern with Direct Current; Magic Lantern with Alternating Cur-

rent; Magic Lantern for Use on House Electric Lighting Sys-

tem; Magic Lantern with Lime Light; Magic Lantern with Petrol-

eum, Gas, Acetylene, and Alcohol Lamps; Magic Lantern with Sun-

light ; Projection of Opaque Objects ; Preparation of Lantern Slides ;

Projection Microscope; Drawing and Photography with Projection

Apparatus; Moving Pictures; Projection Rooms and Screens ; Elec-

tric Currents and their Measurement,—Wiring, Control, Candle

Power, etc.; Optics of Projection; Uses of Projection in Physics;

Normal Vision and Eye defects.

The appendix contains an account of the origin and develop-

ment of projection apparatus ; makers and dealers in this apparatus ;

bibliography ; and an extended index.

There is nothing in print which will so nearly give all he must

have to the worker who must himself install his apparatus from the

ground up, and control its manipulation.

Optic Projection, by S. H. and H. P. Gage. Comstock Publishing Co., Ithaca, N. Y.

Illustrated, 731 pages. Price $3.00, postpaid.

THE EVOLUTION OF SEX IN PLANTS.

Under this title Dr. J. M. Coulter writes the initial number of

a new Science Series to be issued by the University of Chicago.

The expressed purpose of this series is to bring alike to the specialist

and the layman summaries of generally accepted results in various

fields of investigation with a minimum of technical details. Each

issue will limit itself to the statement of a single problem.

The present topic is discussed in eight chapters, as follows :—

i, Asexual Reproduction; 2, Origin of Sex; 3, Differentiation of

Sex; 4, Evolution of Sex Organs; 5, Alternation of Generations ; 6,

Differentiation of Sexual Individuals; 7, Parthenogenesis; and 8,

A Theory of Sex. The book as a whole is a luminous organization

of the facts with which the teachers of Botany are familiar.

Of course plants show the most remarkable persistence of non-

sexual reproduction. The lowest plants have no other method.

60 NOTES, REVIEWS, ETC.

Gradually the sexual method is introduced side by side with the

asexual, and as a modification of it. Later, the sexual method

alternates regularly with the asexual in distinct generations; and

in the Bryophytes about half way up the plant kingdom, the sexual

becomes the dominant generation. In all the higher plants includ-

ing the Pteridophytes, there is a reduction of the sexual stage

amounting to a suppression of almost every thing except the sex

cells themselves, and the subordination of the gametophyte and

its dependence on the asexual sporophyte. In the animals on the

contrary the asexual methods disappear and the sexual reproduction

stands alone in the higher forms.

The diversity of sex first shows itself in the sex cells,—ova

and antherozoids. Next the organs that produce these different

cells differentiate. Finally we find the plants that bear the differ-

ent sex organs come to differ. The influence of the sex function

does not stop, however, with the gametophyte. In the higher plants,

notwithstanding the fact that the gametophytes become much

reduced, the dimorphism related to sex works back into the sporo-

phyte structures. The spores destined to produce the two types of

gametophytes become differentiated; the microsporangium and

microsporophyll differ from the megasporangium and the megas-

porophyll. And in dicecious plants the sporophyte that finally gives

rise to the male issue may differ from that which: gives rise to the

female.

The amateur worker with the microscope will find that this

little book will make more full of meaning his studies of the lower

plants.

The Evolution of Sex in Plants, by Professor John M. Coulter. University of Chi-

cago Press, 1914. Illustrated; 140 pages. Price $1.00 net.

PSY CHOBIOLOGY

The structural biology foundational to psychology is presented

by Knight Dunlap of Johns Hopkins University in a small volume

recently issued. The purpose of the book is to emphasize, for the

psychological student who has not had a thoro course in morphology,

the details of histology and gross anatomy which are of greatest

AMERICAN MICROSCOPICAL SOCIETY 61

psychological significance. This is conceived by the author to in-

clude not merely the customary treatment of the nervous system

and the sensory epithelia, but of the muscles and glands and the

relation of the nerves to these. The chapter headings are:—1, The

Cell; 2, The Adult Tissues of the Human Body; 3, Muscular Tissue ;

4, Nervous Tissue; 5, The Afferent and Efferent Neurons; 6, The

Gross Relations of the Nerves, Spinal Cord, Brain, and other

Ganglia; 7, The Visceral or Splanchnic Division of the Nervous

System; 8, Glands; 9, The Functional Interrelation of Receptors,

Neurons, and FEffectors. ,

This treatment of the subject will be helpful not alone to the

psychologist. The biologist will find it a distinctly valuable reap-

praisal of the psycho-physiological values of the well known struc-

tures. The work is well done. The illustrations are well selected.

If there were no other reason these would make the book a valuable

handbook to the general biologist and student with the microscope.

The concluding chapter is a peculiarly clear and straightforward

statement of the relations of the three main portions of the human

neuro-mechanism, and the general conditions of its proper activity.

An Outline of Psychobiology. By Knight Dunlap. The Johns Hopkins Press, Bal-

timore, 1914. Illustrated; Royal octavo, 121 pages. Price $1.25.

BIOLOGY AND SOCIAL PROBLEMS.

This is the title of the William Brewster Clark Memorial Lec-

tures of Amherst College for 1914. They were delivered by Profes-

sor G. H. Parker. There are four lectures in the series bearing the

following titles: I, The Nervous System; II, Hormones; III,

Reproduction; IV, Evolution. Professor Parker has handled these

interesting themes in his usual exact and lucid manner. Each of

these lectures has a double value: in the first place, each assembles

and summarizes the great underlying discoveries in its field in some-

what the same spirit which this journal has sought to show in its

summaries of progress, altho in a more popular way; secondly, the

lecturer endeavors, in the spirit of the Brewster Foundation, to

apply our knowledge in these fields to the problems of man’s indi-

62 NOTES, REVIEWS, ETC.

vidual and evolutionary responses to the environment of which he

is a part.

This interesting foundation seeks to bring the discoveries of

science to the problem of the conscious control by society of human

improvement.

Biology and Social Problems, by G. H. Parker. Houghton, Mifflin & Co., Boston.

Illustrated; 130 pages. Price $1.10, net.

PROCEEDINGS

of the American Microscopical Society

MINUTES OF THE PHILADELPHIA MEETING

The American Microscopical Society held its thirty-fourth annual meet-

ing in connection with the A. A. A. S. at Philadelphia, Pa. Dec. 30, 1914.

The members met for luncheon in the gymnasium of the University

of Pennsylvania on Wednesday, December 30, and enjoyed greatly the social

gathering for which the Society is indebted to the courtesy of the Univer-

sity of Pennsylvania.

The annual meeting was called to order at 2 p. m. by Vice President

H. L. Shantz, Washington, D. C., in the absence of the President. Profes-

sor Henry B. Ward was elected secretary pro. tem. The letters from the

Secretary, Professor T. W. Galloway, were read in full, giving a record of

the work of the Society during the year, its present condition, and future

possibilities. The report proved of great interest and was discussed in an

appreciative manner by the members present. All were outspoken in regret-

ting that the Secretary was unable to attend the meeting, and in apprecia-

tion of the work which he had done in behalf of the organization.

The report of the Treasurer was read and referred to an auditing com-

mittee consisting of Professors H. B. Ward and H. J. Van Cleave with

instructions to print their report in connection with the report of the treas-

urer in the volume.

The report of the custodian was read by that official and explained

in extenso. It was referred with accompanying documents to an auditing

committee consisting of Doctors B. H. Ransom and R. H. Wolcott who

later reported the account to be correct. This report was accepted and ordered

printed. The discussion of the custodian’s report was extended and inter-

esting. It embraced an analysis of the present condition of the fund the

factors in its growth which had brought it to its existing level, and of the

outlook for its future extension. The Society passed a unanimous vote

of thanks to the Custodian for his careful and continued efforts which have

resulted so satisfactorily in the growth of the fund and expressed the hope

that means might be taken in co-operation with him to increase the sum to

still larger figures.

The Custodian made a full report concerning the official copy of the

publications of the Society, including not only the regular Transactions, but

also the incidental notices and other printed matter given out by the organi-

64 MINUTES

zation. At the conclusion of the discussion it was voted that the official copy

of the Transactions of the American Microscopical Society which was

inaugurated by the Custodian and has been conducted so that it contains the

full history of the organization by reason of including all notices and

circulars issued, be designated the official copy of the Society and be entrusted

to the Secretary for preservation. It was further voted that the copy be

continued by the Secretary in the same manner as has been employed here-

to fore.

Inquiry was made concerning the standard rulings which are the

property of the Society, and the Custodian reported that under vote passed

by the Society the same were loaned to Doctor M. D. Ewell for use during

a limited period. The Custodian was on motion directed to call in this

and other property of the Society.

It was upon motion formally decided that the United States Bureau

of Standards be designated the depository of the standard centimeter A,

and other rulings property of the Society provided they were willing to

accept the trust. The Custodian was directed to correspond with them

with a view to ascertaining their willingness, and in case of favorable reply

to place these standards in their hands with the understanding that the same

may be, under proper conditions as designated by them, available for use

by workers with the microscope when desired.

A nominating committee was appointed consisting of Doctor B. H. Ran-

som, Mr. E. Pennock, Doctor A. M. Bleile, Doctor R. H. Wolcott, and Mr.

Magnus Pflaum. The committee reported the following nominations :—For

President, Professor C. A. Kofoid, University of California; First Vice

President, Doctor L. D. Swingle, University of Utah; Second Vice Presi-

dent, Doctor N. A. Cobb, United States Department of Agriculture, Wash-

ington, D. C.; for elective members of the Executive Committee, Professors

J. P. Campbell, University of Georgia, L. E. Griffin, University of Pitts-

burgh, and Doctor H. L. Wieman, University of Cincinnati; for representa-

tives of the American Microscopical Society on the Council of the Ameri-

can Association for the Advancement of Science, Doctor H. L. Shantz and

Doctor R. H. Wolcott.

On motion the Secretary was instructed to cast a unanimons ballot in

favor of the gentlemen nominated and they were duly elected. On motion

the Society approved the action of the Secretary and authorized him to

proceed with the measures inaugurated for printing and circulating the

annual volume and for bringing the Society to the attention of those likely

to be interested.

The Society adjourned.

Henry B. Warp,

Temporary Secretary.

MINUTES 65

SPENCER-TOLLES FUND.

Custodian’s Report for the year 1914

ReeDOred Atcetianta: Meena oes rcs es yeas owen hah $3,819.83

PRCPTUETT a LECOEVECE ther ae cho hare pepe Bm et $ 234.32

PIRES TOCREUINNG | Ue oc fee et ee a eae oi 75.00

ye aplecetste LSD lth sake Raa, ERAT NEL RaA Ue eatNE RTT Ptr OL Saat 100.00

Expense returned by Treasurer...................... Use 5.50 414.82

$4,234.65

ES Tk a CNRS Cine POP PEE NE a IRGC Senn A A Mya neue 50.00

RO VERTOG Sider aan y ice aang pe are Me a $4,184.65

PU COU ACIORA LOU ALE eas Gaievickes ciao clcledreealy tomes $ 800.27

Pils SALEM Ole DIOCRPOMEN a oy ip oe hes os ood ee ES 758.38

PU ULE ANEMDEL SINGS bao ieluna ts) Reta sect he, cack tae 250.00

MARIO TOST AN MIVINeRUE. boy vp mg. OTA eee eh ee ote 2,566.00 $4,374.65

LESS

PLU REAM UC APE arn) Oak ee dig cod, Eanes ae eae eae 150.00

All life:membershin dies paids i. abs ool oes lack eek 40.00 190.00

DERE UO OC ncn rn ee an hn enna i.) cae $4,184.65

Life members: Robert Brown, dec’d.; J. Stanford Brown; Henry B.

Duncanson; A. H. Elliott; John Hately.

Contributors of $50 and over: John Aspinwall; Iron City Microscopical

Society; Magnus Pflaum; Troy Scientifiic Society.

Macnus Prraum, Custodian.

We, the undersigned committee hereby certify that we have care-

tully examined the foregoing account of Magnus Pflaum, the Custodian, and

found the same to be correct.

B. H. Ransom,

Rosert H. Wo corr,

Auditing Committee.

Dec. 30, 1914.

66 MINUTES

ANNUAL REPORT OF THE TREASURER OF THE AMERICAN

MICROSCOPICAL SOCIETY.

December 24, 1913, to December 24, 1914.

RECEIPTS

Balance on hand: from 1913.0... 66 cc eee oa eee eile eh ee dua wee ss $ 265.11

TSS OF Old HREMDENS | 6c be cc loin ciaiecnss see kd Ripe reid A tone thine dip eels 412.00

Tee bo FFG WAIEIN DENS 2 fk d's Sad last pd bem 's 2d Ww aly Wo rennin Nicaea Oe eee 74.00

Ti Gat OCB bre he Bh nO ER hae ea W ASCE Cag dA aol inate a eee 111.00

Subscriptions for volume 32......-.6.0 6 cece eee cece ete teen ees 79.50

Subscriptions for volume 33...........e cece eee e teen eet e nee 23.50

Subscriptions for volume 34...........0. cece etree een ene ene eens 7.00

Subscriptions for other volumes............ 05 ec eee eee eee eee ees 54.00

Grant from Spencer-Tolles Fund for printing N. A. Cobb’s paper.... 50.00

Advertisers in volume 32........00 cscs ete eee entree een n ete nese ane 228.00

Advertisers in volume 33.2........ 0c. cece cree teed ee eect edenees 75.00 .

fn BL ete Oe Pe LE a ER Ee RTC RO Tt eS $1,379.11

EXPENDITURES

Printing Transactions, volume 32, no. Ao yok Ob bi ae Lae eee $ 268.81

Printing Transactions, volume 33, no. ) Ue ES espe Pare ec me Sey SP | 526.74

Plates for Transactions, volume 32, no. 4..........0.e eee e eee rees 41.38

Same for volume 33, no. 1, 2, and 3......... cece cece eee eens 67.14

Postage and express for Secretary........--.seeeeeee rece seers enes 46.28

The same for the Treasurer.......... ccc ce cece cece ect ences reeenes 14.21

Office expenses of Secretary, stationery stenography,. etc... i sc; teu. 97.38

The same for the Treasurer......-. 06. cee cece reece rent een e eet eeees 17.36

Advertising literature 00.0.0... cece cece eee eet eee eet e nena 19.93

Sundry expenses 6.00... . cece cree reece eee e este tener et eter e eens oF

Contribution to Spencer-Tolles Fund for expenses of the fad EMEA Hee 10.50

Secretary's expenses at the Atlanta elo over) ante Saey See meee ape ee eM i 25.00

Balante On Nand wicks oh’ Soke be nse gn tod Cea ae Pine he 243.61

Tepal iorechits: of. 6 + sic he yvd bis yo Wap aea eet Pale iA ERB Bye Miata syns = ae mn $1,379.11

T. L. Hankinson, Treasurer.

We as a committee have audited the report of the Treasurer and com-

pared it with the vouchers and find it to be correct.

H: B. Warp,

H. J. Van CLEAVE,

Auditing Committee.

WANTED

Microscope Slides and Photomicrographs

Sot ge

POPULAR INTEREST

Just that kind that you show to your non-scientific friends,

and then step back and wait for them to exclaim, “Oh, my! Isn’t

that interesting and beautiful!”

Help create and increase a knowledge and love of the micro-

scope and the world it reveals.

THE AGASSIZ ASSOCIATION

Arcadia:

Sound Beach, Connecticut.

Edward F. Bigelow, President.

A copy of THE GUIDE TO NATURE free, if with the re-

quest you will let us know that you use a microscope.

OPTIC PROJECTION

By Simon Henry Gage, B.S., and Henry Phelps Gage, Ph.D.

This volume of over 700 pages and 400 illustrations deals

comprehensively with the principles and use of all forms of pro-

jection apparatus—the Magic Lantern, the Projection Microscope,

the Opaque Lantern and the Moving Picture Machine.

Special emphasis is given to projection with the microscope

for class-room instruction in Histology and Embryology, and for

drawing with all powers from the lowest to the highest.

POSTPAID, $3.00

THE MICROSCOPE

AN INTRODUCTION TO MICROSCOPIC METHODS

AND TO HISTOLOGY, 11TH EDITION.

By Simon Henry Gage, Professor of Histology and Em-

bryology, Emeritus in Cornell University. 8vo. XVI + 359

pages, 265 illustrations. Postpaid $2.00. :

A Guide for every one who uses the microscope, and for

every student who wishes to learn to use the microscope in-

telligently and effectively.

Send for circulars giving contents, specimen pages and illus-

trations of these and other Nature Study Books.

THE COMSTOCK PUBLISHING COMPANY

Cornell Heights, Ithaca, N. Y.

Members and Friends Will Find Our Advertisers Reliable

INDEX TO ADVERTISEMENTS

Anglers’ Bait & Mfg. Co., Chicago, Ill........---ce eeu eeee sere seeeenes I

Bausch and Lomb Optical Company, Rochester, N. Y.....-.+0+0seee0 es lil

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C. S. Brimley, Raleigh, N. C.... eee ec ce cence cece e rece eneeneneneenress I

Cambridge Botanical Supply Co., Waverley, Mass.......-+++sessesseres I

Comstock Publishing Co., Ithaca, N. Y....-6.. esse ceeee eens Third P. Cover

Guide to Nature, Arcadia, Sound Beach, Conn.........+.++-+- Third P. Cover

The Kny-Scheerer Co., 404 West 27th St., New York......+-.0++s00ee0s IV

Marine Biological Laboratory, Wood’s Hole, Mass.....-.-++++s+s+ee0es I

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Review Printing and Stationery Co., Decatur, Illinois........-..++++.++ IV

Smith: Laboratory Guide for the Study of the Frog.......-.-.++++++: I

Spencer Lens Company, Buffalo, N. Y......0-0sseeee eee eeeeeeeersenees Il

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Members should bear in mind that our advertisers make possi-

ble a larger magazine than could otherwise be issued. We owe them

our patronage as well as our thanks.

SN ———————————————————————————— eee

ORGANIZED 1878 INCORPORATED {1891

VOLUME XXXIV

TRANSACTIONS

OF THE

AMERICAN

MICROSCOPICAL

SOCIETY

PUBLISHED QUARTERLY BY THE

AMERICAN MICROSCOPICAL SOCIETY

DECATUR, ILLINOIS

ANNUAL SUBSCRIPTION $3.00 SINGLE COPY $1.00

DOUBLE NUMBERS $2.00

4h ! a

Nba

tae

cae,

yay

ies

uh .

[sa

TRANSACTIONS

OF THE

American Microscopical

Society

ORGANIZED 1878 INCORPORATED I89Q1

PUBLISHED QUARTERLY

BY THE SOCIETY

EDITED BY THE SECRETARY

VOLUME XXXIV

NuMBER Two

Entered as Second-class Matter December 12, 1910, at the Post-office at Decatur,

Illinois, under act of March 3, 1879.

Decatur, ILL.

Review PrintTiInG & STATIONERY Co.

1915

foe

oh oe gt it fe

OFFICERS.

ETESIOCHE Ae NOFOIDS os oes sick cS > Peres Rede danas Berkeley, Calif.

Perst. Vice Eresutent? TD. SwWINGLEs ii 5c. 2... se en oe os Salt Lake City, Utah

Second ace President: N. A. COBB. oo un vc avs cbs ceeds Washington, D. C.

ELAM CELA VV 2 CFALLOWAY os We coc ny c'r% 06 04 alk eokenc F melee tet Decatur, Ill.

Seeeteverers.-* Fy" 1. CLAN RINSON, © 0G due cos bu anew ay ote Oh an wvioe Charleston, Ill,

Carmodias; | MAGNUS. PRAUM Cos c idevind xia bees oe cide ye ae Meadville, Pa.

ELECTIVE MEMBERS OF THE EXECUTIVE COMMITTEE

Re Eee MOREE S2Oe Care ahs op eA Whee iat ck eran eckts ry Athens, Ga.

Be Kie’ AVMIEMEIC: oboe ese Pees Cele Ors Ass WER ee eee eae Pittsburg, Pa.

Berto VVSRMORN foe e ee oy so ous et Oey Ueno eee oe ant Me AF Cincinnati, O.

EX-OFFICIO MEMBERS OF THE EXECUTIVE COMMITTEE

Past Presidents still retaining membership in the Society

R. H. Warp, M.D., F.R.M.S., of Troy, N. Y.,

at Indianapolis, Ind., 1878, and at Buffalo, N. Y., 1879

ALBERT McCatia, Ph.D., of Chicago, II.

at Chicago, Ill., 1883

T. J. Burritt, Ph.D., of Urbana, IIL,

at Chautauqua, N. Y., 1886, and at Buffalo, N. Y., 1904.

Gro. E. Fett, M.D., F.R.M.S., of Buffalo, N. Y.,

at Detroit, Mich., 1890.

Simon Henry Gace, B.S., of Ithaca, N. Y., .

at Ithaca, N. Y., 1895 and 1906.

A. Ciirrorp Mercer, M.D., F.R.M.S., of Syracuse, N. Y.,

at Pittsburg, Pa., 1806.

A. M. Buere, M.D., of Columbus, Ohio,

at New York City, 1900.

C. H. EiceEnMANN, Ph.D., of Bloomington, Ind.,

Cuartes E. Bessry, LL.D., of Lincoln, Neb.,

E. A. Birce, LL.D., of Madison, Wis.,

Henry B. Warp, A.M., Ph.D., of Urbana, IIl,

at Denver, Colo., 1901.

at Pittsburg, Pa., 1902.

at Winona Lake, Ind., 1903.

at Sandusky, Ohio, 1905.

HeErBertT Osporn, M.S., of Columbus, Ohio,

at Minneapolis, Minn., 1910.

A. E. Hertzier, M.D., of Kansas City, Mo.,

at Washington, D. C., grt.

F, D. Hearn, Ph.D., of Philadelphia, Pa.,

at Cleveland, Ohio, 1912.

. = ’ 4 L

F. CrEIGHTON WELLMAN, of New Orleans, La at Atlanta, Ga., 1913.

_ D. of Li k, Ark, ;

Cuares Brooxkover, Pu of Little Rock, Ar at Philadelphia, Pa., 1914.

The Society does not hold itself responsible for the opinions expressed

by members in its published Transactions unless endorsed by special vote.

TABLE OF CONTENTS

FOR VOLUME XXXIV, Number 2, April, 1915

The Development of Botanical Microtechnique, with Plates I-III, and

12 Text Figures, by Gilbert M. Smith..........--e sees e cere eeereees 71

The Ampulle of Lorenzini in Acanthias Vulgaris, with Plates IV and V,

and 4 Text Figures, by H. E. Metcalf......-..e ees eeeeeeeeeeerceees 131

Notes and Reviews: Protozoology Applied to the Soil, by N. Kopeloff,

H. C. Lint, and D. A. Coleman; Presence of Acidophilous Cells in the

Adrenals of Certain Amphibians, by T. B. Magath; Technic for

Cestodes, by G. R. LaRue; Cultivation of Plasmodium of Badhamia ;

Daphnia without Sexual Forms; Vertebrate Embryology (Prentiss)... 149

TRANSACTIONS

American Microscopical Society

(Published in Quarterly Installments)

Vol. XXXIV APRIL, 1915 No, 2

THE DEVELOPMENT OF BOTANICAL

MICROTECH NIQUE

By Gilbert Morgan Smith

INTRODUCTION

Development of our knowledge of cell structure has been cor-

related in a large measure with the development of methods of micro-

technique, and it is a significant fact that Hooke did not discover the

cell until he prepared sections of plant tissues. We are sometimes

apt to think of the early investigators as having the same advan-

tages we enjoy, but, in order to properly interpretate the relative

value of any early botanical discovery, it is necessary to remember

the means available to the worker of that time. In the present arti-

cle the development of methods of making microscopical prepara-

tions will be treated from the botanical standpoint. The history of

the microscope, and the development of culture methods will not be

considered.

Advances made in microtechnique in Botany and Zoology are

closely related. Discoveries made by zoologists in the art of making

preparations were adopted by the botanist, while botanical methods

were utilized by the zoologist. The microscopists, who were neither

botanists nor zoologists, especially those under the influence of the

London Society of Microscopists, have also played a very appreciable

part in the development of the methods of making microscopical

preparations. Probably for their development of microtechnique,

rather than for their microscopical discoveries, are we indebted to the

greatest extent to the microscopists.

72 GILBERT MORGAN SMITH

In tracing the development of animal microtechnique Apathy

has divided the time into three grand periods as follows:

1. The rule of the Dry Preparation, which lasted from the dis-

covery of the microscope to the end of the 30's.

2. The rule of the compressorium and the razor, 1840-1880.

3. The rule of the microtome, 1880 to the present.

Perhaps a better method would be the discussion of botanical

microtechnique under the following captions:

1. The methods of the early microscopists (from the time of

Hooke’s discovery of the cell to 1800).

2. The technique of the English microscopists (1800-1875).

3. The methods of the German botanists (1800-1875).

4. The development of modern methods of microtechnique (1875

to the present).

Tue METHODS OF THE EARLY MIcCROSCOPISTS

The microscopical laboratory, in the modern sense, did not exist

in the 17th and 18th centuries. There are few illustrations of mi-

croscopists’ work-rooms comparable with those that the chemist,

physicist or pharmacist can show us. Ledermuller shows the way

that a microscopist of that time fitted up a room for the purpose of

showing his friends “‘some of the wonders of nature” by means of

Cuff’s solar microscope. An even better illustration of a laboratory

is found in the head piece in each volume of Joblot’s work (Fig. 1).

This is probably an allegorical picture rather than an actual repre-

sentation, but the microscope before the window, the hand micro-

scope which is being used, the twigs in the vases and various objects

on the table suggest the modern laboratory. The conspicuous posi-

tion of the globe, the telescope and other instruments in the floor,

however, do not fit into our present-day concepts but show the catho-

lic taste of the early investigator.

Up to the beginning of the 19th century the microscope was a

toy rather than an instrument of scientific research. Nelson men-

tions Pepys paying £10 for a microscope in 1664 and thinking it

“a great price for a curious bauble.” The attitude toward micro-

scopy is also shown in the allegorical frontispieces of Ledermuiller

and of Adams. When Wilson says “In viewing Objects, one ought

to be careful not to hinder the light falling on them, by the Hat,

BOTANICAL MICROTECHNIQUE 73

Perruke, or any other Object,” we can easily imagine the casual

manner in which the gentleman of that time looked through his

microscope. Another conception of the purpose of the microscope

is that of Baker (1742) who says ‘“‘And if I can hereby induce any

to pass those leisure hours agreeably and usefully, in contemplating

the Wonders of Creation, which would otherwise be spent in tiresome

Idleness, or, perhaps, some fashionable and expensive Vice, I shall

think these Sheets very happily bestowed.”

The first microscopists had to make their own microscopes as

well as their microscopical preparations and, considering the prim-

itive character of these instruments, it is natural to find them think-

ing the improvement of the microscope more productive of results

than the improvement of the method of making their preparations.

During the 18th century almost all works on the microscope were

written by microscope manufacturers ; so that great emphasis is laid

on description of the construction of the instruments. These “Mi-

crographias” were frequently sold with the microscope and were

therefore written for the benefit of those who desired to dabble in

microscopy. There was little serious use of the microscope, Baker

stating the general attitude in the following: “Many, even of those

who have purchas’d Microscopes, are so little acquainted with their

general and extensive Usefulness, and so much at a Loss for Objects

to examine by them; that after diverting themselves and _ their

Friends, some few Times, with what they find in the Sliders bought

with them, or two or three more common Things, the Microscopes

are laid aside as of little farther Value. . . . .”

Hooke’s microscope, as described in the “Micrographia,” pos-

sessed no stage, the objects being mounted on a point attached to a

pedestal at the base. Since Hooke prepared sections when he dis-

covered the cell the following extract of his description is of interest.

“I took a good clear piece of cork, and with a pen-knife sharpened

as keen as a razor, I cut a piece of it off, and thereby left its sur-

face smooth; then examining it very diligently with the microscope,

but that possibly, if I could use some further diligence,

. L, with the same pen-knife, cut off from the former smooth

surface an exceedingly thin piece of it: and placing it on a black

object-plate, because it was itself a white body, and casting the light

74 GILBERT MORGAN SMITH

on it with a deep plano-convex glass I could exceedingly plainly

see. . . .” A few years later in the Cutlerian Lectures he de-

scribed a method of fastening “Muscovy Glass” to the bottom of the

tube of the microscope in place of the “common Pedestal hitherto

made tse of in Microscopes.” Another method used by Hooke is

quite noteworthy, since it was used but little in the century follow-

ing, and not until about 1820 did the process come into general use.

He says: “But there are other substances which none of these

ways I have yet mentioned will examine, and those are such parts

of animal or vegetable bodies as . . . the Pulps, Piths, Woods,

Barks, Leaves, Flowers, etc., of Vegetables . . . but if the same

be put into a liquor, as water or very clear Oyl, you may clearly see

such a fabrik as is truly very admirable fi

Leeuwenhoek frequently made a microscope for an object that

he wished to view and since these objects were generally fixed to a

point on the microscope we may consider them as a sort of perma-

nent microscopical preparation. Aside from the allegorical figure

holding one of these microscopes in the frontispiece to the “Arcana

Naturae” he has left no figures, while descriptions of his micro-

scopes are known only from other writers. Upon his death a cabi-

net, containing several of these microscopes with their mounts, was

left to the Royal Society of London and described by the vice-presi-

dent, Martin Folks, and by Henry Baker (1753), before they were

stolen from the Royal Society. The only original Leeuwenhoek

microscope known to be in existence today is in the Utrecht cabinet,

the Royal Microscope Society of London having a modern reproduc-

tion of the Utrecht microscope. In Folks’ description we find; “Mr.

Leeuwenhoek, fix’d his Objects, if they were solid to this Silver

Point with Glew; and when they were Fluid, or of such a Nature as

not to be commodiously view’d unless spread on Glass, he first fitted

a little Plate of Talk*, or exceedingly thin-blown Glass, which he

afterwards glewed to the needle, in the same manner as his other

Objects.” The figure of Leettwenhoek’s microscope as given by

Ledermuller (Fig. 2) is particularly instructive since it shows the

fine needle-like point on which the objects were mounted,

“The old usage of the word tale or “talk” is misleading since it refers to mica

and not to the magnesium silicate called talc today.

jecnnenoscets ieee tose

+ RS ER REE SS AST eS

Fig. 1.—Allegorical representation of an eighteenth century microscopist’s lab-

oratory. (Joblot, 1754).

Fig. 2.—A. Leeuwenhoek’s microscope (Ledermiiller, 1768). B. An allegorical fig-

ure showing method of using this microscope. (Leeuwenhoek, 1722).

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BOTANICAL MICROTECHNIQUE 75

In his study of blood corpuscles he first made use of small tubes

for examining liquids. This method of exmaining material in capil-

lary tubes became obsolete about 1800 but during the 18th century

small glass tubes were a part of the equipment of all microscopes

and Baker, Martin, and Adams all figure them. Leeuwenhoek’s

method of preparing these tubes was as follows; (1674) “I did my-

self prepare divers sorts of very slender hollow Glass pipes, of which

some were not thicker than a Mans-hair;. . . . This pipe with

the blood in it, I lay upon a piece of white paper, and with my nail

break a little piece from it, and set it to the pin of my microscope,

having first a little wetted the pin with my spitle, or a little turpen-

tine, to make the pipe stick to it; or else I take the whole Glass-pipe

and with my hand hold it before the microscope.” This use of glass

tubes for mounting material was almost exclusively confined to his

zoological studies, and he later made the tubes larger and larger

even describing one as large as a finger. (1702).

The founders of plant anatomy, Grew and Malpighi, have left

but little record of their working methods. Malpighi says nothing

at all about his methods, while all Grew has to say is; “to do all

this by several ways of section, oblique, perpendicular, and trans-

verse; all three being requisite, if not to observe, yet the better to

Comprehend, some things. And it will be convenient Sometimes to

Break, Tear, or otherwise Divide, without a Section. Together

with the Knife it will be necessary to joyn the Microscope; and to

examine all the Parts.”

In the beginning of the 18th century we find object carriers or

slides, (‘‘sliders” as they were called at that time) coming into gen-

eral use. About the earliest record of a slider that we have is the

figure of one in position for use in the microscope of Philip Bon-

serePte ee thes Deena tory

tha dite tld

AL TOMA A PP

Fig. 3.—An early form of the ivory “slider”. (Wilson, 1702).

76 GILBERT MORGAN SMITH

nani in 1698. Four years later in the Philosophical Transactions,

Wilson shows one of these “sliders” (Fig. 3) describing it as fol-

lows: “EE a flat piece of Ivory, whereof there are 8 belonging to

this set of Microscopes, (tho any one who has a mind to keep a

each of which there are 3 holes fff, wherein 3 or more Objects are

placed between two thin Glasses, or Talks, when to be used with the

greater Magnifyers.” In this connection it should be borne in mind

that material mounted in these sliders was always mounted dry.

Hartzoeker (Fig. 4) used a hinged brass frame, in which the material

was held between two pieces of mica, a process which was used but

little. About the middle of the century glass “sliders” made their

Fig. 4.—Hartzoeker’s frame for holding microscopic objects. (Harting, 1859).

appearance; Martin describing “a long piece of Glass, for moving

the Object this way and that.” The glass slide was not used for per-

manent preparations Adams (1747), Hill, and “Medicus” mentioning

only temporary mounts with the glass “slider.”

In 1742 Henry Baker devoted a chapter of seven pages to the

subject “of preparing and applying objects.” The need of prepar-

ation is seen in his statement that “Most Objects require some

Management, in order to bring them properly before the Glass.”

The first method described, that of dry mounting in the “slider,” is

recommended for use wherever possible. Small concave glasses,

- quite similar to the watch crystals now in general use, are suggested

for examining fluids containing organisms. In mounting these the

material is to be taken up by means of a brush, which is figured

among the microscopical accessories (Fig. 5 A). Baker also suggests

the use of slips of glass, the same size as sliders, so that objects

could be placed on them for examination; the interesting feature

being the recommending glasses of different colors, giving as his

reason; “many Objects being much more distinguishable when

placed on one Color than on another.” “Opake” objects are to be

placed on small slips of colored cardboard, about half an inch in

BOTANICAL MICROTECH NIQUE 77

length and a tenth of an inch in width, and then fastened to the

cardboard with mucilage. For preserving these preparations Baker

A B

Fig. 5.—Apparatus for making microscopical preparations. A. Ivory Slider. B.

Box of tales. C. Camel’s hair brush. D. Brass nippers. (Baker, 1742).

devised a box fitted up with compartments (Fig. 6), this being the

first record that we have of boxes for keeping preparations.

Biter

SOIL

Fig. 6.—Case for preserving microscopical preparations. (Baker, 1742).

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78 GILBERT MORGAN SMITH

Pollen grains are among the microscopic objects recommended

by Baker and in Chapter 22 is found the method of making prepara-

tions of “Farina” (the old name for pollen grains). “Gather your

Farina in the midst of a Sunshiny dry Day, when the Dew is off;

be careful not to squeeze or press it, but shake or else gently brush

it off with a soft Hair pencil upon a piece of clean white Paper.

Then take a single Talc or Isinglass between your nippers, and

breathing on it, apply it instantly to the Farina, which the moisture

of your Breath will make adhere to it. If too great a quantity of

Powder seems sticking to your Isinglass, gently blow off a little; if

there be not enough breath on it again, and touch the Farina with it

as before. Then put your Glass into the Hole of a Slider, and

apply it to the Microscope to see if the little Grains are spread ac-

cording to your liking, and when you find they are, cover them cau-

tiously with another Talc, which fasten down with brass Wire, but

let not the Glasses press hard upon the Farina, for that will destroy

its true Figure, and represent it different from what it is.”

Two further contributions to microtechnique which appeared in

this century are in use to the present day. Ledermuller devised the

use of dipping rods for removing material from a liquid (Fig. 7) ;

while Benjamin Martin gives the first hint of maceration when he

says; “If Leaves are steep'd in Water for Maceration, the Pellicle

or thin Skin of both Sides will easily peel off, which laid on a glass

and view’d with the Light reflected thro them, will discover a most

delicate Texture. . . . .”

Historians emphasize the barrenness of the 18th century, as

compared with the 17th, in the development of the microscope.

With one notable exception, this is also true for microtechnique.

The: great originality which John Hill showed in the manipulation

of the material described in his work entitled “The construction of

timber, explained by the microscope” has not been given due credit.

In his biography of John Hill, T. G. Hill has entirely failed to call

attention to one of the most important features of John Hill’s work,

namely, the superiority of the microtechnique which he employed.

Methods were used which had not been employed up to that time

and which did not come into general use until fifty years later, and

then as rediscoveries by others.

BOTANICAL MICROTECHNIQUE 79

Hill did not rely on any one method but studied the structure

of stems in many different ways. He used a more elaborate method

of maceration than Martin, and was the first to use maceration in

the study of wood, sinking a loose wicker basket containing the

sticks he wished to study into a stream until the tissues were well

Fig. 7.—Ledermiiller’s method for removing material from an aquarium. (1768).

softened. This identical method which was rediscovered by the

younger Moldenhawer in 1812 is considered by Sachs as one of the

great steps in the progress of phytotomy. Hill also makes the first

mention of a method of preservation of material for further study.

The practice of dropping the macerated pieces of wood into a solu-

tion of alum and then transfering them to spirits of wine, after

80 GILBERT MORGAN SMITH

thoroughly drying, resembles in a very crude manner our modern

method of fixing and hardening. The reason for this is seen in his

statement that “Nothing but spirit of wine can preserve these tender

bodies, and, till I found this method of hardening them first, the

liquor often destroyed them.”

Holzner thinks that Sarrabat or Reichel should have the credit

for being the first to use staining methods since they put sticks into

colored liquids and then noted the rise of the color. Apathy has

raised the question as to whether this work should be regarded as at

all comparable to our modern methods of staining microscopical

preparations. Judging by the excerpts cited from these articles by

Holzner they were macroscopical studies only, and it is very prob-

able that Hill was not aware of them, or the work of Bonnet.

Hill is undoubtedly the first to have used staining as an aid

in the study of microscopical anatomy of plants. He prepared an

alcoholic tincture ofcochineal, in which, after it had been filtered, he

placed the stems of plants for a while, discarding that portion

which had been immersed in the fluid when he made his sections.

Another method of staining used was even more advanced since it

involved a mordanting of the tissues before developing the color.

A solution of sugar of lead was prepared, filtered, and put in a tea-

cup and the sticks to be studied were allowed to remain in this

fluid for two days. An essential part of this operation was the

“whelming” of the tea-cup with a wine glass to prevent the drying

of the material. While the tissue was soaking in the lead solution

he prepared a solution of quick lime and orpiment in water and then

transferred the material from the tea-cup to the second solution for

two days. When the sticks were first placed in the second solution

they were colorless, but in a short time they became deep brown.

By means of this staining he was able to demonstrate the existence

of structures invinsible in the uncolored material. A third method,

which was an injection rather than a staining process, was the care-

ful drying of the wood and then boiling it in green sealing wax.

By this procedure the vessels became thoroughly impregnated with

the green sealing wax and the “split pieces resemble striped satins,

- in a way scarce to be credited.”

Fig. 8.—Cumming’s microtome of 1770. (Jour. Roy. Micr. Soe., 1910).

Fig. 9.—Adam’s cutting engine. (1798).

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Fig. 10.—Custace’s cutting engine. (Thornton, 1799).

Inches

Fig. 12.—Pritchard’s microtome (1835).

PrATE. LIT

BOTANICAL MICROTECHNIQUE 81

Other methods of study used by Hill include the placing of tis-

sues of pine in spirits of wine until the resins were dissolved out

and the cells rendered more visible. Another, the placing of tissues

in spirits of turpentine until the contents became clear. Both of

these methods, although extremely valuable in the study of the

anatomy of plants were not used until a much later period. In

this study a slide of ground glass was used in addition to the ivory

“slider”, but both of them were used in the holder devised for the

purpose instead of placing them directly on the stage of the micro-

scope. As to his methods of making mounts with the glass he says:

“it is to be examined, if fresh, in water; if preserved in some of the

spirits in which it is kept; being laid in a little cistern hollowed in

a slip of ground glass.” This form of slide devised by Hill is gen-

erally regarded as being a comparatively modern invention. These

methods of study developed by Hill received but little notice, Adams

seemingly being one of the few to recognize their value as is seen

when we read; “it were to be wished a satisfactory account could

here be given of all the preparations which are requisite to fit for

the microscope the objects of the vegetable kingdom. Dr. Hill is

the only writer who has handled this subject.”

The sections that Hill used’ were cut out on a microtome.

Queckett states that the first cutting machine (microtome in our

sense) was made by Adams about 1770. The instrument that Hill

used (Fig. 8) was one invented by Cummings and was probably

well known at the time since after making two or three Cummings

turned their manufacture over to Ramsden who supplied them to

those desiring “cutting engines.” The body of the instrument (AA)

was made of ivory, while the top was of bell metal. The spiral-

edged cutter was so arranged that the difference between the longest

and shortest radii was greater than the thickness of the largest

piece of wood that the instrument would hold. The handle (F)

was uséd to revolve the spiral cutting blade and after each revolution

of the blade the material being sectioned (H) was raised the de-

sired height by means of a screw (M), each division on the head

of the screw corresponding to an elevation of the material 1-1000

of an inch. Hill was troubled by the sections of wood curling as

they were cut, so a fine spring was used to keep them flat. After

82 GILBERT MORGAN SMITH

cutting, the sections were transferred to spirits of wine. Those

interested in a more complete description than is given here, and

to whom the work of Hill is inaccessible, will find a reprint of

Hill’s description and plate in the Journal of the Royal Society for

1910.

Thanks to the love of the early microscopists for careful de-

scriptions of the minutiae of manipulation we have a good account

of the state of microtechnique at the close of the 18th century given

by Adams. In these “Essays on the Microscope” a description is

given of all the microscopes in use at that time, and, what is of

greater interest from our point of view, he carefully figures and de-

scribes all the accessory apparatus which accompanied those micro-

scopes. All were supplied with ivory sliders which fitted into slider

holders made of brass, while the objects were mounted dry between

pieces of talc and held in the holes of the sliders by brass wires

(Fig. 5 A). The sliders as supplied usually contained objects

ready for examination, but an empty slider or so was sent along, as

well as a box of extra talcs (Fig. 5 B), so that the owner of the

microscope could make preparations if he desired. Other acces-

sories needed in the preparation of objects were camel’s hair brushes

(Fig. 5 C) and brass nippers for adjusting the brass rings that held

the talcs in place (Fig. 5 D). Approximately half a century sepa-

rate the first edition of Baker’s “The microscope made easy”

and the last edition of Adams’ “Essays” yet little improvement is

noted in the accessory apparatus figured, the apparent advancement

shown in a comparison of the figures of Adams and Baker being

due rather to better draughtmanship.

Adams described an improved instrument “for cutting thin

transverse sections of wood.” This “cutting engine” (Fig 9) con-

sisted of a wooden base which supported four brass pillars that

in turn bore a flat plate of brass, in the center of which was a tri-

angular hole. The piece being sectioned was placed in a tri-

angular trough on the under side of the brass plate, and fastened by

a brass screw. A diagonal knife blade, greatly resembling the blade

of a plane, did the cutting. This was moved back and forth by a

handle, its course being governed by two grooves in the top of the

brass plate. The amount that the block was raised was governed by

BOTANICAL MICROTECHNIQUE 83

a micrometer screw. In cutting, only fresh material or well soaked

material was used and this was kept flooded with alcohol to prevent

the curling of the sections. That other microtomes besides those

of Cummings, Adams, and Custace were known may be judged by

the foot-note of Kanmacher (the editor of the second edition of

Adams’ “Essays’’). “Many other kinds of cutting engines have

been constructed, but specimens from them have not yet appeared

_with the perfection which is requisite to this sort of objects ; whether

it lies in the preparations of the woods, or engine, I do not take on

me to determine.” |

In this work also appears the first record of the dealer in micro-

scopical preparations, the last chapter containing a list of “vegetable

cuttings’ which Custace supplied to those interested in microscopy.

His sections were all prepared by means of the microtome that

Thornton has described. Custace was quite famous for the sections

that he made, his preparations being supplied with all the high-

grade microscopes of that time. Even as late as 1852 we find

Queckett saying: “some of his (Custace) preparations have not

been improved on to the present day.’’ Custace, who was a “com-

mon carpenter of Ipswich” kept his methods of making preparations

secret during his life, refusing an offer of £50 from Thornton for

a description of his methods. After his death all his effects were

auctioned off and Thornton “fearful that a monopoly might be made

of the art of preparing vegetable cuttings, as had been successfully

done by Custace,” bid in the two microtomes offered for sale.

Thanks to the generosity of Thornton we have a description of

these “cutting engines.” The outer case of the cutting machine

(Fig. 10) was made of brass, in the form of an oblong box, which

was completely filled with a block of hard mahogany except for the

holes necessary for the mechanism that held and raised the material

being sectioned. The block was raised by a micrometer screw,

which is not shown in the illustration, the screw being operated by

an index wheel (O) at the side of the box. The large screw at the

left of the microtome was used to clamp the block in the brass

“Holdfast’ (D). Especial attention was paid to the designing of

the mechanical means for guiding the knife, and it may be due to

this that the machine cut such good sections. The knife blade was

84 GILBERT MORGAN SMITH

set diagonally on a bed (H) which slid back and forth along a steel

rod (GB). Another device for securing rigidity was the placing of

a spur (u) on the top of the microtome. This spur held the material

in its crotch and was prevented from giving by a screw (v).

In concluding the discussion of microtechnique before 1800 the

following items might be of interest. They are taken from the ad-

vertisement of W. & S. Jones and appeared in Adams’ “Essays.” .

£ s d

Common Microscopes from 2 s. 6d t0.........+-e0es 1 1 0

Compound Microscopes from 2/12/2 to.........+++5. 5 5 0

New Improved ditto with most complete apparatus... 10 10 0

Cutting Engine for slices of vegetable objects......... 3 3 0

Ivory Sliders, for transparent objects (per AOR Ae hak 0 12 0

Custace fine vegetable cuttings in large ivory sliders,

from a set of six sliders to four dozen (per doz.).... 1 10 0

Tue TECHNIQUE OF THE EnciisH Microscopists (1800-1875)

With the beginning of the 19th century we find botanical mi-

crotechnique developing along two distinct lines. With the rise of

the German phytotomists there was a high degree of specialization

along the line of microchemistry while comparatively little attention

was paid to refinements in methods of making preparations. In

England, on the other hand, with the reawakening of interest in the

microscope, attention was largely confined to developing methods

of making microscopic preparations. Non-scientific Englishmen, as

well as the microscopists, were interested in the microscope and so

we frequently find articles on the microscope, or on microscopic

objects in the popular magazines. Examples of these popular articles

on the microscope are those which appeared in the Saturday Mag-

azine or the Mirror. Popular treatises on the microscope also

appeared and through the influence of such works as those of Brew-

ster or Pritchard (1847) there was a recruiting of a body of micro-

scopists. With a few notable exceptions the English microscopist

made no great contributions to botany, that were based on micro-

scopical observation, but they were largely instrumental in advanc-

ing microtechnique. . It has been through the refinements zoologists

have made in the Eiglish microscopists’ methods, and then in turn

their adaptation by botanists that we have the botanical microtech-

nique of today. Indirectly also we are indebted to the microscopists

= Sl

BOTANICAL MICROTECHNIQUE 85

for the great stimulus that their interest in the microscope, and their

willingness to purchase it, gave toward the improvement of the in-

strument ; hence the development of the microscope up to compara-

tively recently has been due almost wholly to demands of the mi-

croscopist for a better instrument.

I have previously mentioned that the early microscopists did

not work in regular laboratories. A great majority of the men in

England who were interested in microscopy in the period under dis-

cussion were not connected with an institution where they would

have regular work-rooms, so worked in their homes. Consequently

writers on microscopy give directions for the best conditions for

work. Goring and Pritchard recommend a separate room, which

they call an observatory, that is always to be kept locked when not

in use. The difficulties with which these workers were beset is

brought out in the following: “Have the fear of the cat before your

eyes, and also those busy, intermeddling, officious, housewives, who,

under pretense of dusting, cleaning, and setting to rights, will sub-

vert and revolutionize the whole economy of your observatory, and

perhaps throw half your tackle behind the fire.”

The use of the ivory and wooden slider continued until the

first or second decades of the 19th century, though they did not be-

come entirely obsolete until about 1860, some of the inferior micro-

scopes at that time being furnished with them. With the general

introduction of the glass slide the methods devised for the ivory

slider were adapted to the glass slider with a few changes. This

modified method is described by Gould who in making permanent

preparations took two pieces of glass, of the same size, and then

pasted on one a piece of paper containing one or two holes. After

the mucilage was dry the material was placed in the holes in the

paper, the other glass pasted on the paper, and the whole held to-

gether until thoroughly dry. That this process became quite gen-

eral is seen in its description by Griffith, von Mohl, Queckett, Hart-

ing, and others.

The publications appearing in the years 1830-1835 mark the

foundation of the English microscopist’s methods. It is impossible to

state who first substituted the glass slide with a cover of talc for the

ivory or wooden slider in making preparations, but that it was a well

86 - GILBERT MORGAN SMITH

known method by 1830 may be judged by its use by Pritchard (1832)

Varley, Solly and Holland. Pritchard's revolutionary method of

mounting dissections in a thick solution of gum and isinglass and

then covering them with a thin plate of talc was described in 1832

but did not come into general use since it was so soon superseded by

mounting in Canada balsam. Credit for the introduction of Canada

Balsam is generally given to J. T. Cooper who suggested its use to

New and Bond, professional microtechnicians of that time, but the

publication of the method is due to Pritchard (1835). Balsam may

have been used before this since Adams states the following about

Swammerdam’s methods of preparing insects for microscopical ob-

servation: “Sometimes he has examined with the greatest success,

and made the most important discoveries in insects he had preserved

in balsam, and kept for years together in that condition.” I have

been unable to find this description in the work of Swammerdam,

but Adams may possibly have taken this from Boerhaave, an article

which I have not personally consulted. However, it was through

Pritchard’s publication that Canada balsam became widely known

as a mounting medium. The first mounts made differed considerably

from those we now use since Canada balsam was taken in the natural

state, instead of dissolving it in some solvent, and after melting a

small piece on a slide the object was mounted before the balsam

hardened. Judging by the space given to the description of the

process in the older works on microscopy there must have been con-

siderable difficulty in making the preparations and all sorts of me-

chanical contrivances were devised to hold the cover in place during

the drying of the balsam, to melt the balsam, to remove air bubbles

from in under the cover glass, etc.

The development of methods of mounting objects in liquids is

of even greater significance. The earliest record I have been able

to find is by Goring and is as follows: “I have neglected to describe

a kind of slider which I use in my microscope; it is composed of a

glass tube, flattened, and drawn out to the size of a common slider,

and polished on one side; its use is to hold microscopic objects

which will not keep in a dry state, such as pieces of finely injected

membrane, petals of flowers, and the like; these little preparations

are introduced into the slider, which is filled with spirits, and cov-

BOTANICAL MICROTECHNIQUE 87

ered at the end with a bit of bladder secured by a wax thread”.

This is the only record of this type of permanent preparation. In

1829 a short anonymous note stated that Holland had covered ma-

terial, which showed the Brownian movement, with a talc and then

hermetically sealed the preparation. Four years later Holland de-

scribed the method of making these “ponds” by taking a piece of

glass and enclosing a space on the glass with a cement composed of

white lead and turpentine and then covering with a talc and sealing.

Although devised to show the Brownian movement Holland stated

that the “pond” could be used for tissues. Previous to the publi-

cation of the process by Holland, Varley had described essentially

the same method for preserving “minute vegetable dissections” and

called this type of preparation a “cell,” the name by which they are

universally known at the present day. White lead cells. were also

made as early as 1830 by Valentine, Solly mentioning preparations

of Valentine’s made at that date. It is possible that this type of

preparation was known even earlier for in 1841 Daniel Cooper said

that Dr. Cook recommended a mixture of salt and water as a mount-

ing fluid “20 years ago” (i. e. about 1820). Pritchard’s process

(1832) is even more important for he is the first to have suggested

mercury bichlorid as-a mounting fluid. Daniel Cooper says that

J. T. Cooper used salt and water with a little acetic acid for mount-

ing vegetable tissues, although he does not give the date on which

Cooper proposed this method.

Goadby is generally credited with being the founder of the

methods of making moist preparations, but the citations just given

show that methods of making moist preparations were well known

before the work of Goadby and any credit due him is more for per-

fecting methods in use than for original discovery. He is best

known for the mounting fluid that bears his name. That this pro-

cess was recognized as being revolutionary is seen by the fact that

the Society of Arts gave him a gold medal in 1841 and raised a

private subscription of £500 to purchase his preparations for the

Hunterian Collection of the Royal College of Surgeons (Goadby,

1852). The formula for the solution was first published by Daniel

Cooper in 1841, but since there seems to have been considerable diffi-

culty in making preparations in this way Goadby described his meth-

88 GILBERT MORGAN SMITH

ods and gave his formulae before the Section of Botany at the

York meeting of the British Association in 1844. The Gannel pro-

cess described by Cooper is unknown except for this single refer-

ence. It was quite similar to Goadby’s method except that ‘“‘super-

acetate of aluminia” was used.

The attitude of the British microscopists towards their dis-

coveries was in marked contrast to that of investigators of other

countries. There seems to have been a great freedom of oral inter-

change of ideas, and one man often devised a really epoch-making

improvement in microtechnique but made no attempt to publish it,

being content to communicate his discovery to his friends by word

of mouth. Michael has explained how microscopists came to see

so much of one another when he says that Bowerbank practically

kept open house for microscopists before the formation of the Mic-

roscopical Society of London. After the formation of the Society

in 1839 there was a formal meeting place for microscopists but the

practice of holding “soirees” at various times favored the oral in-

terchange of ideas. To mention only a few instances, we have

Pritchard publishing Cooper’s method of mounting in Canada bal-

sam, Cooper publishing Goadby’s formule, and Clarke the first

method of clearing, a process which he may possibly not have in-

vented.

Holland is the first to describe the deep cell that is made by ap-

plying successfully several layers of cement. The importance of the

cement cell in microscopy at this time is seen in the number of

———

—— ——

HHH! {

Wii Ah Wi |

WHEY ;

Wit ti)

i WLS Ht

Ul Hd a Lie i

Fig. 11—An early form of turn-table for making cement cells. (Carpenter, 1856).

Hi | ‘ Mt i hi

CARA AAA A

cements described by Griffith and Henfry, Queckett, Carpenter, and

Beale. The chief contributions to the subject of cements were Berk-

ley’s description of Thwaite’s method of using gold size, and Reck-

itt’s recommendation of black Japan on account of its quick drying

properties. At first these cells were made by hand, but the invention

ee ee eee - +

BOTANICAL MICROTECH NIQUE 89

of the turn-table by Shadbolt gave a quick mechanical means of

making them. The first turn-table is figured by Queckett, but it was

soon improved and the instrument we use today shows little advance

over the form given by Carpenter in 1857 (Fig. 11).

Griffith gives the status of microtechnique in the early forties

where he says that the use of ivory and wooden sliders has almost

disappeared but he describes a method of dry mounting very similar

to that of Gould. The method of mounting dry objects in balsam,

and several methods of making moist preparations are also given.

For the latter a syrup and gum mixture, dilute alcohol, water satur-

ated with creosote, and Goadby’s solution are described, the last

named being given the preference. In mounting these preparations

the sealing with some varnish, the use of white lead cells, and built-

up cells are all described.

Goadby’s solution proved altogether too strong for plant ma-

terial and consequently a number of media were devised which had

as their object the avoidance of plasmolysis. Thus Reckitt advised

sealing the tissues in either pure water or a dilute solution of corro-

sive sublimate, while in 1849 Warrington recommended castor oil

for certain fungi since he had found this such a good mounting

medium for crystals a few years previous. The desmids have always

been a favorite object for study with the microscopists and we there-

fore very naturally find several formule of mounting fluids for these

delicate organisms ; among them may be cited Thwaites’ mixture of

1 part alcohol and 12 parts water with as much creosote as could be

dissolved, a proportion which was later changed to 1 part alcohol

to 16 parts of water when the process was described in Ralfs’ work

on the desmids. Ralfs medium for desmids consisted of a grain each

of bay salt and alum dissolved in an ounce of water. Glycerine was

first used by Warrington in 1849 for mounting microscopical prep-

arations, but since glycerine alone caused too much plasmolysis Far-

rants proposed a jelly of equal parts of glycerine, gum arabic and

water, while a formula essentially the same as the glycerine jelly

used today was given by Lawrance in 1859. The idea of using

gelatine as the foundation for the mounting fluid really belongs to

Deane who first proposed a mixture of honey, water, alcohol, creo-

sote, and gelatine.

90 GILBERT MORGAN SMITH

Although all of these methods were described before 1860, we

must bear in mind that they were used only in special cases, Canada

balsam being considered the préeminent medium whenever possible.

The column of questions and answers appearing in Science Gossip

during the sixties contains many more references to Canada balsam

than to any other method. A revolutionary step in the manipulation

of Canada balsam was the dissolving of the balsam before mounting.

This was first used by Griffith in 1843 but does not appear in the

article on microtechnique by Griffith but as an editorial note of a

few lines in the same volume that contains his description of micro-

technique. The use of a solvent for the balsam did not come into

general use in England, until more than a decade later about the

only reference to it being those of Boys and Ralph.

Another great step in microtechnique appearing in the decade

between 1850 and 1860 is the introduction of the process of clearing

tissue before mounting them. In 1851 Clarke, in describing his

treatment of certain animal tissues, stated that he put them in spirits

of wine, then transferred them to turpentine and after they had be-

come quite clear mounted in balsam. It is possible that he did not

originate this method since Farrants remarked in 1857, incidental to

a discussion of the use of glycerine, that he had cleared his material

in turpentine before mounting in balsam since 1850, but makes no

mention of Clarke’s name. From the historical standpoint Clarke’s

publication of the process is important for it was through his de-

scription that it became known abroad and in the hands of the Ger-

man zoologists of 1860-1870 developed into the methods of clearing

that still persist. Evidently the value of this method was not recog-

nized in England since the treatises of Queckett, Griffith and Hen-

fry, Carpenter and Beale all fail to mention it.

The cutting engine as invented by Adams, Cummings, and Cus-

tace was used more or less in England between 1800 and 1870.

After speaking about these 18th century microtomes Queckett goes

on to say “in subsequent times other instruments have been con-

trived for the same purpose, some provided with knives that move

circularly, others with knives fixed in a strong framework of metal,

whilst, in not a few, the cutting is performed by a razor of the ordin-

ary kind, or one ground perfectly flat.” I have been able to find

PS ae ee ee

BOTANICAL MICROTECHNIQUE 91

reference to five microtomes, other than the two described in the

first edition of Queckett in 1848, that were made before 1850 but

judging by the statement of Queckett many more were known.

Michael says that before the foundation of the Microscopical Society

of London George Jackson made “a very servicable cutting-machine

for producing thin sections of wood.” The firm of Charles Baker

of London inform me by letter that they have been making micro-

tomes since about 1840. In 1836 Bowerbank described a microtome

that he had invented which was quite similar to the Adams instru-

ment, the chief difference being that the cutting part of the instru-

ment was a razor ground flat on one side. There are also two old

microtomes in the Science Museum, South Kensington, London, that

were made about 1835*, one bearing the name of Andrew Pritchard,

while the other bears no maker’s name but is of very similar design.

Pritchard’s microtome (Fig. 12) is described as follows in the Sci-

ence Museum catalogue. “The apparatus is made to be screwed to

the edge of a table and consists of a flat plate of brass with a well in

it, in which a kind of piston moves up and down by a micrometer

screw. The wood to be cut is fixed to the piston by a small screw,

and as it is raised a knife drawn along the plane surface takes off

thin sections. Should the piece of wood be too small to be placed

in the triangular chamber, it must be glued to a block of convenient

size.” This is the first record of a microtome fixed to a table and

is the forerunner of the hand microtome.

The instrument of Topping was patterned after the Pritchard

microtome and was the best known microtome of the 50’s, being fig-

ured and described by Queckett, Carpenter, and Beale. It is quite

similar to the hand microtomes of the present day, resembling them

more than the Pritchard microtome. Another microtome is one that

is ascribed to Queckett by Harting and Apathy, although I find no

direct evidence that Queckett invented this instrument. It consisted

of a mahogany base (Fig. 13) that supported four brass pillars and a

top plate. The “well” for holding the material was essentially the

same as has been previously described for the Adams cutting engine.

*TI am under very great obligations to Dr. A. B. Bendle of the British Museum

for his kindness in furnishing me information concerning these microtomes, and to

Capt. H. G. Lyons for the photograph and description of the Pritchard microtome.

92 GILBERT MORGAN SMITH

The knife differs considerably from that of the Adams cutting en-

gine in that it was placed diagonally on a brass frame, the whole

sliding backwards and forwards rahe a guide rod on the top of the

cutting engine.

The great interest in microscopy in the decade of 1850-1860 is

evinced by the appearance of five treatises on the microscope and

microscopic manipulation. With the appearance of these works,

methods which had been but little known outside of a small circle of

HANNAN M Hy

MiG

cece mene Sm il

a — WHE = a

Fig. 13.—Queckett’s (?) microtome. (Queckett, 1848).

microscopists became the property of the world and were taken up

and perfected outside of England. The advances made in micro-

technique by the introduction of the complicated paraffine method

made the science one for the laboratory rather than for the home and

although interest in microscopy continued in England the progress

of the science took place in laboratories of the German zoologists.

The most important contribution of the microscopists to micro-

technique between 1860 and 1875 was the introduction of staining,

a a ee a ao eee

BOTANICAL MICROTECH NIQUE 93

particularly the staining of plant tissues, although the German bot-

anists did not avail themselves of the staining methods of the Eng-

lish microscopists. Since the discussion of this subject will be taken

up later no further mention will be made here.

The French microscopists were dominated to a large extent by

the English school of microscopists and have played a relatively

unimportant réle in the development of microtechnique. Several of

the English works on the microscope were translated into French, Le-

bour’s Galerie Microscopique, for example, being the French edition

of Pritchard’s Microscopic Cabinet. In the works written by the

French themselves English methods were drawn on to a much great-

er extent than the German, Chevalier and Dujardin both showing

quite strongly the influence of the English microscopists. Cheva-

lier deserves credit for being one of the first to substitute thin glass

covers for the talc in making permanent preparations.

BoTANICAL MICROTECHNIQUE IN GERMANY 1800-1875

As far as the art of making microscopical preparations is con-

cerned the technique of the German botanists was far behind that

which the English microscopists used in making preparations of bo-

tanical material during the period described above. This was due to

the German botanist’s belief in the utmost simplicity. To emphasize

the necessity of great dexterity with a few instruments, von Mohl,

Harting, and Behrens all quote Benjamin Franklin’s adage that “a

naturalist must saw with an auger and bore with a saw”. Such

great manual dexterity was developed in cutting free-hand sections,

that mechanical instruments were thought to be only for those who

could not make good free-hand sections. Thus in his review of the

description of the Oschatz microtome, von Mohl held that the Os-

chatz microtome was of real value only when one wished to prepare

large sections for a microscopic cabinet, and that for scientific in-

vestigation the microtome was highly superfluous.

In Germany as in England the dry mount was used exclusively

at first. With the rise of the Phytotomists there is some evidence

of use of water mounts, although it is not clear whether the material

used by Link and Kaulfuss was merely examined in a drop of water

or whether this drop was covered as we do today. The water

94 GILBERT MORGAN SMITH

mount was quite generally used by the later Phytotomists; Meyen

in his directions for manipulating the microscope (1830) telling us

to use a glass slide and a drop of water and then cover it. No per-

manent preparations were made in Germany before 1840, all sec-

tions made with the razor being examined in temporary water

mounts.

Kaiser (1877) states that Germany did not take up microscopy

until about 1839 and that Moser was the first to bring about a de-

velopment of microtechnique, while Behrens thinks that von Mohl

was largely instrumental in creating this interest in microtechnique.

The chief contributions to the technique of making permanent prep-

arations are those of von Mohl, Oschatz, Schact, and Harting. Eng;

lish methods were not drawn on until about 1850 although the

papers of Griffith and Varley had been translated into German. The

year of the translation of Griffith’s paper marks the appearance of

Oschatz’ methods of making permanent preparations. Oschatz be-

ing a microscopist, discussed both animal and plant tissues. For

the latter water alone was not recommended but either a concen-

trated sugar solution, or a sugar solution containing a little acetic

acid was recommended. When the young plant tissues were too

opaque, Oschatz found that they could be cleared to a considerable

extent by placing them in acteic acid before mounting. The follow-

ing year Moleschott described Harting’s process of mounting plant

material in a concentrated solution of iron-free calcium chlorid so

that the technique of making permanent preparations received a great

impetus at this time. Harting took a slide and pasted a strip of

paper on each end and then mounted the material in a drop of the

calcium chlorid solution in the center of the slide. This was covered

with another slide of the same size and the two fastened together

with paste on the strip of paper. There was no necessity for seal-

ing this preparation since the hygroscopic nature of the calcium

chlorid prevented evaporation. An interesting side light showing °

what was considered essential in the study of the cell at that time is

found in Moleschott’s comment on the availability of the method.

He says that apart from the swelling and dissolution of the starch

grains, the dissolution of the nucleus in a few months, and the

shrinkage of the surrounding membrane in many cases (von Mohl’s

BOTANICAL MICROTECHNIQUE 95

primordial utricle), the method of mounting in calcium chlorid so-

lution is a very good one. Immediately following Moleschott’s des-

cription there is a comparison by von Mohl of Oschatz’ and Hart-

ing’s methods in which he thinks Harting’s process is the superior

and he bemoans the fact that it had not been discovered earlier. The

chief objection to mounting in a sugar solution was the inability to

seal the preparation properly, von Mohl’s preparations usually not

lasting over a year. On the other hand Miinter thought Oschatz’

method the best. The “Mikrographie” of von Mohl is one of the

first collections of its kind in Germany and gives a good idea of the

methods in use at that time. It may be well to note that the first

edition of Queckett, which appeared at approximately the same date,

devotes about half the pages to methods of microtechnique, while in

the 277 pages of von Mohl’s work only 27 pages are given to the sub-

ject, thus showing the difference between what was considered es-

sential in microscopy in England and in Germany. Von Mohl

thought that most organic bodies should be studied in water mounts

since balsam rendered them too transparent. A method of mount-

ing dry preparations, essentially like that of Gould, was described.

The technique of Oschatz, Griffith, Thwaites and Reckitt was des-

cribed but scarcely any attention paid to the making of cells, all prep-

arations containing fluids being hermetically sealed by some varnish

after the cover had been placed in position. The calcium chlorid

method of Harting was recommended whenever possible, but the

swelling of the starch grains and the shrinking of the primordial

utricle prevented its universal use. Owing to his strong advocacy

of calcium chlorid von Mohl is frequently credited with being the

originator of this method.

The formation of the “Verein fiir Mikroskopie zu Giessen” in

1856 helped standardize methods. This society adopted, after con-

siderable experimentation, a uniform object carrier for all those

-members who wished to exchange preparations. They rejected the

English size of 1 by 3 inches, a form that came into use in that coun-

try soon after the foundation of the Microscopical Society of Lon-

96 GILBERT MORGAN SMITH

don, and used one 33 by 28 mm. instead.* In the by-laws of the so-

ciety reported by Leuckart and Welcker the following abbreviations

show the mounting materials most generally used in Germany in

1856; Al. alcohol, CB Canada balsam, CC calcium chloride, Gi. gum

arabic, Gl. glycerine, Lc. liq. conservatoire (Paccini’s fluid), WG

water glass, Z. sugar, O. dry mount.

The German botanists were familiar with the publications of

the English microscopists which were appearing about this time and

so we find a gradual abandonment of the calcium chlorid and the con-

centrated sugar solutions as the exclusive mounting media. This

recognition of the English microscopists’ methods seems to have been

due to Welcker’s publication, judging by the statement of von Mohl

in 1857. Unfortunately I have been unable to consult the original

paper of Welcker. Von Mohl here gives the preference to glycer-

ine over calcium chlorid as a mounting medium. Another publica-

tion which greatly influenced botanical microtechnique was that of

Schact, which appeared in 1851. This is the first work devoted ex-

clusively to plant histology. Comparatively little attention is given

to making permanent preparations, only three methods being men-

tioned, namely, the use of calcium chlorid, sweet oil, and Canada

balsam, but because of the minute directions for the anatomical

study of different plants the book was of great value. This work

also illustrates the difference between the microscopical methods of

the English and the Germans. In the case of the English the prep-

aration was the main thing, while with the Germans the preparation

was only a means to an end.

*The proper size for the object carrier was a subject of considerable controversy

in Germany. The following are some of the sizes recommended.

78x26 mm. (London format, 1840)

70x22 mm. (von Mohl, 1840)

30x40 mm. Diameter circular plates. (von Mohl, 1840) —

2/3 x 2/3 in. (Oschatz, 1851)

33x28 mm. (Giessen format, 1856)

55x26 mm. (Frankfort format, cited by Leuckart and Welcker in 1857)

70x20 mm. (Gerlach, cited by Leuckert and Welcker in 1857)

37x22 mm. (von Mohl, 1857)

43x28 mm. (von Mohl, 1857)

48x28 mm. (New Giessen format, date of introduction unknown)

65x25 mm. (Vienna format)

Perhaps the new Giessen format of 48x28 mm. has been the most used in Ger-

many up to the last decade, but at present the English format is in almost universal

use. For other sizes less frequently used in modern times see Behrens.

BOTANICAL MICROTECHNIQUE 97

If the German botanist was not fully abreast of developments

in the technique of making preparations, he more than compensated

for this in the forwarding of botanical microchemistry. By 1800

unorganized studies are found in which nitric acid, hydrochloric

acid, the solvent action of alcohol, and the like were used in an at-

tempt to discover the nature of the cell contents. Perhaps the re-

searches of the French botanist, Girod-Chantrans may be taken as

typical of this blind groping toward a microchemical study of the

plant tissues. One of the earliest definite microchemical reactions

is the discovery by Link in 1807 in which he used iron sulphate for

determining tannic acid in leaves. In the same article Link relied

on warm water, sulphuric and nitric acids as a test for starch. The

macrochemical reaction of starch and iodine (discovered independ-

ently by Stromeyer, and Colin and de Claubry), was used micro-

chemically by Raspail in 1825. Four years later Raspail found that

sulphuric acid gives a purple coloration to albumen in the presence

of sugar. Raspail used this reaction either for the determination of

albumen or sugar, when it was present in large quantities, as in

pollen grains. Schleiden in 1838 and von Mohl in 1840 showed

that after treating the cell wall with sulphuric acid, iodine caused a

blue coloration of the cellulose. Ten years later, in 1850, this meth-

od was largely supplanted by Schultze’s zinc chloride solution. In

the same year Millon devised the test for proteins that bears his

name. Thus we find that by 1850 the microchemical determination

of the constituents of the plant cell was in a fairly satisfactory state.

Although Schleiden gave a list of chemicals for the study of the

cell in 1842, the first serviceable collection of microchemical methods

is that of Schact in 1852, in which the means of recognizing many

different plant products is given. Thus cellulose and xylogen can

be differentiated by the reagents given, protein compounds recog-

nized by their behavior towards iodine, nitric acid (a reaction which

was pointed out by Glauber in 1686), hydrochloric acid, and Ras-

pail’s test. Starch is recognized by iodine, gums and dextrines by

digestion and the formation of a flocculent precipitate in alcohol.

No very sure method is given for sugar or fats, Raspail’s test show-

ing sugar when present in abundance, while fats are determined by

their high refractive power and disappearance under the microscope

98 GILBERT MORGAN SMITH

in the presence of alkalis. Other important advances were the ap-

plication of Troemmer’s reagent to microchemical analysis by Sachs

in 1859 and the use of alcanna tincture by Mueller for the determina-

tion of fats. The growth of microchemistry may be seen by com-

paring Poulson, Behrens, Zimmermann, and Richter.

The work of the Hofmeisterian epoch which led to the founda-

tion of our knowledge of the alternation of generations was carried

out almost wholly on fresh material and with free-hand sections.

Botanical study in Germany between 1840 and 1875 was dominated

by von Mohl, Schleiden, Hofmeister and Naegeli, all of whom

favored the use of as simple a technique as possible, their mastery

of free-hand use of the razor being traditional even to the present

day. It may be that the great skill which they possessed with the

razor made them loath to use the microtome and although Unger and

von Mohl mention the microtome they did not recommend it but

gave the impression that it was an instrument for the dilletante

rather than for the serious worker. Even today this idea lingers in

certain quarters and is expressed in the saying that “a steady hand

is the best microtome.” In certain cases special methods of hold-

ing the material were recommended, von Mohl using pieces of pith

for holding thin leaves and the like, whereas Unger used cork.

Schleiden in 1842 described a method of fastening material to the

thumb-nail with saliva or oil and then rocking the razor blade back

and forth after the manner of a rocking horse. The first attempt

at embedding material appears at this time. Unger tells us that

Fenzl devised a crude embedding process for sectioning seeds by

dropping the material to be sectioned in a hole made by a hot needle

in a piece of stearine, and then the whole mass was cooled and cut.

Griffith and Henfry modified this process by substituting white wax

as the embedding medium. Schleiden devised another embedding

method of immersing minute objects in a thick mucilage of gum ara-

bic which was dried on a small board until glassy. After section-

ing the sections were placed in water to swell them to their normal

size. Staining was not used in the microtechnique, if we exclude

the use of iodine. The article entitled “ovules,” an English des-

cription of German methods by Griffith and Henfry, gives a good

idea of the free-hand methods in use at that time. Their directions

BOTANICAL MICROTECHNIQUE 99

are as follows, “The ordinary plan is to place an ovule between the

thumb and fore-finger of the left hand, and with a very sharp

razor cut it into two unequal parts, in the direction of the axis. The

larger piece is then laid on its flat side on the finger (by the aid

of a mounted needle) and another section made so as to leave a

section preserving all of the central part of the ovule. This ad-

heres either to the finger or to the razor and a drop of water should

be placed on it to free it; then it may be transferred to the slide by

a very fine camel’s hair pencil. Examined under a low power it

will probably be found to require further dissection, with exceed-

ingly fine needles, under a simple lens, sometimes mere pressure is

of service. We have found ovules which have been kept in spirits

easier to dissect; when fresh, the cell membranes are excessively

delicate.”

The compressorium, an instrument much used by the animal

histologists of that time, proved of but little service in botany, while

maceration methods were used to a considerable extent for the dis-

sociation of tissues. The earliest process, introduced by Molden-

hawer, was the decaying of the wood in a manner very similar to

Hill’s method. Various strong acids and alkalis were also used for

this purpose, the recommendations of Schleiden (1842) being typi-

cal, but after the discovery of Schulze’s maceration methods little

else was used. The first fluid Schulze proposed was a mixture of

nitric acid and phosphoric acid but the more recent combination of

nitric acid and potassium chlorate has found widespread favor

among botanists. Chromic acid was also used for this purpose to

some extent, having been introduced by Sanio in 1863.

The mounting media developed by the English microscopists,

especially glycerine and glycerine jelly, were widely used by the

German botanists of the sixties. Mounting media which were or-

iginated by botanists in the latter part of the period under discus-

sion include the use of potassium acetate by Sanio the use of po-

tassium hydroxide by Schulze and Hanstein’s potassium hydroxide

solution. The methods which were used by the German botanists

at that time showed, however, no great advance and between 1860

and 1875 the progress which they made in methods is not at all

comparable to the progress which the zoologists were making.

100 GILBERT MORGAN SMITH

Tue DEVELOPMENT OF THE MODERN MICROTECHNIQUE

To properly understand the development of botanical micro-

technique it is necessary to review the progress that the zoologists

had been making in microtechnique. They soon recognized the

value of Clarke’s process of clearing in turpentine before mounting

in balsam and various improvements were made in the procedure.

As a result we find Kutschin substituting creosote for the turpen-

tine, and two years later Rindfleisch cleared his preparations in

clove oil before mounting in balsam. In the following year, 1866,

Steida made an extensive study of the clearing action of about 30

essential oils, and among others recommended bergamot oil.

Although attempts at staining were first made by microscopists

on botanical material, or by botanists, the development of the tech-

nique of staining is almost wholly due to the zoologists. I have

been unable to find anywhere recognition of John Hill as the real

pioneer in the history of staining. Another process, which is essen-

tially similar to staining, is that of Reade who charred plant tissues

in order to render them more visible when mounted in balsam.

Queckett in 1848 recommended charring or staining with either io-

dine, fustic or logwood extracts to bring out the structure of plant

tissues mounted in balsam. In 1843 Goppert and Cohn used car-

mine for the study of the cell contents of Nitella. A few years

later Hartig (1854), used carmine for staining the cell contents and

noted that the nucleus was not stained until after the death of the

cell. Osborn also used carmine for his studies on the root-tip of

the wheat plant. The data on the history of staining have been col-

lected by Gierke and later by Apathy. Gierke compares the claims

of Goppert and Cohn, Hartig, and Osborn and thinks that Hartig

should be given the credit for discovering the process of staining

tissues with carmine. Apathy thinks that the real credit for the

discovery belongs to Corti, a work which Gierke apparently over-

looked. In my opinion, for reasons given above, Hill should be

credited with having first used staining methods in connection with

microscopical work. Queckett’s work also antedates that of Corti

or Hartig, although Corti should retain the honor of being the first

to apply staining methods to the study of the contents of the cell.

Previous to the study of the early literature, Gerlach was generally

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BOTANICAL MICROTECHNIQUE ; 101

credited with having first used stains. Although this has been

shown to be false, Gerlach’s use of stains is important for it was

through the adoption of his methods that staining came into general

use. Apathy has made a very apt comparison when he says that

Corti and Hartig were the Normans (Norsemen?) of staining while

Gerlach was the Columbus.

Experimentation also began on the value of other substances

for staining tissues. Waldeyer is held by both Gierke and Apathy

as the first to use hematoxylin as a stain. This is incorrect, since

Queckett (1848) and Wigand had both used logwood stains before

Waldeyer. In 1862 Wigand made an extensive study of the be-

havior of plant tissues towards coloring matters. This work was a

study of the phenomena connected with the staining of plant tissues

from the standpoint of the commercial dyer rather than from that

of the microscopist trying to bring out structural differences. Be-

sides cochineal he stained with hematoxylin and certain other

colored plant extracts, as yellow wood (old fustic), cutch, and root

extracts. Negative results were obtained with indigo and various

mineral stains. A selective staining of different parts of the plant

was noted but no use was made of this except to help elucidate the

theory of staining that he proposed. The first hematoxylin stain

of real value was that of Boehmer, who, in 1865 gave a formula

that is in use to the present day.

Soon after the discovery of aniline dyes, we find the applica-

tion of them to the staining of microscopical preparations. The

first to use them was Benecke, although at the present day we do

not know what his “Lila-Anilin-Farbe’ represents. In the next

year (1863) Waldeyer employed Rosaniline (Aniline Red), Anilien

(Aniline Violet), and Parisian Blue (Aniline Blue). In the same

year there was the independent discovery of the staining power of

Magenta (the English name for Fuchsine) by Lynde and by Rob-

erts. Lynde used Magenta for staining the contents of plant cells

and noted that the contents were not stained until after the death

of the cell, while Roberts stained blood corpuscles with Magenta.

In the following year Abbey experimented with a still larger num-

ber of dyes. In studying the contents of the plant cell he used

Mauve, Hoffmann’s Green (Iodine Green), Aniline Brown, Picrate

of Aniline, Magenta, Aniline Green and two blue colors the names

102 GILBERT MORGAN SMITH

of which are not given. Except for the work of Lynde and Abbey

practically all of the early work on staining with aniline dyes was

done by zoologists. The list of discoveries along this line might be

prolonged indefinitely ; for the first twenty years following the intro-

duction of staining with aniline dyes by Benecke, Gierke cites 55

references, each of which contains something that constitutes a

distinct advance over methods known up to that time, and all but

three refer to zoological or medical publications. Among the stains

most used by botanists today may be noted the introduction of

Dahlia by Huguenin in 1874, Eosin by Fischer in 1875, Methyl

Violet, Iodine Violet and Safrain by Ehrlich in 1877, Bismarck

Brown by Weigert in 1878, and Methylene Blue by Ehrlich in

1881. It must be remembered that these stains were used sing-

ly and not in combination as we now use them. The first double

staining is that of Schwarz, who in 1867 stained his material

in carmine and then in picric acid. Other early combinations

of stains were Eosine and Methyl Green, Eosine and _ Dahlia,

and Eosin and Methyl Violet. The method now most generally

used, that of overstaining in a solution of the dye and then de-

staining to the proper intensity became well known through the

work of Flemming (1881), although the process had been pre-

viously used by Beettcher and Hermann. Flemming experimented

with a large number of dyes to find which one gave the best nu-

clear stain, among those used were Safrainin, Magdala Red, Dahlia,

Mauve, Rouge Fluorescent, Solid Green, Ponceau, Fuchsine, Eosine,

and Bismarck Brown. Later Flemming (1884) found another good

stain in Gentian Violet. Safranin and Gentian Violet as a double

nuclear stain was not suggested by Flemming as is frequently

stated, but was first employed by Brazzola; while the much used

triple stain of Safranin, Gentian Violet, and Orange G was com-

bined by Flemming in 1891.

The development of embedding methods is also due to zoolo-

gists. The term embedding should be used with care since it has

been used by investigators in two different senses. In one case

there is merely the surrounding of the material with any medium,

the Fenzl method mentioned above being an example. In the

other embedding process the tissue is completely saturated with

BOTANICAL MICROTECHNIQUE 103

the embedding medium. The first process might well be called an

enclosing method, while Apathy has suggested the term interstitial

embedding for the latter. Paraffine was introduced into micro-

technique, as an enclosing medium, by Klebs in 1869. Other sub-

stances used for the enclosing method of embedding were gum

arabic by Heidenhain, glycerine jelly by Klebs, and albumen by

Bresgen and Calberla. In the enclosing methods of Strickler and

of Born the material was dehydrated, cleared with an essential oil

and ‘then placed in a mixture of wax and oil, while after section-

ing some solvent was used to remove the enclosing medium.

Although Schleiden’s method introduced in 1842 was an inter-

stitial embedding process the first serviceable method is that of

Flemming, who in 1873 used transparent soap. Bergamot oil was

used as the solvent for the paraffine in the older embedding methods,

but it has since been shown that paraffine does not dissolve in berga-

mot oil to any appreciable extent. Turpentine as a solvent for the

paraffine was not generally adopted since it caused plasmolysis.

The independent discovery of cholorform as the solvent for the

paraffine by Griesbrecht and Biitschli, in 1881, brought the method

up to a point where there could be an interstitial embedding of the

most delicate tissues without plasmolysis. The other embedding

medium which is widely used today, celloidin, was introduced by

Duval in 1879. Duval used collodion, but its use was abandoned

after Schiefferdecker showed the greater adaptability of the patent

collodion called celloidin.

Fixing solutions are a comparatively recent development. The

older investigators were more anxious to obtain some substance

that hardened the tissue than to obtain what we now call fixation.

The different editions of Lee reflect well this change in attitude

towards the hardening agents. Chromic acid was one of the earliest

hardening agents introduced, Hannover using it in 1840. The term

fixation came into use in the early eighties and practically all of

our fixing mixtures were proposed in the decade of 1880-1890.

Lang used mercuric bichloride as a fixing medium either alone, or in

combination with acetic acid, picric acid, or alum. Osmic acid,

although introduced into microtechnique by Schultze in 1864 was

not used as a fixing medium until Flesch combined it with chromic

104 GILBERT MORGAN SMITH

acid. Flemming experimented with a number of combinations con-

taining osmic acid and decided that a mixture of osmic, acetic, and

chromic acids gave the best result. The first formula published is

now called the “weak” formula since two years later in 1884, he

gave another mixture of the same Petit in greater concentra-

tion, forming what is called the ‘“‘strong” mixture. These few cita-

tions are not given in an attempt to cover the field but more in an

endeavor to show the period in which methods of fixation came into

general use.

The general opinion seems to be that the microtome is of fairly

recent origin. Thomé states that the greater number of microtomes

go back to two fundamental types, the Ranvier and the Rivet micro-

tome. Minot thinks that the first microtome which resembles the

modern microtome is the instrument of His, the Valentine double-

all

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man: is

tinal

Fig. 14.—Valentine’s double-bladed knife. (Queckett, 1848).

bladed knife being regarded as a forerunner of the modern micro-

tome. In discussing the development of microtechnique before

12800 I have mentioned the microtomes of Adams, Custace, and

Cummings, while it was also shown that the English microscopists

used similar instruments more or less commonly in the years suc-

ceeding 1800. Valentine’s double-bladed knife (Fig. 14), invented

in 1839, has been of limited service in animal histology, and is

even sold to the present day, Another instrument, called a micro-

Fig. 15.—The Straus-Druckheim microtome, (Robin, 1871).

tome, is that of Straus-Druckheim. This, as is shown in Figure

15, is really a pair of dissecting scissors, the blades of which are

prevented from cutting their full length by means of a screw.

BOTANICAL MICROTECHNIQUE 105

In 1843 Oschatz invented an instrument for cutting sections

which he called a microtome. This term was generally accepted

by German histologists but the English microscopists used the term

cutting machine or cutting engine, reserving the name microtome

for the Straus-Druckheim instrument. Robin has protested against

calling sectioning instruments microtomes maintaining that the

term should be only employed in connection with the Straus-Druck-

heim instrument. This is not justified on the grounds of priority

since in 1839 Chevalier called the sectioning instruments of Cum-

mings and Adams, “le couteau micrometrique ou mieux |’ instrument

Fig. 16.—Ranvier’s microtome. (Dipple, 1882).

microtomique.” From this we may judge that the coiner of the

word microtome (Chevalier) used it to describe any instrument for

cutting sections.

The mechanical principle of Cummings and Adams microtomes

is found, with one exception, in all of the microtomes made before

1868. Although varying considerably in detail all of these micro-

tomes have a holder in which the material is clamped and then

rassed through a cylinder by means of a screw. The Oschatz micro-

tome was made in two forms, either a simple hand microtome or

a table form that had a three legged base. Welcker’s microtome

was very similar to that of Oschatz. Other microtomes which were

made in the middle of the last century were those of Smith in 1859,

Schmidt in 1859, and Luys in 1868. The microtomes of Nachet

and Collin appeared some time before 1870 but I have been unable

106 GILBERT MORGAN SMITH

to find the exact date. Other microtomes were also made judging

from Harting’s statement (in 1864) that there was a very old micro-

tome in the Utrecht Cabinet, while Unger mentions using a cutting

machine made by Plossl of Vienna. The microtome of Ranvier

(Fig. 16) presents no further advancement in the construction of the

instrument, in fact being one of the simplest ever devised, but it

was through the use of the instrument by so eminent an investigator

that the microtome came into real favor among zoologists. Gudden

improved the Ranvier microtome by fastening it to the table, but

the fastening of the microtome to the table had been described

years before by Topping and by Pritchard and was well known to

all the English microscopists of the fifties.

Another mechanical principle used in the microtome is the

gradual raising of the object holder by pushing it along an inclined

plane. The first application of this principle is generally described

to Rivet, but Capanema had made use of the principle twenty years

before the invention of Rivet’s microtome. Harting is the only one

who recognized the value of this instrument and he stated that it was

the best microtome which had been made up to that time. Harting

reproduces two of the five figures from Capanema’s plate but the

vertical section is shown upside down so that at a cursory glance

there would appear to be little of value in the machine. The instru;

ment was only about 2% inches long, Fig. 17A, showing a trans-

Figure 17.—Capanema’s microtome. (1848),

verse cross section, the chief point of interest being the object

holder. This consisted of two clamps (ff) which were regulated

by a screw (g), a plate (d) forming the base of the holder. After

the material had been tightened in place by a crank (k), the crank

was removed from the screw. The object holder slid on two lateral

BOTANICAL MICROTECHNIQUE 107

inclined planes (cc in Fig. 17B) and from its bottom a pillar (1)

extended downwards, the turning of another screw (x) moving the

object holder. As the screw (x) turned it caused the holder to

move up the inclined plane and this slowly raised the material held

in the clamp. The sections were cut by means of a knife that slid

along two guide plates (bb).

Rivet’s microtome (Fig. 18) is the fundamental type on which

all improvement of the sliding microtome have been made. Botan-

ists should be interested in this microtome since it was devised for

cutting plant tissues. In the advertisements inserted in the 2nd.

edition of Negeli and Schwendener we find Rivet’s microtome ad-

— ==

—_ _—— cai

Fig. 18.—Rivet’s microtome. (Grénland, 1878).

vertised for cutting plant material, while Ranvier’s microtome is

listed for making animal sections. The microtome as invented by

Rivet was first made entirely of wood by Verick of Paris in 1868.

Minot states that it was described in the Annales des Science Nat-

urelles but the first description that I have been able to find is that

of Gronland. The microtome consisted of three parts a central

block and a separate carrier for the material and the knife. The

central block had a base measurement of 16 by 6 cm, and a height

of 6 cm. On either side of this block there were wedge shaped

grooves so that the middle upper portion was only 13 mm. in thick-

108 GILBERT MORGAN SMITH

ness. The groove at the left had a slope of 1:100, whereas the

right hand groove was parallel to the top of the instrument. On

the top of the middle portion there was a scale parallel to the slop-

ing left hand groove, and this was divided so that each division

corresponded to a vertical elevation of 1:100 of a millimeter. A

block that was fitted in the right hand groove carried the knife

and another in the left hand sloping groove carried the object holder.

The apparatus for holding the material was very simple, being

fashioned after a patent American clothes-pin. This clothes-pin

holder was fastened to the block so that no orientation of the ma-

terial was possible. The microtome was operated by drawing the

knife towards the operator and then shoving ahead the block in

the inclined groove the desired distance, the height which the block

was raised being computed from the scale at the top of the micro-

tome. After drawing the knife forward the process was repeated.

Brandt constructed the sliding microtome of metal instead of

wood, and since this instrument was made by Leyser it is often

called the Rivet-Leyser microtome. Gr6énland considers this sub-

stitution of metal for wood a step backwards. A still more import-

ant improvement in the microtome is the introduction of the mechan-

ical advancing of the object holder by means of the screw, as found

in the Schanz microtome. The other essential improvement was the

discarding of the primitive clothes-pin type of object holder and

the substitution of a holder capable of being rotated in any direc-

tion. Further improvements in this type of microtome have been

chiefly variations in already existing principles used in its construc-

tion. Perhaps the most notable of these has been the introduc-

tion of an automatic device for raising the holder and a mechanical

means of operating the knife carriage.

The Caldwell and the Rocking microtome, both types of auto-

matic instruments, were introduced by the Cambridge Scientific

Instrument Company in 1885. The Caldwell automatic microtome

is of historical interest only, since it was used but little, but in its

simplified form, as the Cambridge rocking microtome, it gained a

widespread popularity. Since this instrument is so generally known

no description of it is necessary. The mechanical error of cutting

sections in a curved plate instead of a flat plate has been overcome

Sor ee

BOTANICAL MICROTECH NIQUE 109

in later models. Ryder’s microtome was invented in 1888 but did

not come into general use since the mechanical principle underlying

the construction of this instrument was also incorrect because the

paraffine block did not pass evenly across the edge of the knife but

moved in the arc of a very short circle. In spite of this defect one

enthusiastic reviewer said that this was “undoubtedly the micro-

tome of the future.”

Microtomes of the rotary type are probably the most generally

used at the present time. They are an American invention, the

idea having been worked out independently by Pfeifer, a mechani-

can at Johns Hopkins University, and by Minot of Harvard. The

first published description is that of the so-called “Johns Hopkins”

microtome in 1886. Only one or two instruments of this type were

ever manufactured, but it is interesting to note that one of them

is still in active service today in the laboratory at John Hopkins

University. Minot’s microtome was made in 1887 by Baltzer of

Leipzig, although the manufacture was soon transferred to Zim-

mermann, who still makes them. The first published description of

these microtomes appeared in 1888.

In discussing the development of the modern botanical micro- .

technique the problem is largely one of finding out at what partic-

ular time methods devised by animal histologists were first applied

to botanical problems. To really appreciate how deeply indebted

we are to the zoologists it is only necessary to read the names of

the fixing solutions and stains used in publications in any botanical

periodical. The almost exclusive appearance of the names of Flem-

ming, Heidenhain, Merkel, Hermann, Pianaese, and many other

animal histologists in connection with descriptions of methods of

study shows this very well.

Mol took 1870 as the date from which he commenced. his dis-

cussion of the modern botanical microtechnique, whereas Strasbur-

ger took 1875 as the starting point for a review of the modern

cell theory; but since there was comparatively little development of

microtechnique in the decade of 1870-1880, it matters little which

date is taken. The article of Strasburger, published in 1875, may be

taken as representative of the most progressive methods in use at

that time. In the study of cell contents no staining methods were

110 GILBERT MORGAN SMITH

used but the necessity of arresting the progress of nuclear division,

in order to allow time for a more detained study, was recognized,

and absolute alcohol was used as the fixing medium. Permanent

preparations were made by transferring the material fixed in alco-

hol to glycerine. Whenever the material was plasmolyzed by the

strong alcohol, a more dilute solution was used for fixation. In

some cases iodine was employed as a stain.

That the methods which were being developed at this time by

the zoologists were not entirely neglected by the botanists as is

shown by the fact that every new method of importance was re-

viewed in Just’s Jahresbericht. Between 1875 and 1880 the most

noticable progress in botanical microtechnique was the gradual

adoption of staining methods. Among the pioneers may be men-

tioned Errera who stained nuclei with Nigrosine, Strasburger who

used Methyl Green and acetic acid for simultaneous fixation and

staining, and the use of Methyl Green by Treub. All of this stain-

ing of the cell contents was with a single stain only.

As shown in the discussion of the history of staining most of

the very early work with botanical material was to bring out the

cell walls. In the same way the first double staining of plant tis-

sues was for the demonstration of fibro-vascular bundles and not for

the structure of the cell contents. The development of double

staining methods for stems and other tissues was due to the desire

of microscopists to make striking microscopical preparations rather

than to bring out morphological structures. Some of the earliest

work was done by the American microscopists in the late seven-

ties. Among the combinations used, hematoxylin and Aniline Blue

by Poole, Crawshaw’s Aniline Blue and Magenta by Barrett, Car-

mine and Aniline Green by Peet, while Rothrock used hematoxylin

or carmine in combination with Iodine Green. Richardson made

the most extensive series of experiments on staining among others

a triple stain of Atlas Scarlet, Soluble Blue and Iodine Green. The

first double staining for the demonstration of cell contents is Mc-

Farlane’s combination of Diamond Fuchsine and Methyl Green.

During the late seventies other methods used by zoologists

were introduced into botanical microtechnique. Leitgeb cleared

preparations with clove oil before mounting in 1875; while Parker

BOTANICAL MICROTECHNIQUE 111

in fixed Chara in a mixture of chromic and osmic acid and after

dehydration in alcohol cleared in clove oil and imbedded in cocoa

butter.

The best index to the expansion of modern botanical micro-

technique is Strasburger’s “Botanische Prakticum”; the different

editions of which cover a period from 1884 to 1913, At the time

that the first edition appeared we find the modern methods of study

fairly well begun. The preéminent fixing fluid recommended is

absolute alcohol. It is true that chromic acid, concentrated picric

acid, and mixtures of chrome-acetic and osmic-chrome-acetic acids

are cited in connection with the study of the algz but since alcohol

is the only medium recommended for the study of the anther, ovary,

and meristematic tissue the technique of fixation may be regarded

as quite primitive at that time. With the exception of MacFarlane's

Diamond Fuchsine-Methyl Green mixture, single stains only were

used for the study of the cell contents. Carmines and hzematoxylins

appear most frequently in the pages of the first edition although

the regressive staining with aniline dyes, so strongly recommended

by Flemming in the early eighties, is described in connection with

the studies on nuclear division, Safranin and Gentian Violet being

considered the best. Double staining is emphasized in connection

with the work on vascular bundles and three different processes are

described, the use of Grenacher’s Alum Carmine-Methyl Green

Picro-Nigrosine and Picro-Aniline Blue.

The process of interstitial embedding in paraffine, celloidin,

or soap was well known to the zoologists when the first edition

appeared and a description of all of these methods is given but

no direct application to the study of plant tissues can be found in

the book. Strasburger refers to Koch (1874) as having used para-

ffine embedding in the study of plant tissues, but this method was

one of enclosing rather than interstitial embedding.

The chief advance as recorded in the second edition is the

use of a much larger number of stains for the study of the plant

cell. There is also a much more complete discussion of the process

of embedding, but, with the exception of a note on the arranging

of serial cellodin sections of anthers there is again nothing said

about the application of embedding methods in botanical work.

112 GILBERT MORGAN SMITH -

‘To Francotte belongs the credit of introducing the paraffine

method into botany. It is true that the notes on the methods for

the study of leaves, stems, anthers, ovaries, and fungi cover but

three pages but this is the first case where complete schedules are

given for the different processes leading.to the embedding of plant

tissues in paraffine. Francotte did not think that paraffine was the

best medium for stems and leaves, but gave the preference to soap,

with cellodin as the alternative. Apparently this work has been

entirely overlooked, since neither Schonland, Moll, nor Koch men-

tion it. The years 1887-1892 mark the establishment of the para-

fine. Attention was called to the usefulness of paraffine through

the publication of Schonland and Moll appearing in the year 1887.

The first method Schonland used was the gradual dehydration with

methyl alcohol and then a transfer to clove oil and from the clove

oil to oil of turpentine; afterwards the tissues were placed in para-

ffine with a melting point of 45 degrees Centigrade. Later (1888)

he substituted ethyl for methyl alcohol and also interpolated a fur-

ther step by allowing the material to remain in turpentine saturated

with paraffine before the transfer to pure paraffine. Moll’s method

differs very little from that which we now use, the chief divergence

from the present schedules lies in a much slower dehydration, sev-

eral hours being allowed for each grade of alcohol into which the

material was placed. Campbell and Koch helped to propagate the

doctrine of embedding, by showing its applicability to large num-

bers of plants. With the appearance of Koch’s work we may con-

sider the availibility of the paraffine method for botanical micro-

technique as well established, although many articles published at

that time continued to give full directions for the process.

Historically the soap method is older than that of paraffine,

but in their introduction into botanical microtechnique the two are

coincident. Pfitzner’s description of the soap method in connec-

tion with plant tissues appeared the same year as Schénland’s and

Moll’s articles, but the greater adaptability of paraffine has pre-

vented the widespread use of soap although Wilcox and Osterhout

are advocates of its use in special cases. The first complete de-

scription of cellodin embedding for plant tissues is that of Busse.

This substance has*always been used to a large extent in section-

BOTANICAL MICROTECH NIQUE 113

ing woods the most generally used method at the present day being

the so-called “Harvard Method” described by Plowman.

The dominance of the school of cytologists led by Strasburger

has been most strongly felt in Germany, England, and America.

The disciples of Strasburger have generally advocated the mastery

of a few methods and after the time of his publication of the pro;

cess of embedding in paraffine and staining with Flemming’s triple

stain (1896) this has come to be regarded as the universal method

in certain quarters. In other centers, notably the French botanists

under the leadership of Mangin and Guignard, there has been the

use of a much wider range of stains. At present it may be said

that there is a general drift towards a more varied attack. This

is perhaps due to the increasing interest in the cytology of the Cryp-

togams, in which there is a less uniform behavior towards the trip-

ple stain of Flemming than there is in the Spermatophytes. This

has led to the use of Pianzsz’s stain, Azur Blue, and several others.

On the other hand the investigation of cellular organization as exem-

plified in the study of mitochondria, has called for the develop;

ment of a special staining technique. With the opening up of these

new fields we may confidently look forward to further develop-

ments along this same line.

Methods of fixation have not multiplied as rapidly as the meth-

ods of staining. A few new fixing fluids have been proposed by

botanists, that of Juel being an example; while the French and

Belgian botanists have taken several mixtures devised by zoologists

and recast the formule so that the fluids can be used for the study

of plant tissues. On the other hand there is also a strong tendency

toward a critical examination of the action of fluids in general use

at the present time, the work of Fischer and Lundegard being

typical examples.

114 GILBERT MORGAN SMITH

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126 GILBERT MORGAN SMITH

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THE AMPULLAE OF LORENZINI IN ACANTHIAS

VULGARIS*

By H. E. Metcalf

INTRODUCTION

In the past thirty years a considerable amount of work has

been done on the lateral line system in the Selachians, in connection

with which the ampulle of Lorenzini have been described. These

ampullze occur in the Selachians only, and for that reason are im-

portant, as other Ichthyopsida have lateral line systems which show

no traces of these ampullz. In the Selachians the ampulle have

almost invariably been classed with the lateral line system. There

is a great difference of opinion among investigators as to the exact

function of these structures; whether they merely do the same

work as the lateral line system, or whether they have a special func-

tion which is lacking in the rest of the lateral line system. These

differences of opinion were not confined to the ampullz alone, but

the lateral line system itself was a basis of dispute. The lateral

line system was at first held to be purely secretory, but later was

believed to have sensory as well as secretory functions. This opin-

ion is at present generally accepted, but the exact function of the

ampullz is still in doubt, and there are many conflicting statements

regarding them.

According to all authors these organs were first described by

Stephan Lorenzini in 1678. Since that time many papers have

dealt with the subject, most of them treating the ampulle only

superficially, being good for the location of the organs only. Leydig

(52), and Boll (68) said that these ampullz should be classed with

the lateral line system. They also saw in them structures which led

them to say that the ampulla were homologous with the electric

organs of the torpedo. But the discovery of ampullz in that fish

soon disproved that theory. In 1852 Jacobson had said that the

*The work for the following paper was done at the Biological Laboratory of

Tufts College, and at the Harpswell Laboratory, South Harpswell, Maine, under the di-

rection of Dr. J. S. Kingsley and Dr. H. V. Neal, to whom I am indebted for many

helpful suggestions during the course of the investigation.

132 H. E. METCALF

lateral line system was sensory in function, and that the ampullze

belonged in the same category. Later upon a closer examination

of the organs he admitted that the old hypothesis of a secretory

function was correct, and that, as the ampullz were so closely re-

lated to that system, they also were secretory in function.

In 1852, both Leydig and Miller published their works on the

anatomy of the Selachians, in which they ascribed a secretory func-

tion to the ampulla. This hypothesis was supported by a new work

of Leydig in 1868. Considering the technical and optical methods

of the time, the results of these two men were truly remarkable,

and for a period of nearly thirty years little or nothing was added

to the subject; while the belief that the ampullz were secretory

- prevailed.

Boll in 1868, and Todaro in 1870 were responsible for the

name of Lorenzini’sche Ampulle as applied to these organs.

The histological structure has never been thoroughly worked

out, except by Forsell (98) who was not certain of his results, be-

cause only material preserved in formalin was available for his use.

His paper has an excellent review of all the literature on the sub-

ject up to 1897. Peabody (97) published a short article on the

innervation of these ampullz as shown by means of methylin blue

preparations. None of the workers have found mucus cells in

the epithelium of the ampullze, and only Parker (10) has done

anything in the way of experimental evidence as to their function.

_ The ampullze in Acanthias vulgaris are arranged in eight defi-

nate groups; two on the dorsal surface (Fig. 1), four on the ven-

tral (Fig. 2), and two on the lateral surfaces. (Fig. 3-A). As

might be expected, these groups are bilaterally symmetrical. The

position of six of these groups is approximately visible in an exter-

nal view of the snout, as the openings of the ducts give an indication

of the internal arrangement of the organs. Thus on the dorsal

side of the snout are a pair of triangular groups of openings. Dis-

section shows these to be connected with a pair of triangular

groups of ampulle (Fig. 1-A), these groups being smaller in ex-

tent than the area occupied by the external openings. From these

ampullz the ducts radiate in all directions, except toward the ven-

tral surface.

AMPULLZ OF LORENZINI 133

On the ventral surface of the snout, in front of the mouth,

the openings of the ampullz are evenly distributed over the entire

ventral surface. (Fig. 2). On dissecting away the skin, however,

it is seen that the three cartilege bars of the rostrum, which run

almost straight forward, divide the ampulle into four rectangular

groups. (Fig. 2—1, 2, 3, 4.) Here as in the dorsal surface the

duct run in all directions, forward, laterally, back and ventrally,

Fig. 1. A diagram of the dorsal surface of the snout showing the distribution of

the openings on the right, and the approximate location of the ampulle on the left.

About natural size.

with the posterior ampullz of the two lateral groups running pos-

teriorly to open at the extreme corners of the mouth. (Fig. 2-B).

On the side of the head, posterior to the mouth, and between

the eye and the first gill slit, occur the spiracular ampulle (Fig.

4-A), all of which have long canals. These open at the posterior

134 H. E. METCALF

region of the mouth, and in the line of openings just anterior to

the first gill slit.

The two groups on the dorsal surface of the snout are inner-

vated by the opthalmicus superficialis (Fig. 1-B), those of the ven-

tral groups by the buccalis (Fig. 2-C), and those of the spiracular

region by the mandibularis externus (Fig. 4-B) branches of the

seventh, or facial nerve. These branches also innervate the lateral

line system in those regions.

Fig. 2. A diagram of the ventral surface of the snout showing the distribution

of the openings on the right, and the approximate location of the ampulle on the left.

About natural size.

The number of ampulle is not constant, there being from

1,200 to 1,900 in each adult fish. The number in the late stages

of the “pup” is approximately the same, so that there is no addi-

tion to the number after the fish is born. There are about 500

dorsal ampulle, 900 ventral and anterior to the mouth, and 200

lateral and ventral, posterior to the mouth, There are no ampulle

posterior to the first gill slit.

All of the ampulla have definate positions in the head, and in

each group the ducts run forward, laterally and backwards as well

AMPULLZ OF LORENZINI 135

as in all intermediate directions. Some of them also point up, and

some down, so that there are ducts radiating in all directions, giv-

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Fig. 3. A diagram of the lateral surface of the snout showing the distribution of

the openings. About natural size.

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Fig. 4. A diagram of the lateral surface of the snout showing the approximate

location of the ampulle. About natural size.

ing a chance for the reception of a stimulus from any side, above

or below. This will be referred to later in discussing the exact

function of the ampullz.

136 H. E. METCALF

This problem of their function is an interesting one. They

are full of mucus, yet no mucus cells have been clearly demon-

strated. Again, typical nerve termination cells which appear to be

sensory in character, have not been found. The ampulle of Loren-

zini may be sensory, secretory, both or neither. The main problem

then of the present investigation is to find out which of these hy-

‘potheses is the correct one. First to see what results might be

had by using the experimental method, and then to see if the his-

tological structure of-the ampulle supports those results.

Tue Gross ANATOMY OF AN AMPULLA.

The external openings of the ampulle are usually single, and

nearly circular, although there are cases found where the open-

ings are double, when the two ducts connect with one ampulla.

When a double opening occurs the two openings are in the form of

semi-circles, so that the size and contour of the double openings

is much the same as that of a single one. In the case of a single

opening, the main or primary duct extends back to the ampulla

itself, but when there are two openings the double ducts are parallel

and closely approximate. (Fig. 16-A). Never did I find a case

where there was a double duct from one ampulla in which the two

tubes ran in different directions to the surface as Forsell (98)

mentions.

Each duct leads directly downwards from the opening for a

short distance, and then widens out quickly to about twice the diam-

- eter of the terminal portion. (Fig. 15-A). This is true no matter

whether the main duct is single or double in character. The length

of the enlarged ducts varies from six to thirty millimeters. They

are longest in the posterior ampullz of the two dorsal groups, and

in the posterior ampullz of the two lateral groups, ventral and

anterior to the mouth.

As the duct approaches the ampullary end the main duct 1s

constricted to about three-fourths its former diameter (Fig. 15-B),

and the duct divides into two secondary tubules. These imme-

diately subdivide into two or more ducts of tertiary order on the

ends of which are the pockets or alveoli (Fig. 15C), these vary-

ing in number on the various tertiary tubes. Some of the alveoli,

however, lead directly into the main duct. A careful study of

AMPULLZ OF LORENZINI 137

those cases in which there are double ducts leading to the surface,

as described above, shows that these are to be regarded as greatly

elongated secondary ducts (Fig. 16-A), as there does not seem

to be any evidence of a branching which would be called tertiary

if these ducts were regarded as primary. The alveoli are not on

the same level, so that a section through any one plane would not

show all of them. This explains why so many of the writers have

stated their numbers to be less than is actually the case. There are

partitions between the alveoli, so that the lumen of the ampulla is

divided into as many compartments as there are alveoli. In about

a dozen ampulle the average number of alveoli was twenty-two,

the range of those counted being between eighteen and thirty-two,

the majority being from eighteen to twenty-six. The alveoli are

elongate in the direction of their major axis, but circular in outline,

with their greatest width near the middle. They differ essentially

from those described by Boll (68) and Peabody (97) in that Acan-

thias has no “centrum” cap to which the partitions run. Here the-

partitions merely separate the alveoli from one another, termin-

ating at the distal end as sharp ridges. The average diameter of

the ampulla in Acanthias is greater than that of six tenths of a

millimeter recorded by Peabody for Galeus, as the approximate

diameter of those in an adult female about four feet long was;

opening one millimeter, duct two millimeters, and ampulla one and

six tenths millimeters. The number of ampullary alveoli is also

greater in Acanthias than in Galeus or Mustelus.

Do THE AMPULLZ OF LORENZINI RESPOND TO OUTSIDE STIMULI?

The experiments carried on by Parker (1910) in his paper on

the influence of the eyes, ears, and other allied sense organs on

the movements of the dogfish Mustelus canis, were repeated in so

far as they related to the ampullz of Lorenzini in Acanthias vulgaris.

The experiments were carried on in a floating car six feet square

and four feet deep. The sides and bottom of the car were covered

with wire netting. The fish were mostly adults which had been

caught with a hook, and carefully brought to the car in a barrel

of sea water. All of the fish in the car which were experimented

on were in good condition, and seemed to be fully normal. They

138 H. E. METCALF

soon got used to their cramped quarters, and seemed in every way

to be fit subjects for experimentation.

Several of the fish were experimented on with more or less

success. The first thing tried was to cut the nerves leading to the

ampulla, and to observe the fish swimming around in the car to

see whether or not their behavior differed from the other unin-

jured occupants of the car in any way. Of a number of fish treated

in this way, including a number of young “garters” as well as adults,

not one of them, as far as could be observed, suffered any incon-

venience from the loss of the use of these organs, or any deviation

from the behavior of other normal and unhurt fish in the car, In

fact, the fish which had had the nerves that lead to the ampulle

cut, could not be distinguished from those that were normal, as

they swam about in the car.

Following this, a variation of the same experiment was tried.

The fishermen in the vicinity of Casco Bay, Maine, have been

bothered so greatly in their fishing by the dogfish, that the custom

has prevailed of mutilating the fish when caught so there will be

no chance of their ever biting a hook again. These fisherman

vouch for the fact that if the tip of the nose be cut off, and the fish

be put back into the water, it is unable to sink and flounders around

on the surface. A number of experiments were performed to see if ©

there was any basis for this statement, and if it had any relation to

the loss of the ampulle which were in that region, but with negative

results. The noses were cut so that all or part of the ampullze were

removed, and in some cases even the brain was exposed, but the

fish seemed to be able to sink at will, and behaved in this respect

as a normal individual. There seems to be no truth in the state-

ment that a dogfish is unable to sink beneath the surface when the

ampullze are removed, or evén when: the entire tip of the nose is

cut away. The explanation of the fishermen’s statement lies in the

fact that a dogfish when brought into the air immediately begins

to swallow air in gulps so that the intestines are filled. Then when

thrown into the water the air inside keeps them at the surface

whether or not the tip of the nose is cut away. All operations on

the fish were carried on under water so that there was no chance

for them to take in air. The ampulle of a single side, both dorsal

AMPULL4 OF LORENZINI 139

and ventral, were removed, but the actions of these fish could not

be observed to differ appreciably from the controls. The openings

of the ducts were also covered with paint so that stimulus from the

outside might be prevented, but without result.

Finally it was decided that the response, if any, needed more

delicate methods to make itself evident. Following Parker, a patch

of skin about two centimeters in diameter was dissected away im-

mediately over a bunch of ampullz, thus removing any possibility

of a response to a stimulus from the sensory nerve endings in the

skin. The fish treated was an adult female, the most active and

well, as also the largest. After a half hours rest given in order that

the fish might be fully recovered from the shock of the operation,

various stimuli were applied to the ends of the cut canals which

had been exposed. A thin delicate needle point was applied to

the canal coming from one ampulla, but no reaction could be ob-

served. Possibly there was a reaction, but it was so small that

it could not be detected. Then the blunt end of a dissecting needle

handle was very carefully applied to a fresh spot, so that there

was no danger of touching the wounded edges of the cut skin, at

the same time trying to touch the fish with the hands as little as

possible, when it was on the surface of the water at the top of the

car. This proved to be a difficult undertaking, as the fish was very

active, and showed a distinct preference for the bottom of the

car. In order that the results could be relied upon, it was thought

best not to try to hold the fish at the top of the water, but after

a little practice the fish could be made to stand still long enough for

its reactions to be seen. Later it was noticed that the reactions

seemed to be the same whether or not the fish was held lightly in

the hands while the stimulus was applied.

‘The normal respiration of the fish was from 42 to 48 spiracular

closures per minute. A number of stimulations were made, using

the blunt point and watching the spiracular valve for an indica;

tion of a résponse. So far as could be noticed this valve did not

change its rate of movement, nor was the rate of respiration re-

tarded as Parker states. Upon turning the fish slightly so that the

mouth could be observed, a very distinct reaction was noticed.

During the normal respirations the mouth is opened, and some of

140 H. E. METCALF

the water is taken in there, as well as through the spiracle. The

distance the mouth is opened in the fish under observation was

normally about one-half inch at each inspiration. The instant a

light pressure was applied to the ends of the cut canals with the

blunt point, the mouth immediately closed, but the respiration con-

tinued without a halt, all of the water being taken in through the

spiracle. The pressure, if applied less than ten seconds caused

the mouth to be closed all during the time that the stimulus was

applied. If, however, the pressure was continued for longer than

ten seconds, the mouth would open after 7 to 8 complete inspirations

through the spiracle, and the water would again be taken in through

both places, although the mouth never opened to its normal extent

while the stimulus was being applied. If the pressure then be

slightly increased the mouth would close entirely again for about

ten seconds, until it seemed that the fish “got used” to the stimulus.

In every case when pressure was relieved the fish gave a distinct

gulp with its mouth, giving the observer an impression that it was

greatly relieved at being rid of the stimulus. All of these responses

to pressure entirely disappeared upon cutting the nerves supply-

ing the ampulle. All of the foregoing shows beyond a doubt that

these organs were sensitive to the stimulus of pressure caused by

the application of the blunt point to the cut canals of the ampulle.

These experiments confirm Parker in that the ampulle of Loren-

zini respond to pressure, but im no case did I get an inhibition of

the respiratory movements which could be noticed, as did Parker

in his experiments. The response was easily seen, and occurred in

other dogfish in the same manner upon later experimentation.

These results would indicate that whether or not there be a secre-

tory function, these organs most certainly have a sensory function.

Tue Histotocy oF THE Duct AND AMPULLA.

The walls of the duct are lined with a single thin layer of

squamous epithelium, with a thin layer of fibrous connective tissue

on the outside. (Fig. 5-C). With iron alum hematoxylin the nu-

cleus and cytoplasm of these cells stain very darkly, even when

extracted so that the nuclei of the other epithelium in the ampull

are clear. This makes it somewhat hard at first to see the nuclei in

the wall of the duct, but careful extraction for that point alone will

AMPULL& OF LORENZINI 141

‘give good results. The cytoplasm of the cell stains very deeply

and seems to be somewhat striated in character. (Fig. 6). Near

the ampullary end of the duct there are protoplasmic projections

extending out from those squamous cells into the lumen. (Figs.

5-G, and 6-B). These were at first thought to be artifacts, and

every possible precaution was taken to prevent any artificial distor-

tion of the cells, but the processes are real and can be seen in all

well fixed preparations such as those fixed in Flemming’s fluid.

These processes arise just above the nucleus, and project a short

distance into the lumen. The protoplasm immediately below them

is vacuolated, and I am sure that these processes are not cilia.

(Fig. 6-C). The number of processes on each cell varies and, as

a rule, the farther away from the ampullary end, the fewer the

number of processes, until finally there is only a convex contour

to the cell, and then a normal squamous epithelial cell with no

traces of any projections. (Fig. 5-H). Material which had been

fixed in Flemming’s fluid was used for studying these processes.

It was stained in iron alum hematoxylin which was extracted ex-

cept for the chromatin in the nucleus and then a sharp counter-

stain with acid fuchsin, much more than would be needed for an

ordinary counterstain, for these processes are not visible with the

ordinary amount of counterstaining. This overstain brings out

clearly the vacuolations in the processes, although the rest of the

cell is overstained. All kinds of methods were used and numerous

stains tried to be sure of these structures, and all showed them to

a more or less degree, even those which gave poor preservation. So

I think that it is fairly certain that these are real structures and

not artifacts. I could get no hint of their function.

These processes continue into the secondary and _ tertiary

tubes, and are on the partitions between the alveoli, but are ‘not

found on the epithelium of the alveoli themselves. (Figs. 5-F, 7

and 8).

At the junction of the ducts and the alveoli, the epithelium

and the connective tissue continues, the latter showing little change

except that it is thicker, a condition continuing over the outer sur-

face of the alveoli. (Fig. 5-B). The epithelium of the alveoli is

best seen in longitudinal sections. (Fig. 5). It is thicker than that

142 ' H. E. METCALF

of the ducts and is composed of two types of cells. At first sight

there are apparently two layers, as described by Peabody (97), but

this is, I think, based on misinterpretation of sections, at least as

far as the evidence in Acanthias shows. (Fig. 5-D). The two

types of cells differ markedly in shape of cells and position of

nucléi. These are (1) larger cells with their approximately cir-

cular nuclei near the basal membrane of the epithelium, (Fig. 9,

A, E, C, and F.) and (2) smaller cells (interstitial cells) with pyri-

form nuclei which lie near the free surface of the epithelium. ( Fig.

9, G, D, and B). It is this stratification of nuclei which conveys

the idea of a stratification of the epithelium, but careful observa-

tion, especially in rather thick sections has convinced me that both

types of cells extend from the basal membrane to the free sur-

face. (Fig. 9, A and B). The cells of the larger type are so large

that there are not many in each section which are cut directly

through the center so that both basal and distal surfaces are shown,

but these are seen in a number of cases in thin sections (Fig. 9-A),

and I think that nearly every cell in the epithelium extends from

the basal membrane to the free surface of the epithelium.

The protoplasm of the larger cells is vacuolated (Fig. 9-E),

and the chromatin in the nucleus forms a rather coarse network

(Fig. 9-C), while the protoplasm in the interstitial cells is rather

striated in character (Fig. 9-G), and the chromatin in the nucleus

forms a finer network with scattered granules, giving these a much

darker appearance, (Fig. 9-D). In no case have I found a cuticle

lining the free surface of the alveoli next to the lumen, as Forself

(98) has described for Acanthias.

INNERVATION.

The different groups of organs are supplied by several branches

of the lateralis componant of the facial nerve, as indicated on page

134, a single twig containing from five to ten medullated fibres run-

ning from the main ramus to each ampulla. These twigs run to

the centre of the ampulla into the space between the alveoli. There

they lose their sheaths, and each breaks up into fibrille. These

pass out between the alveoli, and form a network upon their outer

and anterior surfaces. (Figs. 10 and 11).

AMPULL OF LORENZINI 143

Many methods of impregnation were tried in the attempt to

discover the method of termination but all were failures, with the

exception of intra vitam staining with methylen blue. This, at

best, is uncertain, attacking as it does only certain of the fibres and

fibrille, and one can never be sure that every portion has taken

the stain. Several fairly good impregnations were obtained, and

these indicate that the fibrilla had enlargements in their course

(Figs. 10, 11 and 14), and that they sometimes terminated with

similar end organs. (Figs. 12 and 13). In every case where exact

determination was possible, it was found that these enlargements

were upon the bases of the pear-shaped cells of the alveoli and in no

case were they found connected with the other cells of the epithe-

lium, nor did the nerves extend on to the walls of the ducts.

These facts seem to favor a sensory function for the ampulle.

There are a number of specialized cells which are in a position to

receive stimulation, as the pear shaped cells are in direct connection

with the mucus in the ampulle, and have a connection with branches

of the nerve supplying the lateral line organs which are certainly

sensory in function. The fact that there are no hair cells does not

preclude a sensory function as there are numerous instances in

which distinctly sensory nerves are not connected with hair cells.

The general agreement is that these ampullz, and the ear as well,

are genetically connected with the lateralis system of organs. Hence

the fact that Morrill (97) found sensory cells in the dogfish ear

which had no hairs, is of especial interest in this connection. Then

again there is no need of continuity between nerve and cell, as

Morrill also found in his description of the sensory cells in the ear

of Mustelus.

Tue FUNCTIONS OF THE Mucus.

In many of the accounts of these organs the figures show a

peculiar striation of the mucus contained in the ampulle and the

ducts leading from them, and some authors have discussed the

structure and meaning of this appearance at some length. (Forsell

98). In material taken fresh from the dogfish there is nothing

of the sort to be seen, but it always appears in the fixed material,

but the character of the striation or fibration varies with the reagent

144 H. E. METCALF

used. (Figs. 5 and 6-A). These facts lead me to the conclusion

that the appearance is really an artifact.

The mucus of the ampullz is not needed to lubricate the body,

as there are numerous goblet cells all over the body for that pur-

pose. Also in the adult fish there is no evidence of a discharge of

mucus when alive. The mucus appears when the ampulle are

squeezed, but otherwise it is not seen. That is to say, no more

mucus is found on the surface of the body in the region of the

ampullez than at any other point. Apparently there is little loss

of mucus from the ampullz, all that is lost being worn away by

friction of the water at the opening of the duct. Therefore the

ampullze need but little mucus to keep the tubes full. If this view

be correct it would explain why so little evidence of mucus secre-

tion is found. Treatment with mucicarmin shows very few cells

taking the mucus stain. These cells are isolated and are in the

same position as the pear-shaped cells, but are somewhat narrower,

have smaller nuclei, and have a greater portion of the cell border-

ing on the lumen. They greatly resemble the pear-shaped cells

and in sections stained in the usual way it is impossible to dis-

tinguish between the two. I did not succeed in determining

whether or not they are innervated. Some of the sections of the

ampullz do not show any of these cells, so it is evident that they are

not common, Again it may be said that the blood supply of the

ampulle is very small. This would imply that the secretory func-

tion is very limited—merely enough to replace the slight loss, as

a large blood supply would be needed for an extended produc-

tion of mucus.

The ampulle are situated beneath the skin in the connective

tissue, and are connected with the surface of the body by long

ducts. It is therefore certain, that if any stimulus is to reach the

sensory cells in the pockets, it must either be transmitted through

the overlying integument or it travels to them through the mucus.

This, then may be the function of the mucus in the ampulle—

to act as a conductor, by means of which stimulation from the

outside is to reach the sensory portions of the ampulle.

AMPULLZ OF LORENZINI 145

SUMMARY AND CONCLUSIONS

It therefore appears from both experimental results and from

histological structure:

A. That the ampulle of Lorenzini in Acanthias vulgaris are

primarily sense organs,

B. That they have a secretory function in so far as it is neces-

sary to keep up the supply of mucus in the ampullz and their ducts.

This need is small, as there is little loss.

C. That they respond to the stimulus of pressure, and from

the fact that the ducts radiate in all possible direction—

D. That the pressure may be caused by currents of water

impinging on some of the ampullz harder than others, thus giving

the fish a sense of the direction of the source of the stimulus,

E. That also by means of these ampullz the dogfish may deter-

mine by means of pressure, the depth of water in which it is swim-

ming.

F. That it is also possible that these organs will respond to

the deeper notes as these would cause a vibratory change of pressure

on the ampulle.

G. That these ampulle from their innervation and their func-

tion are rightly classed as sense organs along with the lateral line

system in the Selachians.

BIBLIOGRAPHY

1678. Lorenzini, Stephan.—Osservazioni intorno alle Torpedini. Florence.

1813. Jacobson, Louis——Extrait d’un Mémoire sur un Organe particulier

des Sens dans les Raies et les Squales. Bull. d. Sci. de la Societé

philomatic de Paris. Tome 3.

1851. Miiller, H—Ueber den Nervosen Follikel-Apparat der Zitterrochen

und die sogennanten Schleimkanale der Knorpel Fische. Ver-

handle. Phys.-med. Gesellsch. in Wiirzburg. Bd. II, Nr. 8.

1852. Leydig, Franz.—Beitrage zur mikr. Anatomie und Entwicklungsge-

schichte der Rochen und Haie. Leipzig.

1857. Leydig, Franz.—Lehrbuch der Histologie des Menchens und der

Thiere.

1858. Echhard, C.—Uber die Endigungsweise der Nerven in den Schleim-

kanalen der Zitterrochen.

146

1868.

1870.

1878.

1880.

1888.

1888,

1890.

1892.

1895,

1897,

1898.

1910.

H. E. METCALF

Boll, Franz.—Die Lorenzinische Ampulle der Selachier. Archiv fir

mikr. Anatomie. Bd. IV.

Leydig, Franz—Ueber Organe eines Sechsten Sinnes. Dresden.

Schultze, F. E—Uber die Sinnesorgane der Seitenl. bei Fischen und

Amphibienlarven. Archiv ftir mikr. Anatomie. Bd. VI.

Todaro.—Contrabuzione alla anatomia e alla fisiologia di tubi di

senso dei Plagiostomi.: Messina.

Balfour.—A Monograph on the development of Elasmobranch fishes.

-London. , .

Sappey, Ph. €.—Etudes sur l’appariel Mucipare et le systeme lym-

phatique des Poissons. Paris.

Merkel, Fr.—Ueber die Endigungen der Sensibeln Nerven in der

Haut der Wirbelthiere. Rostock.

Fritsch, G—Uber Bau und Bedeutung des Kanalsystems in der

Haut der Selachier. Sitzungsber. der Berliner Akad. der Wis-

sensch. Bd. 1.

Garman, Samuel.—On the Lateral Line Canal System of the Sel-

achier. Bull. Mus. Comp. Zool. Vol. XVII, No. 2.

Fritsch, G.—Die Electrischen Fisch. Leipsig.

Ewart, J. C—The Sensory Canals of Lemargus. Trans. Roy. Soc.

Edinburgh. Vol. XXXVII, No. 5.

Ewart, J. C., and Mitchell, J. C—The Sensory Canals of the Com-

mon Skate. Trans. Roy. Soc. Edinburgh. Vol. XXXVII, No. 6.

Fuchs, S.—Uber die Funktion der unter der Haut liegenden Kanal-

systeme bei den Selachiern. Pfliigers Archiv.

Morrill, A. D—The Innervation of the Auditory Epithelium of

Mustelus canis (de Kay). Journ. Morph. Vol. XIV, No. 1.

Peabody, J. E—The Ampulle of Lorenzini of the Selachii. Zool.

Bull. Vol. I, No. 4.

Forsell, Gustavy.—Beitrage zur Kenntnis der Lorenzinischen Am-

pullen bei Acanthias Vulgaris. Zeitsch. f. Wissensch. Zool.

Vol. 65.

Nachtrieb, H. F—The Primitive Pores of Polydon spathula (Waul-

baum). Journ. Exp. Zool. Vol. IX, No. 2.

Parker, G. H.—The Influence of the Eyes, Ears, and Other Allied

Sense Organs on the Movements of the Dogfish, Mustelus Canis

(Mitchell). Bull. of the U. S. Bureau of Fisheries. Vol. 29,

oe %

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PLATE IV

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Fig,

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AMPULL OF LORENZINI 147

EXPLANATION OF PLATES

PLATE IV

5. A semi-diagrammatic drawing of a longitudinal section of an ampulla.

Flemming’s fluid, iron alum hematoxylin and eosin. X 200.

6. An enlarged portion of the duct showing the protoplasmic pro-

cesses and the mucus striation. Flemming’s fluid, iron alum hema-

toxylin and eosin. X 800.

7. A cross section of the protoplasmic processes on the partition be-

tween two alveoli. Flemming’s fluid, iron alum hematoxylin and

eosin. X 800.

8 A cross section of the protoplasmic processes on the partition be-

tween the partition between the alveolus and the duct. Flemming’s

fluid, iron alum hematoxylin and eosin. X 800.

9. The epithelium of the alveoli, showing in detail the structure and

relationships of the two types of cells present. Flemming’s fluid,

iron alum hematoxylin and eosin. X 800.

The small figures in the lumen of the ampulla shown in Fig. 5 indi-

the portions which have been figured in Figs. 6 to 9.

148

Fig.

10.

H. E. METCALF

PLATE V

A thick section through the base of one of the alveoli showing

the nerve ramifications over the alveolus. Methylen blue impreg-

nation. X 400.

A cross section showing the nerve extending laterally after hav-

ing lost its sheath. Methylen blue impregnation. X 400.

A thick section showing the method of termination of the nerve

fibre on the base of one of the pear shaped cells. Methylen blue.

X 950.

A thick section showing the same kind of termination on a slightly

differently shaped cell. This difference in shape and the differ-

-ence in shape between these cells and the same cells fixed in

Flemming’s fluid as figured above, may be explained by the fact

that the methylen blue process gives poor fixation. X 950.

A thick section showing two of the pear shaped cells which have

a slight enlargement of the nerve fibre at their base, with no

termination. X 950.

Figures 6 to 14 are camera drawings.

A diagram of an ampulla and duct with a single duct.

A diagram of an ampulla and duct with a double duct.

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DEPARTMENT OF NOTES, REVIEWS, ETC.

It is the purpose, in this department, to present from time to time brief original

notes, both of methods of work and of results, by members of the Society. All members

are invited to submit such items. In the absence of these there will be given a few brief

abstracts of recent work of more general interest to students and teachers. There will be

no attempt to make these abstracts exhaustive. They will illustrate progress without at-

tempting to define it, and will thus give to the teacher current illustrations, and to the

isolated student suggestions of suitable fields of investigation.—[Editor. ]

PrRoTOzOOLOGY APPLIED TO THE SOIL

By Nicholas Kopeloff, H. Clay Lint and David A. Coleman

(From the departments of Protozoology and Soil Bacteriology, Rutgers College,

New Brunswick, N. J.)

In recent years protozoology has materially widened its scope,

as is evidenced by the growing interest in investigations of economic

significance such as those concerning protozoa as parasites, in drink-

ing water, etc. It needed but another step to the realization that the

protozoa in the soil (and they have been observed in as large num-

bers as bacteria) may have a definite function in influencing soil fer-

tility. In fact, Russell and Hutchinson contend that protozoa are

one of the limiting factors in soil fertility because they feed upon,

and consequently limit the numbers of soil bacteria, which are for

the most part, beneficial.

While this and other problems are of primary concern to the

agriculturist, nevertheless their solution depends largely upon the

assistance of the general protozoologist—especially in establishing

certain fundamentals in methodology.

Thus at the very outset the investigator is hampered by the lack

of an adequate method for counting soil protozoa. The four im-

portant methods now in use are:

(1) Rough microscopic examination, which needs no extensive

criticism to point out that it is exceedingly inaccurate; (2) Dilution

method’, which is only approximate by virtue of the fact that the

1Russell and Hutchinson—Jour. Agr. Sci., 3 (1909): 111; 5 (1913): 152, etc.

?Rahn—Centr. f. Bakt. II, 36 (1913): 419.

150 NOTES, REVIEWS, ETC.

differences between dilutions must of necessity be fairly large in

rapid work; (3) Loop method’, where the protozoa in a measured ~

loop are counted microscopically. In the first place the amount of

liquid taken up will vary with a number of samples, because of dif-

ferences in surface tension—especially where various different media

are employed. Secondly, the size of the platinum loop itself will

vary as a result of heating and cooling (on sterilization in a flame)

and chance contact with resistant surfaces; (4) The plating meth-

od‘, is necessarily limited, because some protozoa cannot grow on

solid media.

It thus becomes evident that the above methods do not lend

themselves to accurate work; which fact, led the authors to devise a

new method for the counting of protozoa.

This method is an adaptation of the well-known blood-counting

apparatus—(“Blutkorperzahlapparat”—Carl Zeiss-A. H. Thomas,

Philadelphia). The principles underlying the use of this instrument

is the microscopical observation of a drop of standard size.

The apparatus is designed as follows: A glass disc is cemented

to a glass slide in the middle of a circular cavity slightly larger than

the disc. A thick cover glass, the planes of which are parallel, is

placed over the solution which rests on the glass disc. The excess

liquid is pressed into the reservoir surrounding the disc, thus mak-

ing the film of solution on the disc .1 mm. in depth. It will be seen

that the difference in the depth between the glass disc and the cover

glass is constant, providing the contact between the solution on the

disc and the cover glass is perfect. An area cne square mm. in size

is marked off on the disc, and this is further subdivided into four

hundred squares.

The method of procedure in the use of this apparatus is as fol-

lows: Thoroughly clean both slide and cover glass with alcohol.

With a sterile platinum loop place a few drops of solution to be

examined on the glass disc. It is necessary to have a film of suffi-

cient thickness so that when the cover glass is properly adjusted

and slightly pressed down, perfect contact is ensured. The prepa-

ration is then placed under the microscope and the organisms are

3Miiller—Arch. f. Hyg., 75 (1912): 321.

4Killer—Centr. f. Bakt. II, 37 (1913): 521.

AMERICAN MICROSCOPICAL SOCIETY 151

examined with either low or high power. With the ordinary

Blutkorperzahlapparat cover glass it is impossible to use the oil-

immersion, but we deem it advisable to have a special cover glass

with parallel surfaces ground fine enough to permit the use of that

power, because it has been observed that some very small flagellates

may escape observation even when viewed with the high power lens.

The protozoa are counted in a number of fields and the average

is taken. Examination of each solution was made in triplicate to

ensure accuracy. It was observed that the flagellates and ciliates

maintain practically the same position in the field for the brief

time required for counting. The peculiar movement of each makes

them easily recognizable. However, when they are counted in the

living state the possibility exists of their moving from one field

to another. It depends upon the number of protozoa present, and

the importance of the time element, whether or not they are to be

counted in the living state.

Because of their rapid motility, the large ciliates are killed

before counting. This is done by passing the loopfulls of media

through the vapors emanating from a bottle of osmic acid (as

recommended by Goodey®) before placing them on the glass disc.

If it is desired to stain the organisms, this may be readily done

by using equal volumes of solution and stain (e. g. water solutions

of methylene blue, gentian violet, etc.) without killing the organ-

isms. Or again the organisms may be killed and stained in one

process as with picrosulphuric acid (Kleinenberg); or the same

may be done in two separate steps with osmic acid vapor and other

stains.

The use of low or high power lenses depends largely upon the

kinds and numbers of the organisms present. It is recommended

that more fields be counted with the higher than with the lower

power. }

The results are calculated on the basis of the area under obser-

vation to cubic volume, and then per cc. of solution used.

The advantages of using this apparatus for counting protozoa

are as follows:

5Goodey—Proc. Roy. Soc. Lond., 84 B (1911): 165.

a a NOTES, REVIEWS, ETC.

(1) It is a direct method thus eliminating many errors attending incu-

bation, etc., and the results can be reported immediately.

(2) It is more accurate than any other method in use, because it is a

standard instrument and no mechanical variation is possible.

(3) It is rapid and saves considerable time in contradistinction to

other methods, and the technique is simple. For example, triplicate counts

on any media were recorded in ten minutes,

(4) The counts check more closely than those of the above methods

used.

(5) It can be used to advantage whether the number of protozoa

present be large or small.

(6) It can be used for living, killed, or stained organisms, and permits

of a thorough observation of the individual organisms.

(7) The experimental error is 5%.

Its disadvantages are that the initial cost is greater than that of

other methods, and the sample is too small to be representative. The

error of counts is considerable where the protozoa are very few or

many in number. A number of fields must be counted because of

the uneven distribution, if an accurate count is required.

The starting point for a great deal of experimentation on the

activity of soil protozoa is the choice of a suitable medium for the

development of the organisms. Little work has been done relative

to the comparison of different media, and as a general rule each in-

vestigator has a predilection for some particular one which is seldom

based upon any study of the advantages and disadvantages of the

media adapted to a definite line of research.

Cunningham and Lohnis‘*, Killer*, and others have presented

some valuable information upon this subject, and our experiments

were madeled to a great extent upon the work of the former.

In order to determine which medium would be best adapted to

the large and rapid multiplication of the various kinds of protozoa,

the following media were employed in dilutions of .5%, 1%, 3% 5%,

and 10%. Dried blood, hay infusion, hay infusion plus .5% egg al-

bumen (Goodey )*, peptone, horse, cow, and chicken manures ( Mar-

6Cunningham and Loéhnis—Centr. f. Bakt., II, 39 (1914): 596; ibid. 42 (1914): 8.

4Killer—loc. cit.

5Goodey—loc. cit.

*L_dhnis—loc. cit.

AMERICAN MICROSCOPICAL SOCIETY 153

tin)’, egg albumen, bouillon, and soil extract (Léhnis)* in dilutions

of 400 cc., 600 cc., 800 cc., 1000 cc., 1200 cc., per kg. of soil.

The results obtained in this survey may be condensed in the fol-

lowing table which indicates the maximum numbers of protozoa ap-

pearing on the most favorable medium every day, for five successive

days, as determined by the new counting method previously des-

cribed. It had been previously determined that the count did not

increase materially after the fifth day, consequently there was no

need of continuing the work for any longer period of time.

Since the object was to determine total numbers of protozoa in

the soil, rather than species, no differential count is appended.

Maximum Numbers and Media

Large Large Small Small

Days Ciliates Flagellates Ciliates Flagellates

1 8,520 in Soil 840 in 4,255 in 28,750 in

Ex. 800 cc. 10% Hay 5% D. B. 5% D. B.

2 63,800 in 709 in 5% 9,210 in 282,000 in

Horse 5% Ege Albumen 3% Chicken 5% Horse

3 319,010 in 10,625 in 208,000 in 636,500 in Soil

Hay 10% 10% Hay 3% Chicken Ex. 1000 ce.

4 708,000 in 7,435 in 379,000 in 478,000 in

Hay 10% 5% Cow 3% Ege 1% Horse

5 1,410,000 in 31,900 in 804,000 in 1,878,000 in

10% Hay & Egg 5% Cow 3% Egg 3% Hay & Egg

Summarizing the work outlined above, it was found:

(1) The new method for counting protozoa consists of an adaptation

of the Blutkorperzahlapparat whereby the organisms may be counted directly,

rapidly and accurately. It has been used successfully in the comparison of

media for the development of protozoa and other experiments.*

(2) 10% hay infusion proved to be the most favorable medium for the

development of large numbers of small flagellates, as well as small and large

ciliates. Hay infusion in various concentrations with, and without, the

addition of egg albumen, proved to be well adapted to the development of

the organisms. Hay infusion plus 5% egg albumen proved superior to all

other media for the development of ciliates.

(3) Soil Extract is an excellent medium, though somewhat inferior

to hay infusion plus 5% egg albumen, and with the soil used in this experi-

™artin and Lewin—Phil. Trans. Roy. Soc. Lond., B. 205: 74.

154 NOTES, REVIEWS, ETC.

ment lower concentrations than those recommended by Lohnis developed

protozoa in a shorter period of time.

(4) 3% chicken manure is an excellent medium for the development

of small ciliates.

(5) The numbers and species of protozoa which can be obtained from

a given soil are largely dependent upon the media employed, time of incuba-

tion, as well as the kind of soil used. ;

(6) In general the order of appearance of protozoa was as follows:

small flagellates, small ciliates, large flagellates and finally large ciliates. This

is in accordance with Cunningham and Lohnis’ observations.

*Note: Further results on experimentation and a complete bibliography on soil

protozoa and soil sterilization are awaiting publication.

THE PRESENCE OF ACIDOPHILOUS CELLS IN THE ADRENALS OF CER-

TAIN AMPHIBIANS*

By Thomas Byrd Magath

The adrenal glands of the Amphibians vary, in color, from a

light yellow to a golden reddish-yellow, and extend along the ventral

side of the kidneys in a thin band. The function of these ductless

glands -is not definitely known, altho it is known that an injec-

tion of ‘‘adrenalin” will considerably increase the blood pressure and

that the removal of both glands will result in death, as does an over-

dose of the extract.

Stilling found in the study of the adrenals of the Rabbit, during

the summer, some remarkable cells which stained an intense red with

eosin and appeared very granular in structure. He also found cells

in Rana esculenta which had the same appearance and stained like

those in the Rabbit; because of their appearance in the summer he

called them “Summer cells’.

Patzelt and Kubik, however, found these cells all the year round

and because of their affinity for acid stains called them “acidophi-

lous cells’. The result of their work is as follows:

(a) There is present in the adrenal glands of Rana esculenta,

as in the case of Mammals, Birds and Reptiles, two kinds of cells.

viz., an epithelial portion and a chromaffine portion. The chromaf-

fine portion is made up of large granular cells which turn a brown-

ish-yellow when treated with chromic acid or its salts.

*Contributions from the Biological Laboratory, James Millikin University, No. 12.

AMERICAN MICROSCOPICAL SOCIETY 155

(b) The epithelial portion embraces the common lipoid-con-

taining cells, and among these large granular acidophilous cells.

stainable. They do not change with age, sex activity or nourish-

ment.

(d) These cells are not present in the adrenals of other Anura,

Urodeles, or Reptiles.

(e) <Acidophilous cells appear in some other glands of internal

secretion.

The author, with the purpose of verifying the above results

and if possible of extending them, has made a study of the adrenal

bodies of five Amphibians, viz., Rana pipiens, Rana clamata, S‘peler-

pes bilineatus, Acris gryllus, and Plethodon glutinosus, several ani-

mals of each species, being used. All of these are different species

from those used in the study made by Patzelt and Kubik. It is

found that acidophilous cells appear in Rana pipiens and Rana cla-

mata, as well as in Rana esculenta, as reported by Patzelt and Ku-

bik.

The microscopical technique was on the whole the same as used

by Patzelt and Kubik, and is given below. The tissue was fixed in

potassium bichromate-sublimate-formol, from four to eight hours.

65 cc of 314% aquous solution potassium bichromate ;

35 cc of 5 % aquous solution mercuric chloride ;

100 cc of formol.

The tissue was embedded in paraffin and sectioned five p thick.

The best staining was accomplished by hematoxylin and eosin.

Sections were stained for fifteen minutes in hematoxylin and for

from one to four hours in a very dilute aquous solution of eosin.

The acidophilous cells stained an intense red with eosin, which

showed their granular appearance. These cells stained a reddish-

violet with the Ehrlich-Biondi triacid stain; indeed they stain with

all the acid stains. The chromaffine cells stained a reddish-yellow,

due in part to the chromic salt used in fixing. Another stain which

was used with good results was iron-hematoxylin. The acidophil-

ous cells stained very darkly. The granules stained a deep black

while the protoplasm stained a dark grayish-blue. The chromaftine

cells stained dark grayish-blue and the lipoid cells, black-gray to a

dark gray-blue.

156 NOTES, REVIEWS, ETC.

In general, there are, according to Holmes, two kinds of cells in

the amphibian adrenal glands: epithelium cells which are “cortical”,

are derived from the peritoneum, and chromaffine cells which are

“central cells”, and regarded by Marshall, as modifications of cells

of the sympathetic ganglia. However, in some few species a third

kind of cell is found. These are large spherical cells which have a

large stainable nucleus and are the “summer cells” of Stilling or the

“acidophilous cells” of Patzelt and Kubik.

The results of this study are given as follows:

Rana clamata: Three kinds of cells were distinguished. The

acidophilous cells appeared to be greatly in excess of the other cells

and were very large, being about 15. The lipoid cells were com-

Che acid.

SODr SC

soe - 09 02 Ooo RM,

22 2,00 °° CRS?

0 PF 504°

0° © 02 ?,

e000 :

VOFZI oO

Fig. rt. Adrenal gland of Rana clamata

paratively small, with small nuclei, and scattered thruout the tissue.

The chromaffine cells were arranged in definite patches and ap-

peared about the size of the acidophilous cells.

Rana pipiens: As in the case of Rana clamata, three kinds of

cells were distinguished. The acidophilous cells were less numerous

and smaller, being about 10m. The chromaffine cells were more

scattered and the nuclei of the lipoid cells were much larger than in

the former case.

i an i

AMERICAN MICROSCOPICAL SOCIETY ue

Acris gryllus: Only two kinds of cells were found in this

species. The chromaffine cells were quite numerous and large. The

lipoid cells were quite a little larger and the nuclei smaller than in

the cases of the two frogs given above. No acidophilous cells were

found.

Plethodon glutinosus: In this species the lipoid cells are much

larger than in any of the other examples, being in some cases 72.5 p.

c o QO.

Ro ed

250

C025

200000 0

OLA a a4 One

acid.

Fig. 2. Adrenal gland of Rana pipiens

They are decidedly columnar in shape with large nuclei. The

chromaffine cells are not so prominent and the acidophilous cells are

lacking. :

Spelerpes bilineatus: No acidophilous cells were found in this

species. The chromaffine cells were not so large as in the case of

the other Amphibians and the cell walls of both these and the lipoid

cells stained poorly.

The following conclusions might be made from the observa-

tions :

1. As reported by Patzelt and Kubik, in the case of Rana es-

culenta, there appear in the adrenal glands of Rana pipiens and

Rana clamata, two kinds of cells. An epithelial portion composed

of the common lipoid-containing cells and among them large granu-

lar acidophilous cells, and secondly a chromaffine portion.

2. Acidophilous cells appear in the adrenals of these species all

the year round, and should not be called “summer cells” as named

by Stilling.

158 NOTES, REVIEWS, ETC.

3. In addition to those Anura and Urodeles named by Patzelt

and Kubik, there are no acidophilous cells in the adrenals of Spe-

lerpes bilineatus, Plethodon glutinosus and Acris gryllus.

4. The presence of acidophilous cells in the adrenals of R. es-

culenta, R. pipiens and R. clamata suggests that probably these

species are more nearly related to each other than they are to other

amphibians which do not have the acidophilous cells in the adrenals.

It further suggests that certain physiological activities of these three

species are very similar if not exactly alike.

BIBLIOGRAPHY

Homes, S. J.

Biology of the Frog—pp. 225-226,

MarsHALL, A. M.

Vertebrate Embryology—pp. 387.

PaTzELt, V. AND Kusix, J.

1913. Azidophile Zellen in der Nebenniere von Rana esculenta. Arch.

f. Mikr-Anat. Vol. 81, pp. 82-91.

STILLING, H.

1898. Zur Anatonie der Nebennieren. Arch. f. Mikr-Anat. Vol. 52,

Millikin University, Decatur, Ill. :

June 12, IQI4.

TECHNIC FOR CESTODES

LaRue (Ill. Biol. Monog. Vol. 1: Nos. 1 and 2, 1915) gives

the following account of methods of treating Cestodes:

The following methods have been used by the writer in the

work on this group of cestodes. Toa large extent they may be used

with success on all groups of cestodes, although it should be under-

stood that certain methods which give admirable results with the

relatively small and thin cestodes here dealt with will not give

equally good results if used on the large forms such as Tzenia.

The larger forms were picked out of the intestinal contents,

care being taken to free the head if the worm was attached to the

mucosa. These were then repeatedly dipped in the killing solution

until the worm ceased to contract. The worm was allowed to lie

for 15 minutes to 2 hours in the same fluid. Metallic instruments

are to be avoided if corrosive sublimate solutions are used for fix-

AMERICAN MICROSCOPICAL SOCIETY 159

ation. When the smaller worms were encountered the whole in-

testine slit open was placed in a small quantity of physiological sa-

line solution in a bottle which was then shaken vigorously for about

3 minutes, the killing fluid was added, and the whole then shaken

for one half minute. This is according to the method of Looss.

The fixative was permitted to act 3 to 10 hours.

The killing fluids used were hot 5% solution of formaldehyde,

and hot or cold saturated aqueous solution of corrosive sublimate to

which was added glacial acetic acid to make 1 to 2%. Some other

fluids were tried but nothing gave better results for the purposes of

this study than the corrosive acetic mixture used hot or cold. For

most of the worms the cold solution was preferable to the hot which

sometimes gave rise to artifacts if used at too high a temperature.

In no cases were the worms stupefied before killing.

The usual methods were used for hardening and dehydrating.

Specimens were usually preserved in 85% alcohol after running up

through the grades. Sometimes after the corrosive acetic fixation

5% formalin was used for a preservative with uniformly excellent

results. i

Sections were cut 5 to 10 micra thick for the study of histologi-

cal detail and 20 micra when grosser morphological details were

sought. The sections were stained with hzmatoxylin mixtures,

either Delafield’s hematoxylin or Mayer’s hemalum, and decolorized

in the manner approved for these stains. Methods of staining in

toto followed by sectioning were used with great success at times.

For this purpose Ehrlich’s acid hematoxylin much diluted with 50%

alcohol gave the best results. For a contrast stain eosin in 95%

alcohol was used on the sections. Acid fushion also in 95% alcohol

was sometimes used effectively.

Preparations in toto were much used and were found to be of

great value in mapping out the relationships of the organs of the

proglottids. Frequently these methods showed everything to be de-

sired except the histology of the organs. In some cases even his-

tological details were well revealed by these methods. The stains

which were tried for staining in toto were Mayer’s paracarmine,

Grenacher’s borax carmine, some alcoholic cochineal mixtures, May-

er’s hemalum, Delafield’s hematoxylin, and Ehrlich’s acid hema-

160 NOTES, REVIEWS, ETC.

toxylin. None of the carmine or cochineal stains were very suc-

cessful for none of them show the boundaries of cestode structures

sharply.

The parenchyma in which the genital organs lie always re-

tained too great an amount of these stains to permit a clear view of

the genital organs themselves. The hzematoxylins, however, us-

ually gave wonderfully clear, sharp pictures of the genital organs.

It was at times possible to work out such minute structures as vasa

efferentia almost in their entirety from such preparations in toto.

The three hematoxylin stains were found to be about equally good.

In using these stains it was the practice to dilute the stain with

the proper diluent. Relatively large quantities of the diluted stain

were used for each lot of material. The stain was permitted to act

over night (10 to 15 hours) at room temperature. The excess of

the stain was then removed by washing in distilled water and the

tissue passed through the grades of alcohol to 70% where it was de-

colorized rapidly by adding hydrochloric acid to make a 0.5 to 1.0%

solution. The object was to remove the stain from the peripheral

tissues at a rapid rate and meanwhile leave the stain in the deeper

lying tissues. In this method the duration of the acid bath is us-

ually short, depending upon the size of the piece and the character

of the stain taken by the tissue, and upon the character of the tis-

sue itself. In general it is desirable to decolorize until a light red-

dish blue stain remains and until many of the internal structures

can be distinguished while the tissue is still in the alcoholic medium.

When in the judgment of the operator the proper stain is attained

the tissues are placed in neutral alcohol and then into 70% alcohol

‘rendered slightly alkaline by the addition of a few drops of an

aqueous solution of sodium carbonate.

Preparations were not flattened but were straightened out on a

slide and over this was placed another slide which was supported by

strips of paper of such a thickness that little or no pressure was ex-

erted on the specimen by the slides. Dehydration and clearing were

accomplished while the preparation was thus kept straight. Xylol

and cedarwood oil were used as clearing agents. Preparations were

mounted in balsam.

AMERICAN MICROSCOPICAL SOCIETY 161

The methods outlined above yielded very satisfactory prepar-

ations for the study of these cestodes and they have also been used

by the writer on other cestodes and on trematodes with great suc-

cess. It is noteworthy that the carmine stains give beautiful prep-

arations of trematodes in toto but fail almost entirely for cestodes.

For the cestodes these stains fail because they do not sharply and

clearly outline the sexual organs as they do in trematodes, though

not better than do the hzmatoxylins. In the judgment of the

writer the use of the carmine stains on cestode material has been

responsible for many errors in the interpretation of cestode struc-

tures.

CULTIVATION OF PLASMODIUM OF BADHAMIA

Hilton (Jour. Queck. Micr. Club, Nov. 1914) describes a meth-

od which he has found successful for the continuous cultivation of

plasmodia of the Myxomycete, Badhamia utricularis. He uses

bread which is kept moistened with water.. He finds that it stimu-

lates the growth of the plasmodium to use from time to time, in-

stead of pure water, a mixture consisting of a quart of water to

which has been added half an ounce each of ammonium phosphate

and cane sugar. This seems to give greater vigor to the plasmodium

itself, and also aids it indirectly in that it stimulates the growth of

the filamentous moulds which grow on the bread and are used by

the plasmodium. It would be interesting to know whether this

method would serve for other species.

DAPHNIA WITHOUT SEXUAL FORMS

Banta (Proc. Soc. Exp. Biol. and Med., 1914, p. 180) has

reared Daphnia pulex thru one hundred generations without males

and fertilization. There is no apparent decrease of vigor or vital-

ity, and thus the sexual cycle seems not to be inherently necessary,

altho males have been found in nature at Cold Spring Harbor.

VERTEBRATE EMBRYOLOGY

In this new work on Embryology, Dr. Prentiss undertakes in

one volume to give a working account of the development of the

chick and the pig, together with a description of the stages of human

embryology, histogenesis, and organogenesis. The figures are

162 NOTES, REVIEWS, ETC.

numerous and beautifully presented; the text is clear and satis-

factory. The figures, some of which are in colors, include a large ©

number of whole embryos, dissections, and reconstructions. After a

study of these the serial sections can be related to the whole in such

a way that the student can readily visualize the plane and level of

his section. The book is practically a matrix of human embryology

in which are imbedded the special chapters on the embryology of the

chick and pig. Numerous statements of a comparative character

are included. The treatment is such that the book will be of dis-

tinct value and interest to any student of vertebrate structure and

development.

The arrangement of the chapters is as follows:—Chapter 1,

The Germ Cells ; 2, Segmentation and Formation of the Germ Lay-

ers; 3, The Study of Chick Embryos, of twenty-five hours, thirty-six

hours, and fifty hours; 4, The Fetal Membranes and Early Human

Embryos; 5, The Study of Pig Embryos; 6, Methods of Dissecting

Pig Embryos,—with further studies upon the development of the

head; 7, Entodermal Canal and Its Derivatives (chiefly human, as

are the succeeding chapters); 8, Urogenital System; 9, Vascular

System; 10, Histogenesis; 11, Morphogenesis of the Central Ner-

vous System; 12, The Peripheral Nervous System.

The writer calls attention to the value of a study of Embryology

to the medical student, in that it aids the understanding both of nor-

mal anatomy and of the meaning of anomolies. He also insists that

the view of medical graduates that it is no longer a fruitful field of.

research is false. Aborted embryos and those obtained by operation

in case of either normal or ectopic pregnancies should always be

saved and preserved by immersing them intact in 10 per cent form-

alin or in Zenker’s fluid. An Institute of Embryology has recently

been established by Professor F. P. Mall of the Johns Hopkins

Medical School for the purpose of collecting, preserving, and study-

ing human embryos. Material may be sent there with assurance

that it will be used to the best possible advantage.

An adequate index concludes the book. The mechanical make-

up is all that can reasonably be asked.

A Laboratory Manual and Text-Book of ieee y, by C. W. Prentiss, Ph.D.

Octavo of 400 pages with 368 illustrations. 1915. pit La hin ae Co., Philadelphia and

London. Cloth, $3.75. f

Attention of members of the

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3. Application should be made to Dr. Henry B, Ward,

Chm., Urbana. III.

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TRANSACTIONS

OF THE

American Microscopical

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ORGANIZED 1878 INCORPORATED 1891

PUBLISHED QUARTERLY

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EDITED BY THE SECRETARY

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TABLE OF CONTENTS

FOR VOLUME XXXIV, Number 3, July, 1915

Cell Changes in the Epidermis During the Early Stages of Regeneration

in the Tail of the Frog Tadpole, with Special Reference to Nucleus-

Plasma Relation, with 10 Text Figures, by H. E. Metcalf.......... 167

American Species of the Genus Atractides, with Plate VI, by Ruth

Narahari ges See a ile 94 Gas Ws GOO ee Pee oe oh eee roe 185

Notes and Reviews: A System of Locating Objects in Microscope

Slides, by N. A. Cobb; Stylops and Stylopization; Oogenesis; Size

Dimorphism in Spermatozoa; Termites; Wood-boring Trichoptera;

Egg Concealment; Proventriculus of Hydropsychid Larve; Spider

Poison; Blood-sucking Muscid Larve; Morphology of Thysanop-

tera; Key to Families of Insects; New Orders of Insects; Fossil

Fungus-Gnat; Clearing Bulky and Difficult Objects; Damar as a

Mounting Medium; Penetration of Heavy Cuticula by Fluids; Safety

Razor Blades for the Microtome; Hinges for Lid of Wooden Slide

Boxes; Table for a Microtome; To Remove Balsam from Old

Slides; Modeling Clay for Beginning Classes; Transparent Seal

for Museum Jars; Slide Marker to Use with Microscope; Insect

Mount for Class Study; Double Demonstration Eye-piece; Pre-

serving Plants in Natural Colors; Superior Immersion Fluid; Life

of ‘Inland’ Waters; ; Photomicrography.. 5.5.3: (Gide ges esse nine te ale 189

Notice TO MEMBERS

The Secretary will greatly appreciate notice from members of

any change of address or any failure to receive the Transactions.

T. W. GALLoway,

Secretary.

TRANSACTIONS

American Microscopical Society

(Published in Quarterly Installments)

Vol. XXXIV JULY, 1915 No. 3

CELL CHANGES IN THE EPIDERMIS DURING THE

EARLY STAGES OF REGENERATION IN THE TAIL

OF THE FROG TADPOLE, WITH SPECIAL REF-

ERENCE TO THE NUCLEUS-PLASMA

RELATION?

HERBERT EpMOND METCALF

CONTENTS

lo: Statement: iof. the: problets cds vsidive desi eed 0d Gadp micbeienins ot 167

Tio Materia) cand Mmernodsen ois olie Ho Wena nme eek a eee aaeank et 168

III. Observations.

1. Behavior of the epidermis as a whole.................... 169

2. Behavior of the individual cells in the epidermis........ 174

3. The nucleus-plasma ratio in the migrating epidermis...... 177

PISCE SMT ya oss 5 MEER NFWR Vos ae Sueded lade RONULS ba sep owe at 179

PES OUTAPS Oe. s,s GREE s PETER Pa Gab CONC coats Ke Ea Soh WEEE PRA OHO 183

MIS RE CINOPIRDDY |... as MeRAG Lai) dade oad hw aesivdectelamrneta ey o4 da cdaca lan 184

I. STATEMENT OF THE PROBLEM

The following paper gives the results of a histological study

of the cell changes in the epidermis during the early stages of re-

generation in the tail of the frog tadpole, Rana clamitans. It deals

with the migration of the epidermis which follows the operation

and the accompanying behavior of the epidermal cells, with special

reference to the nucleus-plasma relation. |

1Contribution from the Zoological Laboratory of the University of Illinois,

No. 47.

168 HERBERT EDMOND METCALF

Fraisse (85), and Barfurth (90) both worked on the repar-

ation of the epidermis and other epithelia in vertebrates, and decided |

that in the adult amphibian tail the new epidermis arises from the

elements of the old epidermis, although no evidences of mitosis

were found until the wound surface was completely covered with

new epidermis.

Rand (01) found that also in the earthworm there is an epi-

dermal investment of the wound surface, and in a later paper (04)

decided that the new epidermis arises by means of a migration of

the old epidermal cells, and described to some extent a few of the

cell changes which accompany the migration, as does Emmel (10)

for the crayfish.

Both of the last mentioned authors speak of a cytoplasmic

decrease or an increase in nuclear volume of the cells in the migra-

ting epidermis, either of which would change the nucleus-plasma

relation.

The amphibian tail was selected because of the size and char-

acter of its cells, in that they are large and easily studied under the

microscope. The present paper aims to give an account of the

migration of the epidermis to cover the wound, and to describe the

cell and nuclear changes in the migrating cells.

II. MATERIAL AND METHODS

Two dozen tadpoles of Rana clamitans averaging 75mm. in

length were obtained in September, 1914, kept in an aquarium and

fed on saltine crackers while awaiting experimentation. One fifth

of the tail was removed and the stump allowed to regenerate for

the desired time. When a portion of the regenerated tail had been

cut off for study, the tadpole was put back into the aquarium and

allowed to regenerate until the process stopped, and then used over

again. This method has its advantages, as a section from a regen-

erated tail is much more easily cut than is the original tail, and

makes thinner sections possible because of the softness of the

tissues. By a comparison of sections from a regeneration of the

original tail, and those from a tail previously regenerated, no differ-

ence was found.

o—_" on -ae

NUCLEUS-PLASMA RELATION IN TADPOLE 169

Pieces were taken from tails of 5 minutes, 1 hour, 3 hours,

12 hours, 20 and 24 hours regeneration, fixed in Bouin’s fluid, and

imbedded in paraffin. A number of fixing fluids were tried, and by

far the best results were obtained with Bouin. Sections were

made 5 in thickness, mounted in serial fashion, and stained with

a number of different stains. Iron alum hematoxylin gave the

best results for the determination of mitoses, while Ehrlich’s acid

hematoxylin and eosin furnished the best pictures for general tissue

differentiation, as cell boundaries were well brought out.

III. OBSERVATIONS

1. The behavior of the epidermis as a whole.

The normal epidermis in the tail region of the frog tadpole is

composed of several easily recognizable regions. At the surface I

have not been able to make out a cuticle. The outer layer is com-

posed of a definite layer of cells, cuboidal in section, with almost

spherical nuclei (Fig. 6A). The cytoplasm at the surface stains

slightly darker than that deeper in the cell (Fig. 6E). Next there

are from one to five layers of cells with no regular arrangement

(Fig. 6B), and a bottom layer of slightly columnar cells (Fig. 6C)

with oval or round nuclei which have their long axis, if any, at

right angles to the surface of the epidermis. | Underneath these

cells is a rather thick basal membrane, smooth staining, without

fibrilation or striation of any kind (Fig. 6D). This membrane,

which is drawn dead black in all of the illustrations, is of great

importance in the present study, as it is not regenerated in the early

stages, and therefore the place where the epidermis has been cut

can be easily seen by determining where this membrane stops.

A number of experiments were tried in order to watch the

course of the regeneration in the living material under the micro-

scope, but without material success.

Figures 1-5 show a series of sections near or through the noto-

chord at various times of regeneration, drawn semi-diagrammatic-

ally to show the action of the epidermis. Fig. 1 is a frontal sec-

tion of a tail of a 1 hour regeneration. This shows very clearly

the first process which takes place in the regeneration. The muscles

have contracted both inward and around the tail, thus decreasing

170 HERBERT EDMOND METCALF

to some extent the wound surface. This contraction has the effect

of rounding over the square edges of the cut end, and bends the

epidermis around on the lateral sides so that the epidermis of the

opposite sides touch near the dorsal and ventral sides of the tail

where it is very narrow. At these places the epidermis fuses, and

thus further decreases the wound area. The epidermis has not at

this time begun to move, which is well brought out by the fact that

the basal membrane has about the same relation to the epidermis

as it had at the time of the cut (Fig. 1B). A clot is being formed

to cover the wound, in which black staining fragmenting nuclei of

Figure 1

Fig. 1. Frontal section through the notochord 1 hour after the operation, show-

ing the relation of the epidermis to the basal membrane, and the beginning of the formation

of the blood clot. A, blood clot; B, basal membrane. (200 diameters)

degenerating cells are beginning to be seen. In most cases the

next thing to appear is a complete blood clot over the wound sur-

face, although the time of the formation may be before one hour

has elapsed, or later, but in all cases the clot was formed to a

greater or less degree before 12 hours had past (A-Figs. 1-5).

Figures 2 and 3 show the period of greatest activity in the

epidermis. Figure 2 is a frontal section, and Figure 3 is a sagittal

section of a regeneration of 12 hours duration. As will be seen

in Figure 2 the epidermis has left the basal membrane behind, and

has commenced on its advance over the wound tissues and the clot

(Fig. 2B). This plainly shows the importance of having some

landmark by means of which one may tell whether or not there has

NUCLEUS-PLASMA RELATION IN TADPOLE 171

been an actual advance of the epidermis, or whether this apparent

advance is only a flap of epidermis left by an incomplete cut which

has folded over the wound surface. In the several cases in which

the latter happened, it was immediately noticed because of the ac-

companying basal membrane. In the normal epidermis the cells

seem to stick rather closely to this membrane, and in the normal

individual when the epidermis is torn off, the basal membrane us-

ually comes away with it.

Figure 2A and 3A show the position of the blood clot over the

wound surface. In the tail from which Figure 2 was taken, the

clot covers the entire cut surface including the notochord, while in

Figure 3, the notochord is not covered. This is a very finely fi-

brinated clot with a rather thicker fibranation near its outer surface.

It is for the most part homogeneous, however, and has in its num-

erous fragments of the nuclei of the degenerating cells. These

fragments arise by a process of amitosis, or fragmentation, as it

perhaps had better be called. (Sutherland-15.) At first these

Figures 2 and 3

Fig. 2. Frontal section near the notochord 12 hours after the operation, showing

the advancing end of the epidermis, and the blood clot covering the entire wound. A,

blood clot; B, advincing end of epidermis. (200 diameters)

Fig. 8. Sagittal section through the notochord 12 hours after the operation, showing

the advancing edges of the epidermis and the blood clots on each side of the notochord.

A, blood clot; B, advancing end of epidermis. (200 diameters)

172 HERBERT EDMOND METCALF

fragments do not stain very dark, and seem to come from the cells

near the cut surface, but later they stain almost black, and for some

distance back into the tissue immediately behind the cut edge the

nuclei take the heavy black stain, showing, in all probability, a

degenerative process. One very peculiar thing was found during

the early stages, in that while all of the muscle, connective tissue,

notochordal, and nerve cord cells for some distance back from the

cut show fragmentation and degeneration, not a single nucleus of

the epidermis was found to have a degenerative appearance, as

evidenced by fragmentation and deep staining.

In the early migration the cells seem to travel in two different

ways. In Figure 2, the advancing end of the epidermis is com-

posed of several different layers of cells (Fig. 2B), while the ad-

vancing edge of the epidermis in Figure 3 is composed of only one

layer of cells (Fig. 3B). It appears to me that all of the cell lay-

ers would advance nearly equally as in Figure 2 if the configuration

of the surface ahead of it would allow it to do so. The surface

in Figure 3 is somewhat obstructed, and as it is the outer layer of

cells which is the most active in the migration, the cells below are

stopped, and the outer layer passes over the obstruction.

Figures 4 and 5 show a frontal and a sagittal section of a re-

generation of twenty hours. Figure 4 shows that the epidermis

has advanced over the clot and met the epidermis from the other

side and fused with it. It also shows that the basal membrane is

in the same position as it was when cut (Fig. 4B). The epidermis

here which covers the clot is of normal thickness, and the cells

seem to have returned to their normal position and shape. Figure 5

does not seem to be as far along as Figure 4 as in this case the

investment of epidermis is only a single cell thick. This is the

result of an obstruction of the deeper layers of the epidermis as

mentioned above, and the investment will not reform its several

layers until the beginning of mitosis at about 48 hours.

One of the first things which is noticed in the movement of

the epidermis is the lack of any evidences of cell multiplication in

the epidermis, either near the point of movement, or farther back,

where mitosis or an increase of cells by any means would aid in

pushing the epidermis over the wound. There seem to be even

NUCLEUS-PLASMA RELATION IN TADPOLE 173

fewer mitoses than normally in the epidermis. In all of the sec-

tions which have been examined up to a stage of 20 hours regen-

eration there was only one mitotic figure found. This was near

the region where the epidermis was normal, and had no part in the

movement of the tissue.

I will now sum up briefly the movement of the epidermis as a

whole. As a result of a strong muscle contraction of the tail dur-

ing the first hour, the epidermis from the two sides is brought in

contact above and below the notochord, thus decreasing the wound

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Fig. 4. Frontal section just above the notochord 20 hours after the operation, show-

ing the wound healed by the meeting of the epidermis from the two sides, enclosing the

blood clot. A, blood clot; B, basal membrane, marking position of cut. (200 diameters)

Fig. 5. Sagittal section through the notochord 20 hours after the operation, show-

ing wound healed by meeting of epidermis from the two sides, enclosing the blood clot.

Note that here the epidermis is only one cell in thickness, while in Fig. 4 the invest-

ment is several cells in thickness. A, blood clot; B, epidermal investment. (200

diameters)

area. The epidermis at these places fuses and plays no further

part in the investment of the cut surface. The epidermis surround-

ing the cut notochord and the thick part of the tail then starts to

migrate over the blood clot and wound surface, unhelped by cell

multiplication within the tissue. The epidermis from the two sides

meets near the center of the cut surface, and the cells return to their

normal shape and size. If the epidermis has been able to migrate

with all layers of cells, normal stratification is resumed. If only

174 HERBERT EDMOND METCALF

a single layer of cells has migrated over the wound surface the

epidermis remains a single cell in thickness until mitosis begins.

This action encloses the blood clot which has been formed and is

complete at from 20 to 24 hours. The next process which occurs

is the absorption of the blood clot and when this is completed, at

about 48 hours, regeneration by mitosis begins.

2. The behavior of the individual cells in the epidermis.

As has been stated, the normal epidermis is composed of sev-

eral definite layers, the most conspicuous of which are the upper

layer of cuboidal cells, and the lower layer of columnar cells.

There are several layers of cells between these two limiting layers.

Figures 6 and 7

Fig. 6. Section through the normal epidermis showing the relation of the layers

of cells. A, outer layer; B, intermediate layer; C, columnar layer; D, basal membrane;

E, darker cytoplasm at surface. (1100 diameters)

Fig. 7. Section through the migrating epidermis some distance away from the ad-

vancing end, showing the change in shape of cell and nucleus. A, outer layer; B, in-

termediate layer; C, columnar layer pulling away from basal membrane; D, basal mem-

brane; E, greatly elongated cell at surface; F, space between basal membrane and epi-

dermis. (1500 diameters)

The lower columnar layer is tightly stuck to the basal membrane.

In the twelve hour stage of regeneration, which seems to be the

stage in which the migratory movement is the greatest, as we leave

the normal epidermis and approach the cut edge, the first notice-

able thing is that the columnar cells on the basal membrane lean

over in the direction of the cut surface, so that their nuclei instead

of being at an angle of 90° with the basal membrane, are coming

to be more nearly parallel with it (Fig. 7C). At the same time the

upper layer of cuboidal cells commences to lose its stratification,

NUCLEUS-PLASMA RELATION IN TADPOLE 175 ~

and the cells change from a cuboidal shape to one which is elon-

gated in the direction of the cut surface (Fig. 7E). In following

along the epidermis of a 12 hour preparation, one can find all stages

in the change of shape, from the cuboidal to the greatly elongated

cell at the place of greatest movement.

Immediately after the basal columnar cells start to lean in the

direction of the cut surface, spaces appear between the basal cells

and the basal membrane, so that finally the basal cells are in con-

nection with the basal membrane in only a few places (Fig. 7F).

These spaces are large and do not appear to be in the cytoplasm of

the basal cells, but rather in the region between the cell and the

membrane. The nuclei of the basal cells do not appear to change

Figure 8

Fig. 8. Section through the notochord, showing the advancing end of the epidermis

composed of a single layer of cells. (900 diameters)

their shape to any great degree, merely rotating through 90° so

that their major axis is parallel to the direction of movement. This

is not so, however, with the cells of the upper layer. Here the

nuclei in the normal epidermis were nearly spherical, so that when

the cell elongates in the direction of movement, the nuclei are

squeezed into an elongated shape.

After the cells have passed the place where the cut was made,

most of their nuclei except those of the upper layer have resumed

176 HERBERT EDMOND METCALF

their normal spherical or slightly elongated shape. Those of the

upper layer are still in an extremely elongated condition, as are the

cells. In some of the cases found, it was only this upper layer

which was migrating over the clot surface (Fig. 8). This would

seem to show that the upper layer of cells was the one which was

doing the most active migration, and that they were pulling along

the lower cells. As was mentioned before it is this outer layer

which passes around obstructing portions of the wound surface.

In fact, it is only in this external layer of cells that any change

in nuclear shape and change in chromatin arrangement can be de-

tected. The nucleus changes from a spherical to a greatly elong-

ated shape. This is due, | am convinced, not to any action of the

nucleus itself, but is merely a passive adjustment to conditions

laid upon it by the moving cell. However, there does seem to be a

change of some kind, for the nucleus takes on a rather deeper stain

‘than it did in the normal epidermis, and the chromatin does not

appear to be arranged in exactly the same manner as normally. In

the normal nucleus of the outermost cells, the chromatin is in a

rather coarse reticulum, differing in no way from that of any of

the other cells in the epidermis. After migration starts the chro-

matin appears to be in a much coarser network than formally, with

thick strands of chromatin parallel to the direction of movement.

I do not find any evidences of “polarization” as does Emmel (10)

in the regeneration of the epidermis in the crayfish. The appear-

ance of these cells under the microscope is one of movement, as

they extend their cytoplasm forward toward the cut surface in a

pseudopodial manner. In no case did I find any cells positively

identifiable with epidermal cells wandering loose. Several isolated

cells were found, but were decided to be leucocytes.

After the clot has been covered, whether it be by one or more

layers of cells, these outside cells regain their normal shape, and the

nuclei come back to the spherical form. Their migratory move-

ment is finished with the fusion of the epidermis of the two sides

of the tail. After this fusion and the resumption of the normal

shape, there is a resting period of about 24 hours while the clot is

being absorbed. During this latter period there is no mitosis, and

the epidermal cells have every appearance of normal epidermal

NUCLEUS-PLASMA RELATION IN TADPOLE 177

cells, altho as has been said there may be only one layer. In this

connection it may be said that a count was made to determine

whether or not there is amitosis in the regenerating epidermis after

mitosis has begun, and the resulting ratio was about 100 character-

istic mitotic figures to one dumbbell shaped nucleus, which may or

may not be an evidence of amitosis. In fact, after a careful

study of the later stages of the regeneration, I am inclined to think

that amitosis plays no part in the process. Of course, it is hard

Figure 9

Fig. 9. Curve showing the decrease in the nucleus-plasma relation in the migrating

cells, taken from a frontal section. The abscissa represents the position of the cell in the

epidermis, and the corresponding ordinate the nucleus-plasma ratio for the cell. A,

region of normal epidermis; B, region of the beginning of migration; C, region of the ad-

vancing end and active migration.

to recognize true amitotic figures, but from all the observations

made there was not the slightest evidence that amitosis was char-

acteristic or even common in the regenerating epidermis.

3. The nucleus-plasma relation in the migrating epidermis.

The nucleus-cytoplasm ratio was obtained by a method des-

cribed by Dolly (13) in which the measurements for the axes of

the cells and nuclei are made from camera lucida drawings at a

magnification of about 1000, with a rule graduated to Imm. The

relative volumes are calculated by supposing that the unknown

third dimension is equal to the shortest axis as found in a single

178 HERBERT EDMOND METCALF

section. These three dimensions are then multiplied together, sub-

tracting the nuclear volume from that of the cell. The resulting

figure for the plasma mass when divided by the nuclear volume

gives a figure corresponding to Richard Hertwig’s conception of

the nucleus-plasma ration.

A glance at Figure 9 will show at once some of the results.

The curve was obtained by plotting the cell measurements in the

relative positions the cells held in the migrating epidermis between

the normal portion and the advancing end.

As will be seen from the curve, the nucleus-plasma ratio of

the cells in the normal epidermis is about 3.5. The ratio seems

to increase slightly as the cells begin to leave their regular positions

in the epidermis, and then to steadily decrease until the ratio in

the actively migrating cells is about 1.0. These ratios were ob-

tained from a frontal section.

This reduction is extremely interesting if the results of such

a measurement can be relied upon. Of course it is not necessary

to have an accurate measurement of volume, it being only import-

ant that the calculations apply to all of the cells without a vari-

ation due to the changes in the shape of the cells. It may be in-

teresting to note in this connection that the change in the nucleus-

plasma relation takes place along with the change in shape of the

cell. This might account for the difference in ratio, but I am in-

clined to think that there is in reality a change in the nucleus-

plasma relation, for this method will give the volume of a parallel-

opiped whether it be a cube or otherwise.

Also, to check the results obtained in Figure 9, which was made

from a frontal section, measurements were made from a sagittal

section of a tail at the same time of regeneration. If the entire

cell merely spread out, then the section through this plane would

certainly not show a decrease in the nucleus-plasma relation, but

rather an increase to compensate for the decrease obtained from a

frontal section. This, however, is not so. A curve from a meas-

urement of a sagittal section (Fig. 10) shows essentially the same

characteristics as the curve in Figure 9. These two curves com-

bine all three dimensions of the migrating cells, whereas a curve

from a sagittal or a frontal section alone, will have an unknown

NUCLEUS-PLASMA RELATION IN TADPOLE 179

dimension which has to be estimated. In this way, by measuring

and making curves from frontal and sagittal sections, I think I have

overcome the objection which might be made because of the esti-

mation of the third or the unknown dimension. The proof of this

is that both curves show a decrease in the ratio as they should if

there were a true decrease in the cells.

IV. DISCUSSION

As will be evident from the foregoing, the epidermis which

covers the wound and the clot comes, without a doubt, from the

old epidermis. There is a slight difference in the regeneration in

Z a

Figure 10

Fig. 10. Curve showing the decrease in the nucleus-plasma relation in the migrating

cells, taken from a sagittal section.The abscissa represents the position of the cell in

the epidermis, and the corresponding ordinate the nucleus-plasma ratio for the cell. A,

region of normal epidermis; B, region of the beginning of migration; C, region of the

advancing end and active migration.

this form from that described by Rand (04) for the earthworm,

in that he says “as already pointed out, mere contraction of the

muscular body wall plays an unimportant part in the closing of

the wound, because of the relatively large cross section of the

worm.” In the tadpole the wound is very narrow except at the

region of the notochord, and in one hour’s time the contraction of

the muscles of the tail is sufficient to cause the meeting of the

180 HERBERT EDMOND METCALF

epidermis of the two sides above and below the notochord it was

seen that the basal membrane, and therefore the epidermis, even

overlaps to a slight degree. So it may be said that the first repar-

ation process, that of muscle contration is of some importance in

the early stages of regeneration in this form. There cannot be any

doubt that the new epidermis comes from the old because it may

be seen in various stages on its way, and because there is no tissue

with which it might be confused which has anything like the ia

pearance of the epidermis.

Rand (04) and Emmel (10) differ somewhat as to the in-

ternal cell changes. Rand states for the earthworm “the nuclei,

however, never show marked change in volume” while Emmel says

that in the crustacean “it may be noticed that the nuclei are becom-

ing larger in size, and the chromatin is undergoing certain changes.”

I could find no evidence in either direction in the tadpole, migrat-

ing nuclei being in some cases both larger and smaller than normal

nuclei. Rand does not mention any marked chromatin changes,

while Emmel describes a kind of “polarization” of the chromatin

in the nucleus. I did not find any radical changes in the chromatin

except that it seemed to accumulate in larger bodies than normally,

and that it was more densely packed, due perhaps to the lengthen-

ing of the nucleus.

There can be no doubt that in the tadpole there is a migra-

tion of the individual cells of the epidermis. The extraordinary

change of shape of the cells, especially those of the outermost layer

indicates that the individual cell is moving by lengthening out in

an amoeboid manner. This migration cannot be otherwise, for if

there was a pulling of all the epidermis from the region of the cut

surface, all of the cells would take on the elongated form so char-

acteristic of the outermost layer of cells. This, however, is not so,

and it is this same outer layer which is the most changed in shape,

and which seems to be the most concerned in the process of migra-

tion. The deeper cells have the appearance of being pulled along,

and to support this hypothesis, instances have been noted, where

the deeper layers of cells are obstructed by some of the fragments

of the cut tissues, and have been held back, only the outer layer

traveling over the obstruction.

NUCLEUS-PLASMA RELATION IN TADPOLE 181

The advancing edge of the migrating epidermis clearly shows

then that there is not a passive pushing of the cells over the cut

surface. What is the agency by which the cells are stimulated to

move over the wound? They always move over the surface. In

no case did I find evidences of a migration down into the clot, nor

were there any signs of a free migration, that is, without a solid

support for the migrating cells. Neither were the advancing cells

moving along the fibrils of the clot, as they moved as easily over

the cut notochord. The fibrinations of the clot also were too small

to give anything more than a rough surface over which the cells

might travel. There was nothing like the condition of the spider

web which was used by Harrison (14) in his experiments with

cell migration. The migration here is more like that shown by the

cells which moved along the cover glass in his experiments.

This migration is assigned by the majority of authors to chemo-

taxis, or a chemotactic cell phenomenon, in the sense of Roux (94)

(96). The stimulus may come from the chemical attraction of

the wound surface, or the mutual interaction of the epidermal cells.

The stimulus starts the epidermal cells moving and as the only

direction in which they can move is over the wound surface the

effect of their migration is to heal the wound opening. When the

two sides meet there is no longer any opportunity for migration as

there is no place for the cells to go, so that migration stops. Rand

emphasizes the fact that the stimuli operating there are similar in

nature to those which are said by Herbst (94) to be of great im-

portance in normal ontogeny.

There have been several references to a change in the size of

the cell or nucleus during the migration of the epidermis in various

animals. Rand (04) mentions the fact that the cytoplasm of the

migrating cells in the earthworm appears to decrease, and Emmel

(10) says for the crayfish that the nuclei seem to be slightly larger

than normally. Fraisse (85) states that on the advancing edge of

the migrating epidermis of the salamander the nucleus is sur-

rounded by a small amount of protoplasm, so that the nuclei are

closely packed together. A glance at his Plate I, Fig. 8, shows

this feature, and the change in relation of the cytoplasm to the

nucleus is plainly to be seen without measurement.

182 HERBERT EDMOND METCALF

It seems fairly certain, therefore, that there is a decrease in

the nucleus-plasma ration during migration. The measurements

given in this paper of course are not useful as actual numerical

values of the volumes, being merely to get an approximate curve

of the decrease in the ratio value.

Admitting the fact that there is a decrease in the nucleus-

plasma ratio, what does it signify? If Minot’s hypothesis applies

to all forms of cell phenomena in which there is a decrease in the

nucleus-plasma ratio, then this might indicate a rejuvenescence of

the cell after differentiation. He states “Rejuvenescence depends

on the increase of the nucleus” or in terms of the nucleus-plasma

relation a decrease in that ratio. This was in connection with cell

cleavage of the fertilized egg. He also adds “Reversed cytomor-

phosis is not known to occur, or in other words differentiated ma-

terial cannot be restored to the undifferentiated condition.” Yet

here if the decrease in the nucleus-plasma ratio indicates a re-

juvenescence we have differentiated epidermal cells which are

rejuvenating and afterward returning to the differentiated condi-

tion.

Interesting in this connection is the work of Morgulis (11) on

inanition, in which he describes the effect of starvation on the cells

of the salamander, Diemycytylis viridescens. He concludes that

the volume of both the cell and nucleus decreases as a result of

starvation, but that the rate of diminution of the volume of the cells

is greater than that of the nuclei.

The basal cells of the epidermis in the tadpole pull away from

the basal membrane during migration, and it is possible that this

decreases the interchange of nutrient material between the blood

and the epidermal cells. Therefore they may be reduced to the

condition of starved cells. This, if Morgulis’ hypothesis be cor-

rect would tend to reduce the nucleus-plasma ratio. Therefore the

decrease in the observed nucleus plasma relation may be entirely

due to a starvation of the epidermal cells caused by isolation from

food during migration.

However, Conklin (12) states that rejuvenescence is dependent

on the interchange of material between the nucleus and the cyto-

NUCLEUS-PLASMA RELATION IN TADPOLE 183

plasm, and that anything which facilitates this interchange increases

metabolism and leads to rejuvenescence.

The cells are in active motion, and as the supply of food may

be cut off, and as they are undergoing an unusual activity the rate

of metabolism is without doubt great. This activity is so great

that the cytoplasm is probably used up and therefore the nucleus-

plasma ratio decreases. Therefore, if Conklin’s hypothesis applies

to these cells we have a rejuvenescence as there is a high rate of

metabolism and consequently an interchange of material between

nucleus and cytoplasm.

V. SUMMARY.

In brief the processes which occur in the early stages of regen-

eration in the tail of Rana clamitans are as follows:

1. There is a muscle contraction within 1 hour after the cut

which not only decreases the surface of the wound, but also plays

an important part in the process of healing by bringing the epi-

dermis of the two sides of the tail together both above and below

the notochord, where the epidermis fuses.

2. There is a migration of the epidermal cells over the clot

which is formed at about 12 hours time. This migration is the

result of an active migration of the individual cells of the epidermis,

especially those of the outer layer, without the slightest sign of cell

multiplication of any sort, either by mitotic or amitotic cell division.

3. There is a decrease in the nuclets-plasma ratio in the mi-

grating epidermis.

4. As the cells of the migrating epidermis are pulled away

from their normal position in relation to the blood vessels, this

decrease may be due to starvation.

5. As there is also a very active migration of the individual

cells the rate of metabolism is probably high, and as the cells may

not receive their customary amount of nourishment, the cells may

be forced to rely upon the energy contained in the cytoplasm, and

so use it up during the migration. This would account for the

decrease in ratio.

6. If the views of Minot and Conklin can be applied to these

cells, then this decrease in the nucleus-plasma ratio would indicate

a rejuvenescence of differentiated epidermal cells.

184 HERBERT EDMOND METCALF

7. Migration ceases at about 24 hours and the nucleus-plasma

ratio returns to normal in the epidermal cells.

8. The period of time from 24 to 48 hours is occupied by

the absorption of the clot, and the cells are in a resting condition.

9. At 48 hours regeneration by mitosis begins. No evidence

has been found that amitosis is a factor in the regeneration of the

tail of the frog tadpole.

This problem was suggested by Dr. Charles Zeleny, to whom

I am indebted for many helpful suggestions during the course of

the investigation.

BIBILOGRAPHY

1885. Fraisse, Paul—Die Regeneration von Geweben und Organen bei den

Wirbelthieren besonders Amphibien und Reptilien. Cassel und

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Vol. 37.

1892. Randolph, Harriet—The Regeneration of the Tail of Lumbriculus.

Journ. of Morph. Vol. 7.

1894. Herbst, C—Uber die Bedeutung der Reizphysiologie fiir causale

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1896. Davenport, C. B—Preliminary catalogue of the processes concerned

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1902. Loeb, L.—Uber das Wachsthum des Epithels. Archiv. f. Entw. der

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Anat. Vol. 3, No. 3.

1910. Emmel, V. EA study of the differentiation of tissues in the regener-

ating crustacean limb. Am. Journ. Anat. Vol. 10, No. 1.

1911. Morgulis, Sergius.—Studies of inanition in its bearing upon the prob-

lem of growth. Archiv. F. Entw. der Org. Vol. 32.

1914. Harrison, Ross G.—The reaction of embyronic cells to solid struct-

ures. Journ. Exp. Zool. Vol. 17, No. 4.

1915. Sutherland, G. F—Nuclear changes in the regenerating spinal cord

of the tadpole of Rana clamitans. Biol. Bull. Vol. 28, No. 3.

AMERICAN SPECIES OF THE GENUS ATRACTIDES

By RutH MarsHALL

_ The water mites of the genus Atractides belong to a small sub-

family, the Hygrobatine. About ten species have been described

by European hydrachnologists for Europe; two of these species

have been reported by them also from North America, while one

species is described for Madagascar. Three new species are now

added to the list for the United States. All of the material, thirty-

eight individuals, was found by the author in small lakes in Wis-

consin.

The species comprising this group are easily recognized ; but,

unfortunately, there has been much confusion in the use of the

name Atractides. By some authors it has been used as a synonym

for the genus Megapus of Neuman, and given as the valid name

for the genus Torrenticola of Piersig. The nomenclature used by

Piersig (1901) and by Wolcott (1905) seems to the author to be

logical, and it is used in this paper.

The body of these mites is oval or elliptical; the integument is

usually soft, with a surface covering finely striated or porous. The

epimera are in three groups, with the capitulum lying in close con-

tact with the first pair. The most distinctive feature is the oblique

placing of the third pair and the more or less truncate margin of

the fourth. The genital area is placed about midway between the

epimera and the end of the body; the cleft in both sexes is flanked

on either side by three conspicuous acetabula on a sickle-shaped

plate, another distinctive feature of the genus. In the female the

cleft is long and the acetabula are removed from it; in the male

the shorter cleft is completely enclosed by the plates bearing the

acetabula.

The palpi are about as thick as the legs. The joints of the

palpi increase in length from the first to the fourth, while the fifth

is small and claw-like. A distinctive feature of the genus is the

186 RUTH MARSHALL

presence of two hairs on papille on the ventral side of the fourth

joint, and a sword-like spine on the inner side. This fourth joint

usually bears some fine hairs.

The legs do not vary greatly in length; all of them bear spines

and a few hairs. The first leg is most distinctive; it is often longer

and stouter than the second, and the last joint shows more or less

bending. These features are pronounced in figures published by

European writers on the genus; they are not conspicuous features

of the three new American species here described.

. Atractides parviscutus nov. spec.

Pl. VI, Fig. 1-4

This is a large mite, the largest specimen measuring 1.3 mm.

The epimeral plates occupy an uncommonly small part of the

ventral surface. The spaces between the groups of plates are

large. The surface of the body, including the plates, is very finely

porous. On both dorsal and ventral surfaces the plates marking

the openings of the skin glands are prominent; in balsam mounts

the glands are strongly outlined, and fine hairs may be made out

on the plates.

The legs are much alike; the first two pairs have about the

same length. The first pair are only a trifle the stouter, while the

last joint shows but little bending. The palpi are slender.

Only the female of the species was found in a collection of

twenty-six individuals, all from Wisconsin lakes. Four of these

were found in Nagowicka and Neshota lakes, near Milwaukee, in

the fall of 1908; eighteen in Lake Spooner, in northwestern Wis-

consin, in the summer and fall of 1909; two in Lake Minocqua, in

the northern part of the state, in July, 1912; and two in the Lauder-

dale Lakes, in southern Wisconsin, August 12, 1914.

Atractides phenopleces nov. spec.

Pl. VI, Fig. 5-9

This species is founded upon the examination of a single speci-

men, a female from Lake Spooner, Wisconsin (Aug. 18, 1909). It

measured 1.1 mm. in length. The epimeral plates show fewer of

the irregularities of surface than do those of some species, and the

AMERICAN SPECIES OF GENUS ATRACIDES 187

posterior borders of the fourth plates are sharply truncate. Cer-

tain small characters which distinguish this from related species

are best brought out by the figures. The lateral plates of the genital

area bearing the acetabula are large and somewhat removed from

the rest of the area. The first pair of legs show the bending of

the last joint so characteristic of the genus.

Atracides orthopes nov. spec.

Pl. VI, Fig. 10-12

This new species is represented here by six males, three from

Lake Nagowicka, near Milwaukee (Oct. 15, 1908), and three from

Lake Spooner, Wisconsin (July 16, 1909). It resembles A. gib-

beripalpus Piersig in the character of the epimeral plates, which

show irregular surfaces and a close proximity of the three groups.

The palpi are fairly stout, and the last joint of the first leg is very

long and shows no trace of bending; in these particulars the new

species differs from the related one. The two sword-like bristles

on the distal end of the fifth joint of the first leg are very large.

The surface of the body appears very finely granular, the plates

more so. The length of the body is 0.65 mm.

BIBLIOGRAPHY

KoENIKE, F.

1898. Hydrachniden-fauna von Madagascar und Nossi-Bé.

Abh. Senck. naturf. Gesell., XXI, i11:416, pl. XX VI, fig. 128-130.

1895. Nordamerikanische Hydrachniden. Abh. naturw. Vereins

Bremen, XIII :211-212, pl. III, fig. 58-59.

1909. Die Stisswasser Fauna Deutschlands, XII: 89-94. Jena.

Maciio, C.

1905. Secondo Elenco d’Idracne del Pavese.

Rendiconti del R. 1st Lomb. di. sc. e lett, XXXVIII, 2:152-154.

NeEuMAN, C.

1880. Om Sveriges Hydrachnider. Kongl. Svenska Vet.-Akad.

Hndlgr., X VII :63-65, pl. I, fig. 4.

Prersic., R.

1897. Deutschlands Hydrachniden. Bibliotheca Zoologica, XXII :186-190,

pl. XVIII, fig. 45. Stuttgart, 1897-1900.

188 RUTH MARSHALL

1898.. Hydrachnidenforman aus der Hohen Tatra. Zool. Anz., XXI:;12.

Neue Hydrachnidenformen aus dem sachsischen Erzgebirge.

Zool. Anz., XXI:523-524.

1901. Hydrachnide (und Halacaride). Das Tierreich, XIII :181-185.

1904. Ueber eine neue Hydrachnide aus dem Bohmer Walde.

Zool. Anz., XX VII :453-454.

THor, SIG.

1899. Tredie Bidrag til Kundskaben om Norges Hydrachnider.

Arch. Math. Naturv., XXI, 5 :38-40, pl. XVII, fig. 119-121.

Wo tcortt, R. H.

1905. A Review of the Genera of the Water Mites. Trans. Amer. Mic.

Soc., XXVI:219. (Repr. as Studies from the Zool. Lab., Univ.

of Nebraska, No. 66).

EXPLANATION OF FIGURES

Fig. 1. Altractides parviscutus nov. spec., ventral view.

Fig. 2. Altractides parviscutus nov. spec., dorsal view.

Fig. 3. Altractides parviscutus nov. spec., right palpus, inner side.

Fig. 4. Altractides parviscutus nov. spec., legs, left side.

Fig. 5. Altractides phenopleces nov. spec., ventral view.

Fig. 6. Altractides phenopleces nov. spec., 1st leg, left side.

Fig. 7. Altractides phenopleces nov. spec., 4th leg, left side.

Fig. 8. Altractides phenopleces nov. spec., genital field, female.

Fig. 9. Altractides phenopleces nov. spec., left palpus and capitulum.

Fig. 10. Atractides orthopes nov. spec., ventral view.

Fig. 11. Altractides orthopes nov. spec., 5th, 6th joints, right Ist leg.

Fig. 12. Atractides orthopes nov. spec., right palpus, outer side.

DEPARTMENT OF NOTES, REVIEWS, ETC.

It is the purpose. in this department, to present from time to time brief original

notes, both of methods of work and of results, by members of the Society. All members

are invited to submit such items. In the absence of these there will be given a few brief

abstracts of recent work of more general interest to students and teachers. There will be

no attempt to make these abstracts exhaustive. They will illustrate progress without at-

tempting to define it, and will thus give to the teacher current illustrations, and to the

isolated student suggestions of suitable fields of investigation.—[Editor.]

A SYSTEM FOR LOCATING OBJECTS ON MICROSCOPE SLIDES

By N. A. Coss

All mathematical methods of recording the position of a micro-

scopic object on a slide use a system of codrdinates. Most micro-

scope makers provide some scaled mechanism, usually in connec-

tion with the mechanical stage, which allows of two readings that

locate the position of the object. Sometimes the scales are en-

graved on the two traverses of the stage, sometimes on a special

microscope slide.

The various systems are not in harmony, so that it is often

inconvenient to use on one microscope records made on another.

There are contrivances for overcoming this inconvenience, some of

much merit, but none of them have come into wide use, to say

nothing of universal use.

The object of this communication is to propose a basis for

agreement so simple that it may possibly win universal support.

The proposal is to make one corner of the slide, say the lower left

hand corner, the point of origin for the system of coordinates.

The lower left hand corner of the slide then becomes zero, and any

object is located by telling how far to the right of zero it lies, and

how far above zero. Any system of linear measurement may be

used.

Thus in the illustration the location of the object “x” is re-

corded by the figures 41.5-16.5 millimeters; the object is 41.5 milli-

meters to the right of and 16.5 millimeters above zero.

190 NOTES AND REVIEWS

Against certain small objections may be urged a considerable

number of practical points. First, the point of origin is a natural

one, fixed, not registered, that exists on every rectangular slide

regardless of its size; in a certain sense therefore it is not arbi-

Plutus

__ frets

4I'5 - 165

trary. Second, it is possible to locate objects, approximately at

least, without mechanical apparatus. ‘This is of importance to a

vast number of microscopists who have few accessories. Third,

any point on the slide is indicated by figures having a positive sign.

To one having a plain simple stage without any other acces-

sories than spring clips it is possible by this system to find the

coordinates of an object by the following simple method.

Take a piece of thin paper about ten centimeters long by four

centimeters wide, or larger, and make a pinhole near the middle.

Clamp this paper to the stage with the clips, and bring the pinhole

to the center of the microscope field. Place the slide on the paper,

turn on a strong light, and it will generally be found that the object

can be examined in this fashion. When the object is found, place

it centrally in the field, see that the pinhole is also central, and with

a sharp pencil point trace the lower and left hand edges of the

slide. Finally measure the distances from the pinhole to these two

lines. These measurements will be the coordinates desired. To

find an object given its codrdinates, reverse the process.

ENTOMOLOGICAL NOTES

Stylops and Stylopization—Smith and Hamm (14, Quart.

Journ. Micr. Sci., N. Ser., 60: 435-461) find that in spite of the

fact that active, winged males of Stylops exist, fertilization cannot

occur and development is always parthenogenetic. This parasite

causes a reduction of about three-fourths in the size of the ovaries

of the host bee and no ripe ova are ever produced. The testes are

AMERICAN MICROSCOPICAL SOCIETY 191

usually not affected, and ripe, normal spermatozoa may be formed.

The parasitized female bee usually has a reduced scopa and rarely,

if ever, collects pollen.

Oégenesis—Dederer (’15, Journ. Morph., 26: 1-42) presents

the results of a study of the odgenesis in Philosamia cynthia.

Thirteen chromosomes occur in the pronuclei, thus making the

somatic number twenty-six. No satisfactory evidence as to the

presence or absence of an X-Y pair of chromosomes was secured.

No differential divisions were found in the odgonia. The nuclei

of the future nurse cells show evidences of preparation for division

although the chromosomes disintegrate, forming numerous granules

which arrange themselves near the much infolded nuclear wall.

The nurse cells contribute material to the egg cell by means of

tubes. Amitosis was not observed among the germ cells. During

most of the growth period the egg nucleus is in the spireme stage.

Size Dimorphism in Spermatozoa.—Zeleny and Faust (715,

Journ. Exp. Zool., 18:187-240) report the results of a study of

size differences in the length of the head of spermatozoa from

single testes in fifteen species of animals, of which ten are insects

representing five different orders. Long series of measurements

yield evidence that the population of spermatozoa is composed of

two size groups, due to. the difference in chromosomal content.

These groups are not wholly distinct. The distribution curve in

each species usually showed two high points or modes, and a com-

parison of the degree of separation of these modes with the ex-

pected degree secured by calculations on chromosomal histories

shows a significant correspondence.

Termites—Snyder (715, U. S. Dept. Agr., Bureau of Ent.,

Bull. No. 94, Part II) reports the results of a study of the biology

of the termites of eastern United States. The scope of the inves-

tigation was broad, and important data were secured on communal

organization, castes, life cycle, cannibalism, different forms of nest,

establishment of new colonies, parasites, termitophilous insects, etc.

A few of the important facts revealed by this study are as follows:

The life cycle of Leucotermes is highly variable. New colonies

may be established by three groups of individuals, (1) the sexed

colonizing adults which swarm and leave the colony, (2) neoteinic

192 NOTES AND REVIEWS

royal individuals “produced from nymphs of the second form which

never (?) leave the parent colony, or from young larve, as in

colonies orphaned after the nymphs of the first form have nearly

completed their development”, (3) neoteinic reproductive forms

“supplied to orphaned colonies, which may be derived from nymphs

of the first or second forms, or larve’. A bibliography of about

forty titles is given.

Wood-boring Trichoptera—Lloyd (’15, Psyche, 22:17-21)

describes a new species of caddis-fly (Ganonema nigrum) from

Ithaca, N. Y., the larvae of which have case-making habits which

differ greatly from those of other described American trichopterous

larve. They bore into the solid wood of twigs, heavy bark, or

fragments of broken wood on the stream-bottom and use the tun-

neled wood as portable cases. The food consists of finely pulver-

ized wood. These larve are apparently as active in midwinter

when the temperature is 20° Fahr., as in midsummer. The writer

has found similar cases in streams in Colombia, S. A. Attention

is called to Hudson’s record (1904) of a species of similar habits

in New Zealand.

Egg Concealment—Hegner (’15, Psyche, 22:24-27) reports

results of experiments on the eggs of the potato beetle (Leptinotarsa

decemlineata) to determine the reasons why eggs are laid on the

lower surface of the leaf rather than the upper. The data show

that eggs in various stages of development, if kept in direct sun-

light, are not retarded in their development but are prevented from

hatching, due, probably to partial desiccation. While the advan-

tage of concealment is, no doubt, important, the advantage secured

by protection from the desiccating influence of sunlight is the vital

one.

Proventriculus of Hydropsychid Larve.—Noyes (’15, Journ.

Ent. and Zool., 7 :34-44) finds that in the larve of the genus Hydro-

psyche (Trichoptera) the well-developed proventriculus is unique

in having a large number of distinct comparatively solid, chitinous

teeth in the anterior part. Two valves, continuous with the ceso-

phageal valve, occur in the posterior part. The cesophageal valve

is open along one entire side. The muscle layers in the fore-in-

testine do not conform to the usual arrangement in insects but the

AMERICAN MICROSCOPICAL SOCIETY 193

inner layer is composed of circular muscles and the outer layer of

longitudinal muscles. The anterior portion of the organ triturates

resistant parts of the food while the posterior portion serves the

combined function of a strainer and of a regulator of the passage

of food into the intestine.

Spider Poison—Kellogg (715, Journ. Parasitology, 1:107-112)

summarizes literature and presents discussion of the effects of the

poison of spiders belonging to the genus Latrodectes, the species of

which have been regarded as non-venomous or nearly so by some

students of the Araneida. Evidence is given in support of the

contention that spider poison is formidable and active in character.

Allowing for individual differences in physiological condition in

different people, it appears that the poison of Latrodectes is capable

of producing serious results.

Blood-sucking Muscid Larve—Coutant (’15, Journ. Para-

sitology, 1; 135-150) reports the finding of the larve of Protocalli-

phora azurea, one of the flesh-flies, on the bodies of young crows

as parasites. The blood-sucking habit of these larvae was demon-

strated by a microscopic examination of the contents of the alimen-

tary canals which were full of the corpuscles of the crow’s blood.

Data are presented to support the view that the larvee are intermittent

feeders and the author holds that they do not eat solid flesh, either

fresh or decayed. The paper includes a discussion of the anatomy

of the larve and pupa, the habits, distribution, and chetotaxy of

the adult.

Morphology of Thysanoptera.—Peterson (715, Ann. Ent. Soc.

Am., 7 :20-66) presents the results of a detailed study of the anatomy

of the head and mouth parts of the Thysanoptera. The interpre-

tation of the asymmetrical mouth-parts of this order is significant.

The results of such an intensive morphological study can not be

completely summarized here but the following are a few of the

important general points: The asymmetrical mouth-parts of thrips

are arranged in the form of a cone and are fitted for sucking.

Mandibular and maxillary parts are uniformily present and show

asymmetry. The greatly reduced tentorium is homologized and ren-

ders important aid in the interpretation of the piercing organs. The

piercing organs in the two suborders are homologous. The sucking

194 NOTES AND REVIEWS

apparatus of the pharynx, the salivary glands and the head glands

have been critically studied.

Key to Families of Insects—Brues and Melander (1915) have

published a book entitled “Key to the Families of North American

Insects”. A brief complete key of the dichotomous type has been

constructed and leads to the identification of 483 families. The

system of orders adopted is essentially that of Handlirsh, more

recently established orders being also included, thus making a

total of thirty seven. The chief characters of each order are given.

The eighteen plates containing 437 figures of representative insects

or their morphological details, the glossary, the index, the pronun-

ciation of the names, and the inclusion of synonyms are useful

characters of the book. In spite of the fact that the new system

of orders adopted involves a considerable departure from the one

commonly used and necessitates a certain amount of readjustment,

persons interested in taxonomy of insects will find the book a useful

source of information.

New Order of Insects —Silvestri (’13, Portici Boll. Lab. Zool.

Gen. Agr., 7:193-209) describes three species of a very interesting

new genus, Zorotypus, from the Gold Coast, Ceylon, and Java. A

new order, Zoraptera, is proposed for the reception of these species,

the characteristics being as follows: minute; terrestrial; wingless ;

agile; predaceous. Body depressed. Antenne moniliform, nine seg-

ments. Eyes vestigial. Mandibles strong. Thorax as long as abdo-

men. Abdomen with ten segments. Cerci short, conical, one seg-

ment. Legs similar; hind femora enlarged; tarsi with two seg-

ments. The specimens on which this new order is based are only

about 2 mm. long. They occur in rotting, vegetable debris.

Fossil Fungus-Gnat.—Cockerell (‘15, Can. Ent., 47:159) de-

scribes the first genuine fossil Mycetophila found in America. The

general appearance and structure are said to be exactly as in the

living species, Mycetophyla punctata Meigen, but differences in

certain details of structure led to the disposal of the form as a new

species, described under the name of Mycetophyla bradene. This

fossil was collected from the Miocene shales of Florissant, Colo-

rado. The studies of the European species from Baltic amber of

Oligocene age and the American form show that the fossil forms

AMERICAN MICROSCOPICAL SOCIETY 195

are of the same type as the living ones and it appears that some

of the Mycetophylide have continued through the long lapse of

time without evolutionary progress except for the changes in minor

specific characters.

Kas. State Agricultural College,

Manhattan, Kas. PauL S. WELCH.

NOTES ON BIOLOGICAL METHODS FROM UNIVERSITY OF ILLINOIS

At the request of the Secretary of the American Microscopical

Society the following notes on Biological Methods in use at the

University of Illinois have been gathered with an aim toward offer-

ing suggestions that might be of value to the readers of the Tran-

sactions who are looking for shorter cuts and improved methods

in their Biological work. The writer makes no claim of discovery -

or of originality for the greater part of the methods offered here,

in truth many of them are old facts revived while some of them are

but modifications of standard biological methods.

For clearing bulky and difficult objects—The use of synthetic

oil of wintergreen as a clearing agent for microscopic mounts of

whole objects greatly increases their transparency. Objects which

appear opaque after treatment with cedar oil, xylol, or toluol, by

the use of synthetic oil of wintergreen become highly translucent.

This is especially true in the case of solid parenchymatous animals

such as large trematodes, leeches, and planarians. Violent diffusion

currents accompanying the transfer from absolute alcohol to the

oil may be avoided by running the specimens through a series of

mixtures of absolute alcohol and the oil. In most cases three or

four such mixtures are sufficient, though for animals with thin body

walls and large internal cavities the writer has found as many as

six grades necessary at times. From the oil of wintergreen transfer

to Canada balsam or damar in xylol on a slide and add coverglass.

Damar as a mounting medium.—Damar dissolved in xylol has

many advantages over Canada balsam. Balsam turns a dark brown

with age. In mounts of embryos or other objects where considerable

volume of the mounting medium is used this change of color soon

196 NOTES AND REVIEWS

impairs the value of a mount. Damar retains its light color and

perfect transparency indefinitely. In preparing either damar or

balsam it is well to dissolve in an excess of xylol. This gives a very

thin liquid which should be run through filter paper. After filter-

ing evaporate the excess of oil as rapidly as possible to prevent

chances of contamination by acid fumes or by dust from the air.

This is best accomplished by placing the damar or balsam over a

steam bath, though by using care the oil may be driven off by

placing the liquid in a porcelain dish over a gas flame.

Lo facilitate penetration of fluids in organisms with a heavy cuti-

cula—Many animals covered with an unbroken cuticula strongly

resist the penetration of fluids. In many cases where there are

large internal cavities this causes the specimens to turn black or

sometimes opaque chalky white when brought into the nrounting

medium, damar or balsam. Some avoid this by cutting the animal

into pieces. This allows free penetration through the cut surfaces

but is almost sure to lead to confusion in interpreting the relations

of parts. The writer has found that several small punctures through

the cuticula while the animal is still in alcohol allow the clearing

and mounting media to enter freely. By performing this operation

under a binocular the holes can be made with a very fine needle in

regions of the body where they will least interfere with internal

structures.

For best results with the oil immersion objective a drop of

immersion oil, or a drop of water, should be placed upon the upper

surface of the substage condenser and brought into contact with the

bottom of the slide. This prevents refraction of light by the air be-

tween the condenser and the slide, thus producing stronger illumin-

ation of the field. |

Safety razor blades for the microtome.—In using an adapter for

safety razor blades in a rotary microtome it has been found that a

fairly heavy double-edged blade, such as the Durham-Duplex, gives

less vibration and for that reason more uniform and better sections

than are possible with the extremely thin blades. The blade men-

tioned also has a much longer cutting edge.

To hinge the lids of wooden slide boxes—Lids of the ordinary

wooden slide boxes very readily become separated from their prop-

AMERICAN MICROSCOPICAL SOCIETY 197

er boxes and unfortunately are not ofter interchangeable. A strip

of gummed cloth tape answers very well as a means of binding these

lids and at the same time prevents interchanging the covers.

A table for the microtome.—A suitable table for a microtome

that is used by several persons in different rooms often becomes a

problem. In the local Department of Zoology this has been solved

by using a typewriter cabinet manufactured by the Toledo Metal

Furniture Company. This cabinet consists of a table mounted upon

wheels. The microtome is bolted to the table. When in use the

top is rolled back and the two hinged sides are lowered, forming

ample working space. By throwing a lever the stand is raised off

the wheels onto a set of legs which gives greater stability. This

arrangement serves admirably for ordinary histological work, but for

perfect series of thin sections the stand is not rigid enough to give

great precision with a rotary microtome.

To remove balsam from old slides and coverglasses—Balsam

may be removed from old slides and coverglasses by boiling them

in a strong solution of “Gold Dust”. All paraffin should be removed

before putting slides to soak in this mixture, otherwise a film which

is hard to remove forms over the surface of the glass.

The use of modeling clay in a beginning class—tThe difficulty

of inducing the beginner with the microscope to interpret miscro-

scopic objects in three dimensions may be eliminated to great extent

by requiring each student to model some animal such as the Par-

amecium. Modeling clay may be purchased from any artists’

supply house.

A transparent seal for museum jars.—Celluloid wet with acetone

makes a transparent seal for the covers of museum jars. Celluloid

strips, or a ring in the case of a round jar, should be cut just a little

wider than the ground glass surfaces to be cemented. The celluloid

is then dipped in acetone, placed upon the edge of the jar and the

cover applied before the acetone has evaporated. Slight pressure

should be applied until the celluloid has set.

A slide marker for use with the microscope—In dealing with

gross structures carbon ink is frequently used to direct attention

to a certain section of a series on a slide. Frequently finer details

198 NOTES AND REVIEWS

need to be marked for future reference. For this work the pen is

too uncertain. The Leitz Microscope Company have an object

marker which meets the demand for a marker of any sized area

admirably. A diamond is fitted eccentrically upon a dummy objec-

tive. The spot to be marked is brought into the exact center of

the field and the marker is swung into place instead of the objective.

By bringing the diamond point into contact with the coverglass and

rotating the marker a circle is scratched upon the coverglass around

the field desired. The diameter of the circle may be regulated at

will by means of a set-screw. In making class demonstrations with

the microscope a series of slides once marked may be referred to at

any time without the necessity of hunting over an entire collection

or even over the slides that are known to contain the demonstrations

desired.

An insect mount for class study.—The Department of Entomol-

ogy has been devising mounts for insects to be used in large classes in

economic entomology where it becomes impracticable to furnish

each student with several specimens of every form studied. One of

the most successful mounts is the one for Lepidoptera which re-

quires for its manufacture: two thin pieces of glass (preferably cov-

erslips for lantern slides), a sheet of heavy cardboard or picture

mounting board, and passepartout paper. A square hole of proper

size for the specimen to be mounted is cut from the center of the

card. An insect either spread or with wings folded may be held

in the chamber formed by placing a sheet of glass on each side of the

card. The edges of the mount are then bound with passepartout

paper. Protection against Dermestidae is secured by cutting one or

more holes in the edges of the card and filling these with napthaline

crystals.

A double demonstrating eye piece for the microscope.—lIt is

ofter difficult to make sure that the beginning student sees the thing

he is supposed to see under the microscope. A double demonstrating

eye-piece which fits in the microscope in place of the ordinary oc-

ular enables the instructor and the student to observe the same field

at the same time. The adjustable needle pointer in this instrument

makes it possible for the instructor to point out the organism to be

AMERICAN MICROSCOPICAL SOCIETY 199

studied and under other circumstances he may require the student

actually to point out structures which he thinks he has seen. This

instrument is manufactuured by E. Leitz.

Preserving plants in their natural colors —In the Botany depart-

ment of this Institution extensive use is being made of a method for

preserving green plants in their natural colors. Fifty per cent acetic

acid in water is saturated with copper acetate as a stock solution.

To one part of the stock solution add four parts of water and in this

boil for ten minutes the plants to be preserved. After this treatment

put the plants into four percent formalin for preservation. Fungus

spots and insect pests show much more naturally in materials pre-

pared in this way than they do in materials that have been bleached

out in the preservative.

To seal vials and bottles—Gutta percha and paraffin melted

together give much better results than paraffin alone for sealing

corked vials and bottles to prevent evaporation. The gutta percha

prevents the mixture from becoming friable.

Ripening hematoxylin—Many text books on histological

methods still hold to the idea that hematoxylin stains must ripen at

least three weeks before they are ready for use. By adding a small

amount of hydrogen peroxide to a stock bottle of newly prepared

hematoxylin the stain becomes usable in a very few hours. The

minimum time required for ripening by this process has not been de-

termined though the writer has obtained perfect results from Ehr-

lich’s hematoxylin six hours after it was prepared.

University of Illinois.

Urbana, Ill. H. J. VAN CLEAVE.

SUPERIOR IMMERSION FLUID

For 4 years V. Jensen, (Hospitalstidende, Copenhagen. Dec.

2, LVII. No. 48) has been using a mixture of alpha brom-naphthelin,

24 parts, with 76 parts liquid paraffin, and has found it extremely

satisfactory as an immersion fluid. He tests it to get the refraction

index the same as that of the ordinary immersion oil. Its advantage

over the latter is: It flows easily ; keeps perfectly, even uncovered ;

can be easily removed at any time, early or late; it does not dry

out, and the specimen keeps its color perfectly.

200 NOTES AND REVIEWS

THE LIFE OF THE INLAND WATERS.

Under this title Messrs. Needham and Lloyd are issuing a book

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Chapter I treats in an introductory way some of the conditions

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the life within them. Chapter IV gives an account of the aquatic

organisms themselves :—algz, colorless water plants, moss-worts,

fernworts, and seed plants; protozoans, lower invertebrates, arthro-

pods, and vertebrates. Chapter V discusses the individual adjust-

ments of organisms to the conditions of aquatic life and to one

another. Chapter VI treats aquatic societies and Chapter VII,

inland water-culture.

Aside from its educational value in connection with school

courses, the book has interest to the general student because the

waters are a source of a considerable part of our food supply,

_are important in determining the sanitary conditions of our exist-

ence on the earth, and are mixed up vitally with our recreation and

pleasure.

The Life of Inland Waters. James G. Needham and J. F. Lloyd. Illustrated

1915. Comstock Publishing Co., Ithaca, N. Y.

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TABLE OF CONTENTS

For VoLUME xxxiv, Number 4, October, 1915

North American Frog Lung Flukes, with Plates VIJ-IX, by W. W. Cort.. 203

Elytral Tracheation of the Tiger Beetles (Cicindelide), with Plates

a MET DY Via ee CLE OTOL NSIT cleo 6 Wdv le dng «ates he Ok tee eee 241

A Study of the Scales of Some of the Fishes of the Douglas Lake Region,

BV GASTAUT V1, eV ATs Sas As Ao eic tn ain se MERE ETE Pied sooner pa he orn Re 255

Notes and Reviews: A Reliable Method for Obtaining Amoeba for Class

Use, by Charles A. Kofoid; Notes on Laboratory Technique from the

Zoological Laboratory University of Michigan, by George R. LaRue;

Entomological Notes, and Notes on Oligocheta, by Paul S. Welch;

The Olfactory Sense in Insects, by E. W. Roberts; Arachnoidiscus in

Maryland Diatomaceous Earth, by B. C. Welsh; A Satisfactory

Dissecting Board, by Newton Miller; Mechanism of Mendelian

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COnstrution (and oS yal ws 5 ibs sa Od ee cals be tien Re ELE ee sents 295

Necrology

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Jonathan Deuell Hyatt (with Plate XVIII)....................008. 306

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DROS eer st iy ree Sie Linu oe kak WN alaiia Siaighn BME Se se meh k's C6 wire rete eee 319

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TRANSACTIONS

American Microscopical Society

(Published in Quarterly Installments)

Vol. XXXIV OCTOBER, 1915 No. 4

NORTH AMERICAN FROG LUNG FLUKES

By WILLIAM WALTER Cort*

Trematodes from the lungs of anura were first reported over

a century ago by Zeder (1800:160). In 1819 Rudolphi (1819:99,

378) described a lung fluke from Rana esculenta under the name

Distomum variegatum. Altho after Rudolphi a number of work-

ers added to the knowledge of this form, it was not until the work

of Looss (1894:71) that an adequate description was given. Later

the same author (Looss, 1899:600) proposed for the frog lung

fluke a separate genus, Hematolechus, and upon re-examination

of his old Distomum variegatum material found that it included

three distinct species. The largest of these he designated the type

of the genus under the name H. variegatus and the others he

named H. similis and H. asper. Stiles and Hassall (1902 :20)

called attention to the fact that in their opinion Looss (1899 :602)

in mentioning the name Distoma simile in connection with his

preparations had given this name to the second of these species

before the name Hematolechus similis. The name Distoma simile

which is preempted they call a “still born” homonym and propose

for the species the combination Hematolechus similigenus. Since

the only name proposed by Looss was Hematolechus similis this

specific name in my opinion should not be superceded. Heam-

*Contributions from the Zoological Laboratory of the University of Illinois, under

the direction of Henry B. Ward, No. 53.

204 WILLIAM WALTER CORT

atolechus asper Looss labels as a species inquirenda since he had

only two specimens, but later work has placed the standing of this

species beyond any doubt. In 1902 on account of Stal’s hemipteron

genus Hematolecha established in 1874, Looss (1902 :732) changed

the generic name Hematolechus to Pneumoneces. He did this

influenced by Braun’s (1901:55) contention that if family or sub-

family names were formed from generic names which differed

only in ending, they would be identical. This seems to me to be a

logical application of the rule of priority and I shall accept the

later name Pueumoneces.

Leidy (1851 :207; 1856:44) was the first to notice lung flukes

in American frogs, but his descriptions are too brief to make

precise diagnosis possible. In 1902 Stafford (1902:895) described

five new species of this genus from Canada, and in a later publi-

cation (1905:687) he designated them Pneumoneces longiplexus,

P. breviplexus, P. varioplexus, P. similiplexus and P. medioplexus.

In regard to P. varioplexus Stafford (1902 :906) writes:

“T have obtained it in Toronto and Montreal, but I find that

I have only few mounted specimens and imperfect notes; conse-

quently I shall not describe it at length but shall trust to the draw-

ing to illustrate its chief characters. I shall be glad to come upon

it in numbers for greater assurance as to its claims for specific

distinction.”

It has not been possible to obtain from Stafford this or any

other of his type specimens for examination. Except in the size

of the eggs the characters of the above species agree with those

given for P. similiplexus. I have never collected any lung flukes

which might be assigned to this species and at present would feel

inclined to consider its distinctness as very doubtful. I shall there-

fore treat it as a species inquirenda. Pratt’s (1903:29)Ostiolum

formosum Stafford (1905:687) considered to be a synonym of

Pneumoneces medioplexus. A study of Pratt’s type material

has confirmed this view. The only other account of frog lung

flukes from North America is Seeley’s (1906:248) description of

P. complexus, from Rana pipiens in North Carolina. The present

paper adds a new species found in Colorado, for which the name

Pneumoneces coloradensis is proposed. ‘Two other species of

ene

—— ee ee lle”. Le

—— ee ee ee

Se ee. |?

NORTH AMERICAN FROG LUNG FLUKES 205

the genus have been described, one by Klein (1905:60) from

India as P. capyristes and the other by S. J. Johnston (1912 :320)

as P. australis from Australia.

Work on the trematodes of North American frogs was under-

taken by the writer in the fall of 1909 while a graduate student

at the University of Illinois. Two previous publications (Cort

1912, 1913) have covered part of this work. In addition to the

studies on the lung flukes at that time collection and observations

have been made of these forms from a number of different lo-

calities. Material has been studied of Pneumoneces longiplexus,

P. breviplexus, P. similiplexus, P. medioplexus, P. complexus and

of the new species from Colorado, P. coloradensis.

I wish to express my appreciation to Professor Henry B.

Ward for his guidance during the early part of this work, for the

use of his library and for material from his private collections.

Thanks are due Dr. George R. La Rue for help in tecnic, and to

Dr. H. S. Pratt, Mr. Hermann Douthitt and Dr. M. C. Hall for

material.

TECNIC

The worms for sectioning were usually killed after shaking

according to Looss’ method (Looss, 1901) in saturated corrosive

sublimate plus: 1 to 3% acetic acid. The specimens which were

destined for toto mounts were usually transferred into distilled

water before killing, in order that part of the eggs might be evac-

uated. After this treatment they were killed without shaking. All

specimens were left in cold killing fluid from 6 to 18 hours, but

when hot fluid was used less time was required. For toto staining

Mayer’s Paracarmine, Mayer’s Hzmalum and Delafield’s Hama-

toxylin were found most useful. The worms were much over-

stained in dilute solutions and then very rapidly and completely

destained in 2 to 4% HC1 in 70 to 85% alcohol. This method

of differentiation has been found useful for most toto mounts of

trematodes, since in this way almost all of the parenchymatous

tissue is cleared of stain, so that the internal organs stand out

clearly. For staining sections hematoxylin with eosin as a coun-

ter was my favorite combination. Much time can be saved by

206 WILLIAM WALTER CORT

staining the worms in toto in Ehrlich’s Acid Haematoxylin, differ-

entiating on the slide after sectioning and then counterstaining in

the higher grades of alcohol. By this method it is not necessary

to run the sections on the slide thru a lower grade of alcohol than

85%. The only difficulty in the tecnic of the frog lung flukes is

due to the presence of great masses of eggs. In some of the

largest specimens sectioning became almost impossible on account

of these great masses of eggs. When possible all species were

studied alive.

GENERAL DIscussIon

OccurRENCE. Altho reports of the occurrence of North Amer-

ican frog lung flukes cover an area including Canada and the north

eastern and central portions of the United States, they are rela-

tively so few and scattered that little idea can be gained of the

range of the genus and the distribution of the several species.

Host. All reports of North American representatives of the

genus Pneumoneces are from Bufo or Rana. There seem how-

ever to have been few if any examinations of the smaller anura.

A given host may harbor even in a restricted locality several

species, and one species may infest several different hosts. Pneu-

moneces longiplexus and P. breviplexrus have been found in Rana

catesbiana; Pneumoneces longiplexus, P. similiplexus, P. medio-

plexus, P. complexus and P. coloradensis in Rana pipiens; and

Pneumoneces medioplexus and P. breviplexus in Bufo americanus.

In several specimens of Rana pipiens which were examined from

near Chicago there were found represented three species, Pneu-

moneces medtoplexus, P. similiplexus, and P. longiplexus, in sev-

eral cases individuals of two different species occurring in the same

frog and even in the same lung. Stafford found P. longiplexus to

be the most common species in Rana catesbiana. In my collections

from Rana pipiens, Pneumoneces medioplexus is by far the com-

monest form. All degrees of infection have been found, varying

with the locality from a few flukes in a small proportion of the

frogs examined to a heavy infection in a large proportion. The

heaviest infection of frog lung flukes ever reported was in a speci-

men of Rana pipiens from Oshkosh, Wisconsin. This frog contain-

NORTH AMERICAN FROG LUNG FLUKES 207

ed twenty full grown specimens of Pneumoneces medioplexus in one

lung and twenty-two in the other. The parasites almost filled the

lung cavity, their actual bulk being greater than that of the lung

tissue.

Size. Within the limits of a given species there is found

great variation in the size of sexually ripe individuals, i. e. indi-

viduals in which the uterus up to the genital pore contains fully

developed eggs. In those species in which a considerable number

of individuals were available for study, the largest sexually ripe

forms were two and even three times the size of the smallest. Be-

tween the extremes were all gradations. Some difference in size

seems to be due to differences in environment. The average size

of the specimens of P. longiplexus which I have collected from

Rana pipiens is considerably less than that of those collected by

Stafford from Rana catesbiana. From a region in northern Minne-

sota all the specimens of Pneumoneces similiplexus found, altho

crowded with eggs were considerably below the average size for

the species. In cases however where different sizes were found

in the same locality or host, it seems probable, as Stafford (1902:-

896) suggests, that these flukes continue to grow in size after they

have reached sexual maturity. Such variations in size within a

species make any statements of average size either for the whole

animal or for its organs of little value. Suckers and reproduc-

tive organs usually vary in size in proportion to the size of the

individual, only the size of the eggs and the spines remaining con-

stant. For this reason measurements will always be given for

individuals, and mere size comparisons, except in the case of the

eggs and spines, will be given little weight in specific diagnosis.

However ratios in size such as between oral suckers and aceta-

bulum, and ovary and testes, are found to be quite constant within

the species and give some of the most important criteria for spe-

cific determination.

Spines. Great variation in regard to the spines is found

among the species of this genus (Fig. 4). Some species are

entirely covered with them, some are only partially covered, and in

some of the cuticula is entirely smooth. It would seem from the

general small size of the spines and their complete absence in

208 WILLIAM WALTER CORT

certain species that in the genus Pneumoneces they are of little

value to the individual and are in the process of degeneration.

That there may be variations in this character within a species is

suggested by the difference in Stafford’s and my own account in

regard to the presence and absence of spines in P. longiplexus and

P. breviplexus (see specific accounts). In the individuals which

I have examined there have been no variations within a species in

the character of the spines.

SucKERS. The acetabulum is always smaller than the oral

sucker; in several species it shows degeneration and is apparently

of no functional value to the adult.

Dicestive System. The characters of the digestive system

appear to be of generic value and show specific difference of value

for diagnosis only in the ratio in size of the oral sucker to the

pharynx and in the length of the esophagus, altho this character

is almost entirely obscured by differences in the contraction state

of the anterior ends of different individuals.

MALE REPRODUCTIVE SysTEM. Variations in the size, shape

and position of the testes give one of the easiest ways of identifying

the species of the frog lung flukes (Figs. 2, 3, 8, 9 and 10). The

testes are located diagonally in the body usually back of the middle,

with the posterior testis on the same side as the ovary. From the

anterior tips of the testes pass forward the vasa efferentia which

extend near the dorsal surface of the body forward on each

side of the seminal receptacle and unite just in front of the ovary

to form the seminal vesicle. The seminal vesicle is a long narrow

sac filled with sperms and enclosed for its entire length in a thin

walled cirrus sac. Just back of the genital pore the seminal vesicle

narrows into an ejaculatory duct, surrounded by a group of unicel-

lular prostate glands, and a short cirrus with thick walls. The |

cirrus varies with the species from sausage to pear shaped. Both

the ejaculatory duct and the cirrus are enclosed in the cirrus sac.

When the cirrus is not protruded the male passage opens with the

metraterm at the common genital pore. Figures 11 and 12 show

the relations of these parts for two of the species. In one speci-

men of P. medioplexus which had been placed in distilled water,

the cirrus was extruded and a quantity of sperms ejected. The

NORTH AMERICAN FROG LUNG FLUKES 209

length of the extended cirrus was equal to about one-half the

width of the body at this region. In one toto preparation of P.

_longiplexus the cavity of the cirrus contained ripe eggs. This

shows a continuity of passage between the metraterm and the

cirrus, and suggests the possibility of self-fertilization. This case

is parallel to the one Looss (1894 :61, Taf. VI, Fig. 127 ) notes for

Distomum cygnoides.

FEMALE REPRODUCTIVE ORGANS. The general position of the

ovary and the ducts near it are characters of generic rank. Specific

differences are to be found in the size and shape of the ovary

and slight variations in the size and position of the ducts. The

ovary which is rounded, oval, or irregularly lobed, according to

the species, lies to one side of the middle of the body just back

of the acetabulum. In all the species the condition of sexual

amphitypy is found, the ovary being sometimes on one side of the

body and sometimes on the other, and the other genital organs in

like relation. Lying in the midline of the body and dorsad of the

ovary, covering a considerable area, is a group of unicellular glands,

the so-called “shell gland.” This gland surrounds the female

ducts of this region. After reading Goldschmidt’s (1909) paper

on the yolk and shell glands of trematodes, in which he ascribes

the shell producing function not to the so-called ‘“‘shell gland” but

to the vitelline glands, I was struck by the absolute inadequacy of

such a gland as the “shell gland” of the frog lung flukes to furnish

the material for the shells of the great numbers of thick shelled

brown eggs which distend the uteri in these forms. I agree with

Goldschmidt that this gland cannot be the shell gland and will follow

his usage in calling it Mehlis’ gland. In the ducts of the vitellaria

could be distinguished the same shell droplets described by Gold-

schmidt. These facts force me to the conclusion that in the frog

lung flukes as well as in the forms described by Goldschmidt the

vitelline glands produce the shell material. The seminal recep-

tacle is round or oval in shape near the mid line of the body ven-

trad of the ovary and Mehlis’ gland and overlapping the posterior

portions of both. It is a thin walled sac as large or larger than

the ovary and is full of disintegrating sperm cells in the larger

specimens. No Laurer’s canal is present. The oviduct starts as

210 WILLIAM WALTER CORT

a narrow tube from a point on the median dorsal side of the ovary.

It passes immediately into Mehlis’ gland and receives the duct from

the seminal receptacle, which is very short and passes up from

the dorsal side of that organ. Just after having been joined by this

duct the oviduct is met by the medium duct of the vitellaria from

the vitelline reservoir. It then passes thru Mehlis’ gland as the

odtype which is recognizable by its heavy walls and becomes the

beginning of the uterus. The uterus runs forward a ways along

the inside of the ovary before it turns backward in its course. The

vitellaria are much divided, extending for a considerable distance

along each side of the body near the dorsal body wall. At either

or both ends there are median groups of acini. The groups are

composed of numbers of small acini and are connected by longi-

tudinal ducts. These are gathered from each side just back of

the ovary into two median ducts, which join just back of the ovary

into the median vitelline reservoir (Figs. 13 and 14) VD, VR.)

While there is much individual variation in the number and arrange-

ment of the groups of acini and the ducts, each species is constant

within certain limits. Also the size of the acini and the number in

a group vary with the species.

Uterus. Differences in the arrangement of the uterine coils

give a character by which certain species can be readily distin-

guished. The general course of the uterus is from the ovary to

the posterior end and back to the genital pore, filling especially in

the larger specimens all the available space between the organs.

There may or may not be longitudinal folds from the posterior

end outside of the intestinal ceca (Figs. 2, 3, 8, 9, 10). Three of

the American species have no lateral longitudinal folds of the

uterus outside of the intestinal ceca. These forms agree in hav-

ing a narrow post-acetabular region, so that the genital organs force

the intestinal ceca close to the sides of the body. Those species

in which lateral longitudinal folds are present have a distinctly

widened posterior body and considerable space between the re-

productive organs and the lateral margins. P. longiplexus in which

these folds reach furthest forward has a body distinctly widened

clear up to the pharynx. Since in their development the folds of

the uterus force their way into all available space between the

NORTH AMERICAN FROG LUNG FLUKES 211

organs, the course of the folds would depend to a considerable

extent on the size of the body and the size and shape of the organs.

In the development of the narrow forms there is no space outside

of the intestinal ceca for the posterior folds of the uterus to grow

into, altho one very large specimen of P. medioplexus showed con-

trary to the usual condition in this species short folds outside the

intestinal ceca.

Eces. The uteri in these forms are crowded throughout their

lengths with enormous numbers of small eggs. When fully de-

veloped they are dark brown and thick shelled. and when evacu-

ated contain an embryo in a very early stage of development.

Altho in some species the largest eggs may be as large as the

smallest or even the average for another species, the average sizes

of large numbers of eggs for each species show very distinct dif-

ferences (Fig. 1, 4, B, C, D, E, F). In giving the egg size for a

species both the range of variation and the average will be record-

ed. A considerable deviation in the egg size for the species P.

similiplexus was found in the eggs of three specimens from a sin- |

gle specimen of Rana pipiens from Oshkosh, Wisconsin. This

variation which has been already noted in another connection (Cort,

1915) is of interest in showing that there may be variations within

a species in a character which has up to this time proved so uni-

versally constant.

LirEe-History. Practically nothing is known of the life-his-

tory of the frog lung flukes. No account is to be found in the

literature of the intermediate host and the development of the

cercarie. Ssinitzin (1907:34) found stages of P. variegatus free

in the body cavity of both nymphs and adults of the damsel-fly,

Calopteryx virgo. By feeding experiments he was able to infect

frogs with these forms, showing that the insect here serves as

the transfer or secondary intermediate host. Stafford (1902)

gives some data on immature forms in frog lungs but makes no

attempt to follow out the development of organs. For the species

P. coloradensis I obtained from a frog’s lung a considerable series

of different ages which will be considered in connection with the

description of that species. A very immature specimen (Fig. 5)

suggests from the character of its excretory and digestive systems

that the cercariz of the lung flukes belong to the Xiphidiocercariz.

212 WILLIAM WALTER CORT

SYSTEMATIC SECTION

Pneumoneces Looss 1902

Syn. Hematolechus Looss 1899, preoccupied

Dracnosts. Body flattened, elongate, tapering anteriorly;

cuticula delicate, wholly or partially covered with spines, or

smooth; acetabulum smaller than oral sucker, sometimes very

small and of little functional value; digestive system with large

oral sucker, no prepharynx, good sized pharynx, short esophagus

and long intestinal ceca extending to the posterior end of the body ;

excretory vesicle Y shaped, with long median stem extending up

to the region of the ovary and short branches; genital pore in mid-

ventral line just back of the oral sucker; long cirrus sac extending

from the acetabulum to the genital pore and enclosing a long semi-

nal vesicle, a short ductus ejaculatorius surrounded by prostate

glands, and a short protrusible cirrus; ovary with or without lobes

just back of the acetabulum; testes round, oval to elongate, with

or without lobes, on opposite sides of the body, back of the ovary ;

Laurer’s canal absent; large seminal receptacle present; vitellaria

divided into groups of small acini arranged along the sides of the

body from in front of the acetabulum to back of the posterior

testis; uterus much coiled extending to the posterior end of the

body, in some species with longitudinal folds outside the intes-

tinal ceca; eggs very numerous, small with dark brown shells ;

habitat in the lungs of Anura.

Pneumoneces longiplexus Stafford 1905

Syn. Hematolechus longiplexus Stafford 1902

Dracnosis. Characters of genus; large thick species; cuticula

covered in all parts of the body with a dense coating of small

spines; oral sucker twice as large as acetabulum; ovary somewhat

lobed; testes long and narrow, the posterior extending almost to

the posterior end of the body; cirrus pear-shaped ; vitellaria of very

small acini, with from 16 to 26 in a group; uterus with longitudi-

nal folds outside the intestinal ceca extending to the pharynx; eggs

average 0.0248 mm. in length and 0.015 mm. in width; from lungs

of Rana catesbiana and R. pipiens.

NORTH AMERICAN FROG LUNG FLUKES 213

Description. Pneumoneces longiplexus was found by Staf-

ford (1902:901) to be very common in Rana catesbiana Shaw, in

Ontario, Quebec, New Brunswick, and Nova Scotia. I have found

this species infrequently in R. pipiens from near Chicago, Illinois.

In several hundred frogs from this region it occurred to the num-

ber of fifteen specimens in ten hosts. Also four specimens of

this species were found in a bull-frog, R. catesbiana, caught near

Urbana, Illinois, and others from the same host from North Jud-

son, Indiana.

The specimens of Pneuwmoneces longiplexus from Rana pi-

piens are on the average smaller than those described by Stafford

from the bull-frog. Their position in the frog’s lung was different

from that of any of the other lung flukes found. Instead of hang-

ing by the oral sucker they were coiled up among the folds of the

lining of the lung and pushed against the wall so that they were

visible externally. As soon as the infected lung was opened under

saline solution, they floated away without the tearing of tissue

necessary in the loosening of the other species. The freed worms

showed considerable power of movement but no locomotion. The

pre-acetabular region especially was capable of extension into a

long slender neck which was swayed backward and forward.

Well extended toto mounts of this worm are in the form of

an elongate ellipse (Fig. 10) with the anterior end bluntly nar-

rowed. The smallest of the mounted specimens was much dis-

tended with eggs and had a length of 2.64 mm. and a width of

1.17 mm. An averaged sized mount was 4.6 mm. long and 2 mm.

‘in width. Stafford gives the size of mounted specimens of this

species from the bull-frog as 7 to 8 mm. in length and 2 mm. in

width. He also notes one living specimen which measured up to

15 mm. in length. This species had a thickness greater than half.

its width. In one series cross sections measured in the pre-ace-

tabular region 0.93 mm. in width and 0.59 mm. in thickness, thru

the ovary 0.96 mm. in width and 0.54 mm. in thickness, and thru

the middle of the testicular region 0.86 mm. in width and 0.43 mm.

in thickness.

The cuticula in Pneumoneces longiplexus for the specimens

which I have examined is 0.008 to 0.01 mm. in thickness and is

214 WILLIAM WALTER CORT

covered in all regions of the body with a very dense coating of

spines, about 0.001 to 0.002 mm. apart and 0.004 to 0.006 mm. in -

length. The spines point backward and are set only part way thru

the cuticula (Fig. 4, A). Stafford’s account of the cuticula in

this species is entirely different from the description given above.

He writes (1902:902) of the cuticula of P. longiplexus:

“The cuticle in this species is thick and perfectly smooth,

there being no trace of spines either in the fresh worms or the

preserved sections.”” Whether this difference is due to a variation

within the species or an error in Stafford’s account is impossible

at the present writing to determine.

The ratio in size in this species of the oral sucker to the

pharynx varies from 2:1 to 5:3, and that of the oral sucker to the

acetabulum from 5:2 to 2:1. In a large mount 7 mm. in length

the oral sucker was 0.36 mm. in length and 0.42 mm. in width, the

pharynx 0.22 mm. in length by 0.18 mm. in width and the aceta-

bulum 0.17 mm. in diameter.

In P. longiplexus the ovary is just back of the limits of the

first third of the body length, and on account of the extreme length

of the testes the reproductive field fills practically all of the pos-

terior two-thirds of the worm (Fig. 10) The testes (Fig. 10, T)

are very characteristic. They are much elongated, and extend

almost to the ends of the intestinal ceca, with the one on the same

side as the ovary reaching slightly posteriad and being longer than

the other. They are irregular cylinders tapering to both ends and —

more than twice as long as wide. At their anterior ends they taper

for about two-fifths of their lengths to the tip from which the

vasa efferentia are given off, while at the posterior end they are

bluntly pointed. In a mount 4.4 mm. long the posterior testis ex-

tended posteriad 0.4 mm. farther than the anterior, within 0.54 mm.

of the posterior end of the animal. The posterior testis of this same

specimen was 1.26 mm. long by 0.32 mm. wide and the anterior was

1.08 mm. in length and 0.27 mm. in width. In a cross section

0.9 mm. wide by 0.54 mm. thick thru about the middle region of

the testes of another worm, the posterior testis measured 0.23 mm.

in width and 0.43 mm. in thickness and the anterior 0.20 mm. in

width by 0.36 mm. in thickness.

NORTH AMERICAN FROG LUNG FLUKES 215

The male ducts show characteristics of specific value in the

cirrus which when not protruded is a short thick pear-shaped

organ, and in the shortness of the ejaculatory duct (Fig. 12, C).

The ovary (Fig. 10, O) is a somewhat oblong body irregularly

lobed, lying a little to one side of the middle of the worm, with

its anterior end dorsad of the acetabulum. In a mount 6.8 mm.

long the ovary was situated 2.2 mm. from the anterior end with

its long axis slightly diagonal to the long axis of the animal, and

measured 0.85 mm. in length and 0.72 mm. in width. It is not

as thick as in the other species of lung flukes studied. In a cross

section thru its middle measuring 1.3 mm. in width and 0.63 mm.

in thickness the ovary measured 0.65 mm. in width by 0.22 mm.

in thickness.

Lying a little behind and ventrad of the ovary in almost the

exact center of the animal is the seminal receptacle (Fig. 10, SR),

a regularly oval organ, slightly elongated with its long axis coin-

ciding with the long axis of the worm. It is slightly larger than

the ovary and fills the space between it and the ventral body wall,

overlapping it for about three-fourths of its length. In a frontal

section thru the middle of a specimen 3.6 mm. in length the seminal

receptacle measured 0.51 mm. in length by 0.38 mm. in width, and

in a transverse section 0.98 mm. in width by 0.37 mm. in thickness

it measured 0.32 mm. in width by 0.27 mm. in thickness. The con-

nections of the female reproductive organs of P. longiplexus are

shown in figure 14.

The vitellaria (Fig. 10, Y) of this form are characterized by

the large number of acini in a group, from 16 to 26, and by the

small size of the individual acini, which in a specimen of medium

size varied from 0.064 mm. to 0.096 mm. in length and from

0.040 to 0.064 mm. in width. The vitellaria extend from a point

about half way between the anterior tip and the acetabulum to

within a short distance of the posterior extremity. There was

to be found considerable individual variation but this could not

be worked out in detail, because in most of the specimens avail-

able the vitellaria were hidden by great masses of eggs. Figure

10 gives an idea of their arrangement in one specimen.

216 WILLIAM WALTER CORT

The uterus (Fig. 10, U) passes forward from the odtype

around the anterior end of the ovary and then thru the region

back of this organ in a series of transverse folds. From just in

front of the testes it passes directly back between these organs

to the posterior end of the animal, where it makes two very long

and voluminous longitudinal folds outside of the intestinal ceca

up to the region of the pharynx. From the posterior end of the

animal it then passes forward in a series of median coils, more or

less voluminous depending on the quantity of eggs present, which

are ventral in position and for the most part transverse in direc-

tion. Toward its anterior end it narrows into a metraterm about

0.45 mm. in length (Fig. 12, M). The uterus is characterized by

its large caliber, the simpleness of its folding by the great length

of the lateral longitudinal coils outside the intestinal ceca and by

the general longitudinal direction of its coils.

Fully developed eggs in this species (Fig. 1, E) vary from

0.022 to 0.027 mm. in length and from 0.014 to 0.017 mm.

in width. The average of a large number of counts from several

different individuals was 0.0248 mm. in length by 0.015 mm. in

width.

The above account supplements Stafford’s original descrip-

tion of the species. Differences were found in the size of the

animals and the presence of spines in my specimens.

Pneumoneces breviplexus Stafford 1905

Syn. Hematolechus breviplexus Stafford 1902

- Dracnosts. Characters of the genus; largest American frog

lung fluke; thickness almost half width; cuticula thick, entirely

smooth ; oral sucker twice the size of acetabulum; ovary very deeply

lobed; testes elongate, usually lobed; acini of vitellaria 12 to 20

in a group; longitudinal folds of uterus outside intestinal ceca

reaching in front of posterior testis; eggs average 0.0225 mm.

in length by 0.0144 mm. in width; from lungs of Rana clamitans,

R. catesbiana and Bufo americanus.

Description. Stafford (1902:904) described P. breviplexus

from Canada from the lungs of the bull-frog, Rana catesbiana. He

found this species not nearly so common in this host as P. longi-

plexus and sometimes in the same host as that species. The first

NORTH AMERICAN FROG LUNG FLUKES 217

specimens of this species which came to my notice ~were from the

common toad, Bufo americanus, from Oklahoma. Later several

were taken from the lungs of specimens of Rana catesbiana and

R. clamitans from North Judson, Indiana.

This is the largest American representative of this genus, be-

ing approached only by Pneumoneces longiplexus. The largest

mount which I have is of one of the specimens from Bufo ameri-

canus which in a somewhat contracted condition measures 9.4 mm,

in length and 2.74 mm. in width. In a normal state of contraction

the body is spindle shaped, tapering slightly to the blunt posterior

end and with the pre-acetabular region somewhat more tapering

(Fig. 9). The smallest specimen of this species examined had the

uterus in complicated folds crowded with eggs. It had a length

of 5.8 mm. and a width of 1.3 mm. in the region of the anterior

testis. This mount was used in making the drawing for this species

since the structures were less obscured by the uterine coils than

in the other specimens mounted. The thickness was about equal

to half the width. In a series of cross sections the width at the

region half way between the anterior end and the acetabulum was

1.1 mm. and the thickness 0.7 mm.; thru the ovary the width was

1.7 mm. and the thickness 0.85 mm.; and in the region of the an-

terior testis the width was 1.8 mm. and the thickness 0.7 mm.

The ratio of the oral sucker to both the pharynx and the ace-

tabulum averages about 2:1. The actual size of these structures

is of little value in specific diagnosis since they are subject to so

much individual variation. In a specimen 9.4 mm. long by 1.4 mm.

wide the oral sucker had a transverse diameter of 0.28 mm. and the

pharynx 0.145 mm. while in the smallest specimen studied which

had a length of 5.8 mm. the oral sucker had a transverse diameter

of 0.4 mm. and the pharynx of 0.19 mm. The ratio was found in

all cases to vary but slightly and the size usually varied directly

with the size of the individuals.

The cuticula in all the specimens of this species which I have

examined is very thick and entirely without spines. In a place

where there is an average extension the cuticula is from 0.25 to

0.30 mm, in thickness. Stafford (1902 :904) writes of the cuticula

and spines of this species:

218 WILLIAM WALTER CORT

“The cuticule of this species is thick, but unlike the preceding

species (P. longiplexus) is beset with numerous backward pro-

jecting spines.” Either the presence of spines is a variable factor

or Stafford confused his observations.

In P. breviplexus the genital field, without the vitellaria takes

up about half the length of the body and extends to within about

one-fifth of the body length of the posterior end. The ovary (Fig.

9, O) is to one side of the median line and is very deeply lobed on

its lateral margin. This deep lobing of the ovary is in marked

contrast with the other species of the genus in which this organ

is unlobed or very slightly lobed. The seminal receptacle is an

unlobed structure ventrad of the ovary and about the same size

as that organ. The testes (Fig. 9, T) are elongate structures ir-

regularly lobed. Sometimes the lobing is along the median margin

and sometimes along the lateral margins. The testes overlap for

a part of their length. In the specimen 9.4 mm. long and 2.74 mm.

wide the ovary had a length of 1.57 mm. and a width of 0.92 mm.

The testes overlapped for the distance of 1 mm. and the posterior

extended within 2 mm. of the posterior end. The anterior had a

length of 1.71 mm. and a width of 1.03 mm. and the posterior had

a length of 2.31 mm. and a width of 1.1 mm.

The vitellaria in this species (Fig. 9 V) are very like those of

P. longiplexus. In a specimen of average size the acini measure

0.08 mm. to 0.12 mm. in length and 0.056 mm. to 0.096 mm. in

width. Since the size of the vitellaria varies with the size of the

animals these measurements are only of value in broad general

comparisons. The number of acini in a group varies from 12

to 20 with an average nurhber of 15 of 16. In a few specimens

in which all the vitellaria could be made out the number of groups

varied from 16 to 20. The number of individuals studied were too

few to make any statement of individual variations or average

conditions for these structures of much value. However the gen-

eral condition shown in Figure 9 held for the five individuals in

which these structures could be made out.

The folds of the uterus in P. breviplexus are large, as in the

previous species. From the odtype the uterus passes posteriad be-

tween the testes to the posterior end. From here long longitudi-

NORTH AMERICAN FROG LUNG FLUKES 219

nal folds extend outside the intestinal ceca. There is considerable

variation in the length of these folds apparently correlated with

the number of eggs present. In the individuals where they were

shortest they extended in front of the posterior testis and in one

case where the worm was much distended with eggs the fold on

the same side as the ovary extended to the middle of that struc-

ture and the one on the other side in front of its anterior margin.

This approaches rather closely to the condition found in P. longi-

plexus. Stafford must have made his description of this species

from a few individuals since he describes the longitudinal folds

of the uterus as short and from this character picks the specific

name breviplexus. From the posterior end the ascending part of

the uterus coils forward filling the available space between the

ovary and the testes. In front of the ovary it passes forward in

short transverse folds up to the genital pore, in very large speci-

mens forming a black mass in this region.

The uterus is for’ its whole length crowded with very small

eggs (Fig. 1, F). Measurements of fully developed perfect eggs

give a variation in length from 0.0205 mm. to 0.026 mm. and in

width from 0.013 mm. to 0.016 mm. with an average length of

0.0225 mm. and a width of 0.0144 mm. This is the smallest egg

size recorded for any of the species of Pneumoneces.

P. longiplexus and P. breviplerus are very closely related.

They are the largest and heaviest of the frog lung flukes. Both

show lobing of the ovary, altho that of P. breviplexus is much more

pronounced, and both have elongate testes. The vitellaria of both

show similar arrangement and have small numerous acini in the

groups. Further the lateral longitudinal folds of the uterus outside

the intestinal ceca are longer in these two species than in any others

of the genus and the eggs are very nearly the same size, being the

smallest for the genus. Slight differences between the species show

in almost every part. The most useful for distinguishing them are

the differences found in body shape, length of the lateral longitudi-

nal folds of the uterus and shape of the ovary and testes.

Pneumoneces similiplexus Stafford 1905

Syn. Hematolechus similiplexus Stafford 1902

220 WILLIAM WALTER CORT

Dracnosis. Characters of genus; cuticula with scattered

spines which thin out back of acetabulum and are entirely absent

back of the posterior testis; ratio of oral sucker to acetabulum

4:3; ovary and testes without lobes, small round or oval; acini of

vitellaria large with 6 to 13 in a group; longitudinal folds of uterus

outside of intestinal ceca reaching beyond posterior testis; eggs

average 0.0376 mm. in length by 0.0184 mm. in width; in lungs of

Rana pipiens and Bufo americanus.

Description. The original description of Pneumonaces sim-

iliplexus is by Stafford (1902:907). He reports this species from

Canada from the lungs Rana virescens Kalm and from Bufo lenti-

gmosus Shaw. I have found it only in Rana pipiens, sometimes

occurring with P. medioplexus, but never as common as that form.

This fluke (Fig. 3) is spindle shaped, widest just back of the mid-

dle of the body and tapering gradually toward both ends, with the

pre-acetabular region somewhat long and narrow. Stafford notes

that this species is more inclined to be cylindrical than the other

species he studied of this genus. However, all the specimens which

I have examined are distinctly flattened, having a thickness less

than one-half the width. In the size of sexually mature individuals

there is a great variation. In the smallest worms examined the

folds of the uterus were crowded with fully developed eggs. One

of these specimens measured only 1.8 mm. in length and 0.67 mm.

in width at the region of the testes. One of the largest mounts

measured 5.8 mm. in length by 1.96 mm. in greatest width. Figure 3

shows an individual of about medium size. Measurements of a series

of cross sections show the relation between width and thickness. A

cross section thru the middle of the pre-acetabular region had a

width of 0.94 mm. and a thickness of 0.48 mm.; at the acetabulum

the width was 1.1 mm. and the thickness 0.48 mm.; at the anterior

testis the width was 1.22 mm. and the thickness 0.54 mm.; at the

posterior testis the width was 1.25 mm. and the thickness 0.54 mm.;

and back of the posterior testis the width was 1.1 mm. and the

thickness 0.44 mm.

The acetabulum in this species is located about one-third or

one-fourth of the distance from the anterior to the posterior end.

The ratio of the oral sucker to the acetabulum averages 4:3 and the

NORTH AMERICAN FROG LUNG FLUKES 221

oral sucker to the pharynx about 2:1. The ratio in specimens of

different sizes varies but little altho the actual size of the suckers

varies with the size of the animal.

The cuticula of P. similiplexus has a thickness of 0.007 to

0.012 mm. The region in front of the posterior testis is set with

small spines which vary from 0.009 to 0.013 mm. in length (Fig.

4, B). These spines are more numerous around the anterior tip

and become very scattered back of the acetabulum, and in the

region back of the posterior testis the cuticula is entirely smooth.

The above description differs considerably from Stafford’s account

(1902 :907) of the cuticula and spines in this species. His de-

scription is as follows:

“The cuticule in preserved specimens is about 0.018 mm. thick

and is regularly and thickly beset with spines, about 0.022 mm. in

length, leaning backward with a slight curvature and extending

thru the whole thickness of the cuticula, with the points project-

ing beyond. Viewed from the surface where one can see

great numbers of them together, they appear to be in longitudinal

and transverse rows, often 0.015 mm. to 0.02 mm. apart, but

sometimes even less, or more, depending .on the region and the

state of contraction of the animal. As is common they are most

abundant at the anterior end.”

The genital field (Fig. 3) not including the vitellaria occupies

about the third and fourth fifths of the body length. The ovary

and testes are round or oval, small compact unlobed structures,

which are definitely separated from each other. The small size,

regular outline and position of these organs are characteristic of

the species. The seminal receptacle is an oval structure ventrad

of the ovary in the median line of the worm and overlapping for

part if its length the posterior portion of the ovary. In a small

sexually mature specimen 1.9 mm. long the ovary measured 0.29

mm. in length by 0.21 mm. in width, the anterior testis 0.34 mm.

in length and 0.34 mm. in width and the posterior testis 0.46 mm.

in length by 0.34 mm. in width. In a very large specimen 6.4 mm.

long the ovary measured 0.46 mm. in length by 0.43 mm. in width,

the anterior testis 0.48 mm. in length by 0.45 mm. in width, and the

posterior testis 0.56 mm. in length and 0.58 mm. in width. The

222 WILLIAM WALTER CORT

posterior testis was in every case larger than the anterior. While

the ovary and testes were actually larger in the larger specimens

than in the smaller, in proportion to the size of the animal they

were always larger in the small individuals. In a section thru the

ovary 1.1 mm. wide and 0.54 mm. thick the ovary was 0.26 mm.

wide and 0.26 mm. thick. The testes fill almost the entire thick-

ness of the posterior body region. In a cross section thru the

posterior testis 1 mm. wide by 0.37 mm. thick the testis had a thick-

ness of 0.33 mm.

The vitellaria (Fig. 3) in P. similiplexus are characterized by

the large size of the acini and the small number in a group. In

average sized specimens the acini have a length varying from

0.096 to 0.14 mm, and a width from 0.06 to 0.1 mm. The number

in a group varies from 6 to 13 with the average number about 10.

The groups (Fig. 3, Y) extend from about half way between the

anterior end and the acetabulum to within a short distance of the

posterior tip. There are always at least two groups behind the

posterior testis between the intestinal ceca, and two or three median

groups at the anterior end. In five specimens where the complete

arrangement could be made out there was found to be consider-

able variation. The total number of groups varied from 16 to 19,

with six or seven on a side beside the median groups. Stafford

notes that the vitellaria in this species extend to the ends of the

intestinal ceca. His drawing however shows them extending only

to the region of the posterior testis. Klein (1905 :64) at the end

of his description of a new species of this genus, sums up the spe-

cific differences of all the species of the genus Pneumoneces in

a table for comparison. As one characteristic of P. similiplexus

in order to bring it into close relation to P. similis Looss he gives

the following:

“Dotterstocke reichen nicht bis zum hindern Rand des hindern

Hodens ;” but he adds this note:

“Stafford gibt in seiner Arbeit 1902 an, dass die Dotterstdcke

bei Pneumoneces similiplexus bis ins Hinderende des Korpers,

jedenfalls bis zum Ende der Darmschenkel reichen; diese Bemer-

kung stimmt jedoch nicht mit seiner eingnen Abbildung iiberein,

da letzere gleiches Verhalten zeigt wie bei Pneumoneces similis.”

NORTH AMERICAN FROG LUNG FLUKES 223

That Klein should have chosen this characteristic from Staf-

ford’s description rather than his drawing is shown by the extent

of the vitellaria in the specimens of this species which I have

examined.

The folding of the uterus is characteristic. From its beginning

in the shell gland the uterus passes for a short distance forward,

and then goes to the right side and loops down in a slightly dorsad

position to the region back of the posterior testis where it fills the

space between the intestinal ceca. From the posterior end of the

body it forms two longitudinal folds outside the intestinal ceca,

which reach beyond the anterior margin of the posterior testis. The

ascending part of the uterus passes forward ventrad of the ‘descend-

ing loops and between the testes to the right side of the body

where it runs longitudinally for a short distance. Next it passes

to the left side just behind the ovary dorsad of the acetabulum and

fills with short transverse folds the region between the intestinal

ceca up to the genital pore. The above description holds good for

those individuals in which the ovary is on the right side of the

body. The course of the uterus is exactly reversed when the ovary

is to the left.

The eggs of this species are the largest of the American species

of the genus (Fig. 1, A). Fully developed eggs vary from 0.034

to 0.04 mm. in length and from 0.017 to 0.21 mm. in width with an

average of 0.0376 mm. in length and 0.0184 mm. in width.

Pneumoneces medioplexus Stafford 1905

Syn. Hematolechus medioplexus Stafford 1902

Ostiolum formosum Pratt 1903

Diacnosts. Characters of genus: long slender worms; cuti-

cula covered with small spines; ratio of oral sucker to acetabulum

greater than 4:1; ovary and testes unlobed ; acini of vitellaria large

with from 6 to 13 in a group; no longitudinal folds of uterus out-

side intestinal ceca; eggs average 0.032 mm. in length and 0.0186

mm, in width; in lungs of Rana pipiens.

Description. P. medioplexus was first reported from Canada

by Stafford (1902:908) from the lungs of Rana virescens Kalm.

One year later the same form was described by Pratt (1903 :34) as

Ostiolum formosum. This species has proven to be by far the

224 WILLIAM WALTER CORT

most common fluke in the lungs of specimens of Rana pipiens —

which I have examined. I should expect to find it widely distrib-

uted throughout the range of Rana pipiens. Infection is some-

times very heavy, records showing as many as thirty or forty

flukes in one frog. The worms are firmly attached by the oral

sucker to the walls of the lung, and their bodies hang free in the

cavities. They are very active either when attached or freed, show-

ing great power of extension and contraction of the body, and a

very, free movement of the pre-acetabular region.

P. medioplexus is an elongate slender worm (Fig. 2) widest

at the region of the anterior testis. One of the largest mounts

had a length of 7.8 mm. and a width at the region of the anterior

testis of 1.2 mm. From the ovary back to the limit of the pos-

terior testis the width is about uniform, the pre-acetabular region is

considerably attenuated and the region back of the posterior testis

narrows to a bluntly pointed posterior tip. The smallest sexually

mature specimen examined had a length of 3.9 mm. and a greatest

width of 0.59 mm. Stafford’s notes that living specimens of this

species may reach a length of 15 or 16 mm.

This worm is distinctly flattened, the thickness being on the

average about two-fifths of the width. Measurements of a series

of cross sections of a single worm show this relation. In a sec-

tion in the middle of the pre-acetabular region the width was 0.66

mm. and the thickness 0.33 mm.; at the acetabulum the width

was 0.93 mm. and the thickness 0.38 mm.; at the middle of the

ovary the width was 0.94 mm. and the thickness 0.42 mm.; thru

the middle of the anterior testis the width was 1.1 mm. and the

thickness 0.45 mm.; thru the posterior testis the width was 1.12

mm. and the thickness 0.46 mm.; and half way between the pos-

terior testis and the posterior end of the body the width was 0,94 ©

mm. and the thickness 0.38 mm.

The genital field without the vitellaria occupies a little more

than the second third of the body length.

The acetabulum is separated by quite a little distance from the

anterior margin of the ovary and is in front of the first third of

the body length. It is very small being only about one-fourth

or one-fifth the size of the oral sucker. Ina specimen in which the

. a

NORTH AMERICAN FROG LUNG FLUKES 225

oral sucker measured 0.39 mm. by 0.37 mm. the acetabulum had

a diameter of 0.08 mm. The pharynx is about three-fourths the

size of the oral sucker. In a specimen 7.8 mm. in length the oral

sucker had a length of 0.40 mm. and a width of 0.35 mm. and the

pharynx had a length of 0.29 mm. and a width of 0.26 mm.

The cuticula of this species measures on the average 0.007 mm.

in thickness and is densely covered with a thick coating of spines

about 0.01 mm. in length (Fig. 4, C). Stafford very aptly describes

them as looking under low power “like a dense coat of short hair.”

They cover the entire surface of the body and thin out but little

toward the posterior end. Pratt (1903:36) makes the absence of

spines an important diagnostic character of Ostiolum formosum in

fact using it as one of the distinguishing characters of his new

genus. The examination of his type specimens showed that the

cuticula was entirely sloughed off. This would account for the

absence of spines in his description. 4

The testes (Fig. 2, T) are unlobed round or oval with some-

what squared corners and separated sometimes by a distance equal

to half the length of one of them. In the large specimen previously

mentioned, 7.8 mm. in length the anterior testis is 0.56 mm. long and

0.64 mm. wide and the posterior testis has a length of 0.64 mm.

and a width of 0.6 mm. The thickness of the testes is but little

less than their width and is practically equal to the thickness of

the body. In a cross section thru the middle of the anterior testis

1.1 mm. wide by 0.41 mm. thick the testis had a width of 0.45 mm.

and a thickness of 0.36 mm. The relations of the male ducts in

this species show little of specific value. Figure 11 shows the

ends of both the male and female ducts.

The ovary (Fig. 2, O) is elongate oval, lying a little distance

back of the acetabulum either to one side or the other of the body,

with its long axis diagonal to the long axis of the worm and its an-

terior end toward the midline of the body. The shape of the ovary

varies considerably and in several cases it was slightly lobed. In the

same specimen for which the measurements of the testes were given

the ovary had a length of 0.56 mm. and a width of 0.28 mm. This

organ has a thickness equal to its width. In a cross section through

the middle of the ovary 0.96 mm. wide by 0.40 mm. thick the ovary

226 WILLIAM WALTER CORT

had a width of 0.29 mm. and a thickness of 0.30 mm. In about half

the specimens examined the ovary was to the right side, in the

other half it was to the left. The seminal receptacle is larger than

the ovary and lies in the midline of the body posterior and dorsad

of the ovary and overlapping it for about half its length. The rela-

tions of the ducts of the female organs are shown in Figure 13 and

their characters are of generic rather than specific value.

The acini of the vitellaria (Fig. 2, Y) in P. medioplexus are

large, about the same size as those of P. similiplexus, and there

are from 6 to 13 in a group. The groups extend from a point

about half way between the anterior tip and ovary to the region

back of the posterior testis. It was possible to work out in detail

the arrangement of the vitelleria in twelve specimens. Altho con-

siderable variation was found in the number of groups and their

position, in all specimens the general grouping was the same.

Most of the groups are located in lateral lines along the sides of

the body. The most anterior of the groups in each line were al-

ways about on a level. The line on the same side as the ovary was

the shorter, extending only to the posterior limit of the anterior

testis. The longer line on the opposite side always extended be-

yond the posterior testis with usually a median group connected

with the last two posterior groups to form a triangle (Fig. 2, Y).

One or two median groups were always present in the pre-ovarian

field. In the twelve specimens studied the total number of vitellar-

ian groups varied from 18 to 23. The variations in arrangement

are to be found in the number in each lateral line and in the num-

ber of the median groups. The following table gives the details

of arrangement for the twelve specimens.

te ote Oe. Or een ea ee are ORE i oe

FPotal number. -2sr. aii te are. 18°20 20 2b} 22 2b eae ee eee ie eee

Number on Long Side....... See Ss 10 2 11 10b 8 10 MIs oa

Number on Short Side...... 6518 iG10.8 Bo: B28 ec CA oe i Bae ease

Number of Anterior Median

RSP 935 yt on cn eae Rn 7 ieke eee es tne ERO ae Ie ae 9 |

Number of Posterior Median

SrOups? LLCs eee ewe eee 1 ah aS Cease Rm hocks cout bet) PERS PLA oY

There is some question in regard to the value of the character-

istics of the vitellaria of the trematodes for specific diagnosis.

ee ee a

NORTH AMERICAN FROG LUNG FLUKES 227

Barker (1907) finds considerable variation in these structures

among certain species of the genus Opisthorchis. I have insuffi-

cient material for any detailed analysis of the vitellaria for the

genus Pneumoneces. In the case of P. medioplexus where the

largest number of specimens could be studied in detail the varia-

tions of the vitellaria fall within narrow limits. The general size

of the acini, their number in a group and the number of groups

are items which, though somewhat variable, yield definite points of

specific difference. It may be suggested that the characters of the

vitellaria of a trematode must not be given too much weight in the

differentiation of species unless the variations have been worked

out in a number of individuals.

The arrangement of the folds of the uterus of this species is

very characteristic (Fig. 2, U). The uterus at its beginning runs

ventrad for a short distance and then courses backward in short

transverse folds between the testes, and down one side of the post-

testicular region and back the other, filling all the space inside of

the intestinal ceca. From the posterior limit of the testes the as-

cending folds pass between these organs ventrad of the descend-

ing folds, then forward ventrad of the seminal receptacle and ovary

and up to the genital pore, filling the pre-ovarian space in the larger

animals with short transverse folds. The general course of the

folds is transverse and at the posterior end there are no longitudinal

folds outside of the intestinal ceca. In two very large specimens

examined there was a variation in this respect, for on each side in

these specimens very short longitudinal folds extended outside of

the intestinal ceca. These specimens showed the greatest mass of

eggs and the most complicated coils of any of the specimens of this

species examined. This variation emphasizes the tendency of the

uterus in its development to crowd into every available space. The

smallest specimen of this species examined was not half the length

of the largest individuals but had mature eggs in the uterus up to

the genital pore. In this individual the general course of the uterus

was like that described above, but the transverse folds were shorter

and much less complex. |

The measurements of over two hundred eggs from several

different individuals give a variation in length from 0.022 to 0.029

228 WILLIAM WALTER CORT

mm, and a range in width from 0.013 to 0.017 mm. The average

length was found to be 0.0255 mm. and the average width

0.015 mm. (Fig. 1, D). Stafford gives the size of the eggs in this

species as 0.028 mm. by 0.018 mm, while Pratt gives the measure-

ments of the eggs for his Ostiolum formosum as 0.039 mm. by

0.017 mm. This measurement proved to be an error since in the

type specimen of O. formosum which I examined the size of the

eggs was found to fall within the limits for the species as given

above. Pratt has since re-measured the eggs for O. formosum

with the same results. He ascribes his original mistake to a con-

fusion of notes. .

The study of Pratt’s type specimen of O. formosum establishes

its identity with the species Pneumonaces medioplexus. Since

Stafford has priority his specific name stands. There remains to

be considered the further question of whether this species should

be separated from the genus Pnewmoneces and be made the basis

of a new genus as Pratt has done. At the time of Pratt’s descrip-

tion of this form he knew only of the three European species of

the genus Pneumoneces. The increased knowledge of these forms

in my opinion invalidates Pratt’s genus Ostiolum and forces us to

include the form which he would place in this genus in the genus

Pneumoneces. Pratt separates the new genus Ostiolum from

Pneumoneces on the basis of the following characteristics. “It

[Ostiolum formosum] differs principally from the genus Hem-

atolechus |Pneumone ces] in the position of the acetabulum, which

is further forward.than in that genus, the size of the testes, which

are much smaller than in Hematolechus, in the arrangement of the

uterine folds, which have a general longitudinal direction in Hem-

atolechus, and in the length of the excretory vesicle which extends

much further forward than in Hematolechus. In that genus also

the worms are also often covered with. spines, while in Ostiolum

these structures did not appear in any of the specimens examined

by me.”

An analysis of the above characters shows that if they are to

be made the basis of generic distinction, the genus Pneumonaces

would need to be split into three or four different genera, which

would be absurd in view of the fundamental resemblances. The

NORTH AMERICAN FROG LUNG FLUKES 229

position of the acetabulum varies slightly in the different species of

this genus depending on the amount of attenuation of the anterior

end. Variations in the size and the shape of the testes are more

properly specific than generic differences. The arrangement of the

coils of the uterus seems to depend on the size and shape of the

organs and the shape of the body since it grows into all the avail-

able space between the reproductive organs. The difference in the

length of the median stem and branches of the excretory vesicle is

not as great as Pratt’s emphasis on that point would suggest and

seems to depend on the length and amount of attenuation of the

body. In his description of the excretory vesicle of Ostiolum for-

mosum Pratt (1903:35) states that the median portion is close to

the ventral side of the animal and that the branches are dorsad of

the intestinal ceca. The fact is that in all the species of the genus

Pneumonceces the median stem of the excretory vesicle is near the

dorsal body wall and the branches ventrad of the intestinal ceca,

and in Pratt’s type specimen mentioned several times already there

was no variation from the usual condition. The final point of

difference made by Pratt in regard to the absence of spines has

been shown to be based on an error. But at any rate since in the

different species of the genus Pneumonecces there is great variation

in the character of the spines the distribution of these structures in

this genus must be considered to be of specific rather than generic

value.

Pneumoneces complexus Seeley 1906

Dracnosis. Characters of the genus; cuticula entirely with-

out spines; ratio of oral sucker to acetabulum about 4:3; ovary and

testes irregular in shape and may be slightly lobed ; acini of vitellaria

large with 12 to 17 in a group and four to six median groups in

front of acetabulum; average size of eggs 0.032 by 0.018 mm.;

no longitudinal folds of uterus outside of intestinal ceca; habitat

lungs of Rana pipiens. -

DescripTION. Seeley (1906:248) described Pneumoneces

complexus from the mouth of Rana pipiens from North Carolina.

Since that time I have obtained several specimens of this species

from the lungs of Rana pipiens from Raleigh, North Carolina.

These specimens were very much distorted and crowded with eggs

230 WILLIAM WALTER CORT

so that the internal organs were obscured and so preserved that it

proved impossible to section any of them. However enough can be

made out to add several points to Seeley’s description, correct some

errors and to give this form a place as a distinct species of the

genus Pueumoneces. I will be glad to obtain better material so

that the description can be carried further. 3

The presence of the specimens found by Seeley in the mouth

of the host was without doubt an abnormal circumstance due to the

fact that the frogs had been killed with chloroform, giving the

worms a chance to migrate from the lungs. The normal shape

cannot be determined from the distorted specimens at my disposal.

The largest of my specimens had a length of 5.2 mm. and a width

of 2 mm. Seeley’s specimens were not so contracted. He gives

measurements of 5.8 mm. in length, 1.7 mm. in width and 0.71 mm.

in thickness. In shape he records them as being widest just in

front of the middle of the body, tapering slightly to a blunt posterior

end and rapidly toward the anterior end. The oral sucker is about

twice the size of the pharynx, and bears a ratio to the acetabulum

of 4:3. In the specimen 5.2 mm. long the oral sucker had a length

of 0.39 mm. and a width of 0.38 mm., the pharynx a length of 0.22

and a width of 0.21 mm., and the acetabulum a diameter of 0.31 mm.

The cuticula in this species is entirely smooth. The ovary and testes

are rather irregular in shape and slightly lobed. The testes over-

lap for part of their lengths. |

The vitellaria have from 12 to 17 acini in a group and the

acini themselves have a length in average sized individuals of 0.08

to 0.14 mm. and a width varying from 0.056 mm. to 0.096 mm.

The groups reach from just a little way back of the pharynx to

near the posterior end. They show a greater massing of groups in

the anterior end than in any of the other species studied, there be-

ing 9 or 10 groups in front of the ovary, 4 to 6 of which are

median. The total number of groups in two specimens in which

they could be completely made out was 22.and 23.

The uterus in this species has no lateral longitudinal folds

outside the intestinal ceca.

NORTH AMERICAN FROG LUNG FLUKES 231

The eggs of P. complexus (Fig. 1, C) have a length varying

from 0.030 to 0.035 mm. and a width from 0.017 to 0.020 mm. The

average length is 0.032 mm. and the average width is 0.0186 mm.

Strange to say, Seeley makes no special mention of three rather

important characteristics of P. complexus as described by him.

According to his description and figure (Seeley, 1906:248) this

worm has no cirrus or cirrus sac, the genital pore to one side of

the median line and the yolk glands on the ventral side of the body.

Since Seeley seems to ascribe no particular value to the differences

noted above, since he very evidently had insufficient and poor ma-

terial and since his paper gives evidence of superficial observation

and lack of grasp of the subject, I should be inclined to ascribe

these differences to mistakes in observation. The specimens I have

examined of this species are like the others of the genus in these

regards.

Pneumoneces coloradensis n. sp.

Dracnosis. Characters of genus; elongate slender worms;

thickness greater than half width; spines present only on anterior

tip; ratio of oral sucker to acetabulum 5:4; field of genital glands,

without vitellaria, less than one-third body length; ovary and testes

round to oval to slightly irregular, but unlobed; testicular zones

usually abut; groups of vitellaria in front of acetabulum few; no

longitudinal folds of uterus outside intestinal ceca; eggs average

0.0344 mm. in length and 0.0195 mm. in width; habitat lungs of

Rana pipiens.

Description. Pneumoneces. coloradensis n. sp. was first

brought to my attention by a mounted specimen given me in Sep-

tember 1913 by Dr. M. C. Hall from material collected from

Boulder, Colorado. Later the same fall I came upon this species

again in the lungs of specimens of Rana pipiens from a small ditch

east of Colorado Springs, Colorado. At this time in all forty-seven

specimens were obtained from nine out of seventeen hosts examined,

In the summer of 1914 further examination was made of the frogs

from the region of Colorado Springs, § owing that.in Rana pipiens

this new species was comparatively common. “Infact it is the only

Zz,

frog lung fluke found in this region. In one of the frogs examined

in the late summer of 1914 fifteen flukes of this species were found,

232 WILLIAM WALTER CORT

showing gradations in size from a little beyond the cercarial stage

to very large adults. As in other species of the genus there is con-

siderable variation in the size of sexually mature individuals, the

largest being as much as twice the length of the smallest. The

largest mount examined measured 8.1 mm. in length and 1.55 mm.

in width, while the specimen shown in figure 8 with a fully devel-

oped uterus and mature eggs up to the genital pore, measured

3.3 mm. in length and 0.54 mm. in width. In shape this species is

most like P. medioplexus, being narrow for its length but tapering

less toward the ends. The region from the anterior groups of the

vitellaria almost to the posterior end has a nearly uniform width.

The thickness of the body is-greater than half the width in every

region except back of the posterior testis. A series of sections of

a medium sized worm gave the following measurements: a section

through the acetabulum was 0.7 mm. wide and 0.49 mm. thick;

one through the region half way between the oral sucker and the

acetabulum was 0.56 mm. wide and 0.48 mm. thick; through the

middle of the anterior testis the width was 0.77 mm. and the thick-

ness 0.48 mm.; through the middle of the posterior testis the width

was 0.8 mm. and the thickness 0.5 mm.; and a section through the

region half way between the posterior testis and the posterior end

had a width of 0.64 mm. and a thickness of 0.3 mm.

The average ratio of the oral sucker to the pharynx is 10:7

and to the acetabulum 5:4. The ratio in both cases is quite con-

stant. The ratio of the oral sucker to the pharynx varies from 8:5

to 5:4 and that of the oral sucker to the acetabulum from 7:6 to

4:3. The size of the suckers and the pharynx in sexually mature

- worms varies directly with the size of the worms, and there was no

relation between the variations in size and of ratio of these struc-

tures.

The cuticula in P. coloradensis is smooth except at the very

anterior tip in the region over the oral sucker and the pharynx.

The spines (Fig. 4, D) covering this region are thickly set over the

oral sucker and thin out rapidly backward, only a few scattered —

spines showing back of the pharynx. These spines are the largest

for any of the species studied, averaging between 0.009 and 0.013

if °

ee ee

‘aie |

NORTH AMERICAN FROG LUNG FLUKES 233

mm. in length and from 0.003 to 0.005 mm. in diameter at their

bases. They point strongly backward and have their points curved

backward.

The ovary (Fig. 8, O) of this species is just behind the acetabu-

lum which if further back than in any of the other forms studied,

being just behind the limit of the first third of the body length.

The field of the genital organs, without the vitellaria, is charac-

terized in being further back and shorter than in any of the other

American representatives of this genus. The distance from the

anterior margin of the ovary to the posterior margin of the posterior

testis is less than one-third of the total body length.

The ovary is a round or oval unlobed organ which lies just be-

hind the acetabulum to one side or the other of the body. Ventrad

of the ovary but somewhat posteriad and overlapping for about

one-half its length is the seminal receptacle, which is a round or

oval organ lying in the midline of the body, and a little larger than

the ovary. The testes (Fig. 8, T) are round or oval to almost

rectangular structures—close together with the two testicular zones

usually abutting. They occupy approximately the fourth fifth of

the body length and are only a little to the side of the median line.

This position as in P. medioplexus depends on the narrowness of

the body in this region. The posterior testis is the larger of the

two. The size of the reproductive glands varies directly with the

size of the individuals and will be given for two different specimens.

In the mount 8.1 mm. in length the ovary had a length of 0.49 mm.

and a width of 0.42 mm., the anterior testis was 0.64 mm. in length

and 0.60 mm. in width and the posterior testis 0.74 mm. in length

and 0.86 mm. in width. In a much smaller specimen 4.4 mm. in

length the ovary had a length of 0.32 mm. and a width of 0.22 mm.,

the seminal receptacle had a length of 0.32 mm. and a width of

0.24 mm., the anterior testis had a length of 0.42 mm. and a width

of 0.46 mm., and the posterior testis had a length of 0.46 mm. and

a width of 0.44 mm. The testes have a thickness but little less than

their width and almost equal to the thickness of the body at the

testicular region. In a cross section thru the middle of the

posterior testis having a width of 0.9 mm. and a thickness of 0.48

mm. the testis was 0.55 mm. in width and 0.46 mm. in thickness.

234 WILLIAM WALTER CORT

The arrangement of the ducts of the reproductive organs is similar

to that of P. medioplexus and offers no points of importance for

specific diagnosis.

The vitellaria are almost entirely limited to the lateral zones,

there being at most only one median anterior group and one or two

posterior median groups which are always considerably to one side

of the median line. The acini have about the same average size

as those of P. medioplexus and there are from 8 to 14 ina group.

The coiling of the uterus is similar to that of the other narrow

forms, P. medioplexus and P. complexus, there being no longitudi-

nal folds outside of the intestinal ceca and the direction of the folds

being transverse.

The eggs vary in length from 0.032 to 0.039 mm. and in width

from 0.018 to 0.021 mm., with an average length of 0.0344 mm. and

an average width of 0.0195 mm.

A considerable series of immature forms of this species was

taken from the lungs of the frogs. The youngest specimen found

(Fig. 5) which is by far the youngest specimen of the genus ever

reported, was found with a number of other specimens of the same

species in the lung of a specimen of Rana pipiens. There is no

question in my mind that this specimen belongs to the species Pneu-

moneces coloradensis since it was found with immature and ma-

ture individuals of this species, and no other frog lung fluke has

been found near Colorado Springs where this was collected. Since

this immature form gives some hint of the character of the cercaria

of this species an extended description of its structure will be given.

This specimen (Fig. 5) has a length of 0.4 mm. and a width

of 0.15 mm. The acetabulum is back of the center of the body and

has a diameter of 0.063 mm. The mouth opening is slightly sub-

terminal. The oral sucker has a length of 0.079 mm. and a width

of 0.074 mm. and the pharynx has a length of 0.0496 mm. and a

width of 0.039 mm. It is interesting to note that the ratios of the

oral sucker to the acetabulum and pharynx fall within the limits of

the ratios found for these structures in the adults. The esophagus

is very short and in the figure entirely contracted behind the

pharynx. The intestinal ceca run almost to the end of the body of

the animal and contain blood, showing that the worm had been in

NORTH AMERICAN FROG LUNG FLUKES 235

the frog’s lung long enough to be feeding on the blood. The ex-

cretory pore is at the middle of the posterior tip. It connects with

the main part of the excretory bladder by a short narrow tube, 0.016

mm. in length. The bladder consists of the wide median part and

two wide lateral divisions extending up to the region of the oral

sucker dorsad of the intestinal ceca. In the living animal all parts

of this bladder were crowded with highly refractive concretions like

those which gather in the excretory systems of cercarie. This is

the only immature stage in which I found those concretions. The

excretory bladder resembled very closely the condition found in

encysted Xiphidiocercarize and this character together with the

structure of the digestive system suggests that the cercarie of the

species of the genus Pnewmonecces belong to this group. The loco-

motion of this immature specimen was watched on a substratum.

With the acetabulum firmly attached the anterior end would stretch

forward and the oral sucker would take hold. The acetabulum was

then loosened and the body contracted. The acetabulum then took

hold and the movement was repeated. By a rapid series of these

movements this larval form could make considerable progress. This

movement is exactly like that of a cercaria on a substratum. Dor-

sad of the acetabulum are irregular masses of nuclei which are

probably the anlage of the reproductive organs. In the drawing

(Fig. 5) they are hidden by the acetabulum.

Several larger immature specimens of this species were avail-

able for study. The form shown in figure 6 shows considerable in-

crease in size over that shown in figure 5. The intestinal ceca are

much distended with frog’s blood hiding the excretory vesicle. The

anlage of the reproductive system shows definite differentiation into

organs. In a larger form (Fig. 7) which appears about ready to

produce eggs, but having none in the uterus, the organs are fully

developed and differ only in size from those of the sexually mature

specimens. The folds of the uterus in this specimen are much less

complicated than in those individuals where a quantity of eggs is

present.

P. coloradensis is most closely related to P. complexus. It has

the same shape of the body and general arrangement of the uterus

as that species. The size of the eggs and the general relations of

236 WILLIAM WALTER CORT

the reproductive organs are more like that species than any other.

Our slight knowledge of P. complexus makes comparison of the

two species in certain characters impossible. Enough is known

however to make sure their distinctness. Differences between these

two species are noted in the ratios of the oral sucker to the acetabu-

lum and pharynx, and in the size of the eggs. P. complexus is en-

tirely without spines, while P. coloradensis has spines at the an-

terior tip. The most noticeable difference is in the arrangement of

the vitellaria. In P. complexus there are eight to ten groups of

vitellaria in front of the acetabulum of which four to six are

median groups, while in P. coloradensis there are not more than

five groups in front of the acetabulum and in none of the specimens

examined was there more than one median group.

a> tS aa » —

NORTH AMERICAN -FROG LUNG FLUKES 237

KEY TO THE AMERICAN SPECIES OF PNEUMONCECES

1(2) Longitudinal folds of the uterus present outside the intestinal ceca

reaching at least in front of the posterior testis..........+-+.+..- 3

2(1) No longitudinal folds of the uterus present outside the intestinal

FE a Wee IRE A NA, pA AAD IE ei tea a eR. ceagto elae F RR eh 7

3(4) Testes elongate, length at least twice width...........-...se+eeees 5

4(3) Testes round or oval, length but little greater than width...........

Pneumoneces similiplexus Staf.

5(6) Longitudinal folds of the uterus outside the intestinal ceca reaching

to the pharynx.

Pneumoneces longipplexus Staf.

6(5) Longitudinal folds of the uterus outside the intestinal ceca never

reaching in front of ovary.

Pneumoneces breviplexus Staf.

7(8) Acetabulum very small, not one-fourth size of oral sucker.

Pneumoneces medioplexus Staf.

8(7) Acetabulum three-fourths size of oral sucker.............-.++.+5 9

9(10) Not more than two median groups of the vitellaria in front of the

acetabulum.

Pneumoneces coloradensis Cort.

10(9) Four to six median groups of the vitellaria in front of the acetab-

ulum.

Pneumoneeces complexus Seeley

238 WILLIAM WALTER CORT

LITERATURE CITED

Barker, F. D. 1907. Variations in the Vitellaria and Vitelline Ducts of

Three Distomes of the genus Opisthorchis. Trans. Amer. Micr. Soc,

27 :99-110.

Braun, M. 1901., Zur Verstandigung iiber die Giltigkeit einiger Namen

Fascioliden-Gattungen. Zool. Anz., 24:55-58.

Cort, W. W. 1912. North American Frog Bladder Flukes. Trans. Amer.

Micr. Soc., 31 :151-166.

. 1913. Notes on the Trematode Genus Clinostomum. Trans.

Amer. Micr. Soc., 32 :169-182.

. 1915. Egg Variation in a Trematode Species. Jour. Parasitol.

2 :25-6.

Goldschmidt, R. 1909. Eischale, Schalendriise und Dotterzellen der Trema-

toden. Zool. Anz., 34:481-498.

Johnston, S. J. 1912. On some Trematode-Parasites of Australian Frogs.

Proc. Linn. Soc. N. S. W., 37 :285-362.

Klein, W. 1905. Neue Distomen:aus Rana hexadactyla. Zool. Jahrb., Syst.,

22 :58-80.

Leidy, J. 1851. Contributions to Helminthology. Proc. Acad. Nat. Sc.

Phila., 5 :205-209. *

1856. A Synopsis of Entozoa and Some of their Ectocongeners

Observed by the Author. Proc. Acad. Nat. Sc. Phila., 8:42-58,

Looss, A. 1894. Die Distomen unserer Fische und Frésche. Biblioth. Zool.,

No. 16, 226 pp.

. 1899. Weitere Beitrage zur Kenntniss der Trematoden-Fauna

Aegyptens, Zool: Jahrb., Syst., 12 :521-784.

» 1901. Zur Sammel—und Conservierungstechnik von Helminthen.

Zool. Anz., 24 :302-318.

. 1902. Ueber neue und bekannte Trematoden aus Seeschildkréten.

Zool. Jahrb., Syst., 16 :411-891.

Pratt, H. S: 1903. Descriptions of Four Distomes. Mark Anniv. Vol., pp.

25-38. .

Rudolphi, C. A. 1819. Entozoorum synopsis cui accedent mantissa duplex et

indices locupletissimi. Berlin. 811 pp. 3 pls.

Seeley, L. B. 1906. Two Distomes. Biol. Bull., 10:249-254.

Ssinitzin, Th. 1907. Observations sur les métamorphoses des Trématodes.

Arch. Zool. Exp. et Gen., 7 :21-37.

NORTH AMERICAN FROG LUNG FLUKES 239

Stafford, J. 1902, On American Representatives of Distomum variegatum.

Zool. Jahrb., Syst., 16 :895-912.

_ 1905. Trematodes from Canadian Vertebrates. Zool. Anz.,

28 :681-694.

Stiles, Ch. W. and Hassall, A. 1902. Eleven Miscellaneous Papers on Ani-

mal parasites. Bur. An. Industry. Bull. 35.

Zeder, J. G. H. 1800. Erster Nachtrag zur Naturgeschicte der Eingwei-

dewiirmer, Leipzig. 320 pp. 6 pls. (Cited after Looss).

Macalester College, Saint Paul, Minn.

ABBREVIATIONS USED IN PLATES

A, acetabulum OT, odtype

C, cirrus P, pharynx

CS, cirrus sac PG, prostate glands

DS, duct of seminal receptacle S, seminal vesicle

E, excretory vesicle SR, seminal receptacle

G, genital pore T, testis

I, intestine U, uterus

M, metraterm VD, vitelline duct

MG, Mehlis’ gland VR, vitelline reservoir

O, ovary Y, vitellaria

OS, oral sucker

240 WILLIAM WALTER CORT

EXPLANATION OF PLATES

All drawings were made with a camera lucida. For the sake of clear-

ness the eggs are omitted from the uteri in the toto drawings.

Fig. 1. Eggs of the American species of Pneumoneeces, average size.

A. Egg of Pneumoneces similiplexus.

ne ay coloradensis.

complexus.

medioplexus.

longiplexus.

breviplexus.

& cs “

¢é

JIMOOw

Fig. 2. Pneumoneces medioplexus, dorsal view.

6c ‘ec

Fig. 3. os similiplexus,

Fig. 4. Cuticular spines of American species of Puewmoneces.

A. Spines of Preumonaces longiplexus.

B. 3 ic similiplexus.

i : . “ medioplexus.

D. "3 - o coloradensis.

The cuticula of Pneumoneces complexus and P. breviplexus is with-

out spines.

Figs. 5, 6, 7. Stages in the development of Pneumoneces coloraden-

SiS.

Fig. 8. Fully developed Pueumoneces coloradensis, ventral view.

Fig. 9. Pneumoneces breviplexus, dorsal view.

‘ce ee

Fig. 10. Be longiplexus, sy oe

Fig. 11. End passages of the reproductive system of Pneumoneces

medioplexus.

Fig. 12. End passages of the reproductive system of Puneumoneces

longiplexus.

Fig. 13. Diagramatic reconstruction of the connections of the female

reproductive organs of Pueumoneces medioplexus.

Fig. 14. Diagramatic reconstruction of the connections of the female

reproductive organs of Pneumoneces longiplexus.

Va mm Ud

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ELYTRAL TRACHEATION OF THE TIGER BEETLES

(CICINDELID/:)*

By V. E. SHELFORD

I. INTRODUCTION

In course of a study of the relation of the various structures to

the color pattern of Cicindela a large number of elytra were mounted

in hot balsam and the trachea examined as was described in an

earlier paper (Shelford 713). The studies embraced all of the fam-

ilies and subfamilies of the Cicindelide of earlier authors. The

group is world wide and. some of the material has been obtained only

with expense and difficulty. The studies have involved the drawings

of elytra of many individuals and the results as a whole show the

tracheation of the elytra of a large heterogeneous group. A large

number of drawings is presented because they are much more satis-

factory than description, and some idea of variation and the form

of the elytron can be obtained. Fully three times as many individ-

uals as shown, were drawn outside of Cicindela.

The nomenclature used is from Horn’s “Index der Cicindeli-

den”. Elytra of Cicindela and Tetracha have been studied fresh,

and the amount of asymmetry and variation of different individuals

noted.

II. GENERAL STRUCTURE OF THE ELYTRON

I. Chitinous Columns

The two walls of the sac like elytron are held together by chiti-

nous pillars or columns which in the adult appear in cleared

elytra as shown in figure 1. The different layers of cuticula show

here as rings around the original central spindle. These are often

proportionately larger and occupy more of the space than shown

in this figure.

*Contributions from the Zoological Laboratory of the University of Illinois, No. 54.

242 V. E. SHELFORD

2. Hairs

Hairs which in a primitive insect usually cover the wing en-

tirely are present in nearly all tiger beetle elytra. In the Manti-

choras the hairs are of two sizes and are usually just in front of

the chitinous columns. Hairs in this group are usually located on

the posterior side of backward projecting tubercles. Under the

microscope the hairs may be located on the pigmented area of the

elytra by the light area which is produced by the thin cuticula at the

base of each hair. Hairs appear on the whole to be less common

in the unpigmented areas and when present usually are surrounded

by a narrow rim of pigmented cuticula. Hairs occur in practically

all groups though they have been lost from the majority except for

a few at the base of the elytron and scattered along the trachea.

(See fig. 6, 21-31, 32-42, and 45.) In Platychila pallida I found no

indication of hairs. This is the only representative of the Platy-

chilide. Mantichora (Mantichoride) (Fig. 2) which stands in

close relation to Platychila has the dorsal portion of the fused elytra

covered very fully with hairs and the epipleuron has only scattered

hairs. One of the Megacephalide, Megacephala (Tetracha) equin-

octialis has the elytron completely and uniformal covered with small

hairs. The other species of Megacephala which I have examined

klugt and lwmata show only a few hairs at the base of the elytron

(Fig. 43 to 45).

In the only observed representative of the Pognostomide which

stand at the beginning of the series in W. Horn’s classification,

FIGS. 1-7. STRUCTURE OF THE ELYTRON AND THE MORE

GENERALIZED TYPES OF TRACHEATION

Fig. 1. Mantichora tuberculata DeGeer (Africa) ; Arrangement of columns

and hairs.

Fig. 2. Mantichora tuberculata DeGeer (Africa); Distribution of hairs.

Fig. 3. Pognostoma chalybeum Klg. (Madagascar); Distribution of hairs.

Fig. 4. Amblychila cylindriformis Say (Kansas); Six fully developed .

trachea.

Fig. 5. Omus Dejeani Bite (California) ; Degenerate radius and media.

Fig. 6. Collyris sp; Showing reduced cubitus and anal.

Fig. 7. Platychila pallida F. (S. Africa) ; Reduced anal.

PALAEOMANTICHORIDAE CTENOSTOMIDAE

MANTICHORA. MANTICHORA re POGNOSTOMA.

NEOMANTICHORIDAE COLLYRIDAE —PLATYCHILIDAE

Phtthaae!

Fe ent PE a mr way,

AMBLYCHILA, OMUS COLLYRIS. PLATYCHILA

PLATE X

244 V. E. SHELFORD

the elytra was covered with hairs. In the Collyride (Collyris and

Tricondyla are arboreal) the hairs are confined to a few at the base -

and a few scattered along the lines of the trachez.

3. Pits and depressions

The elytra of many species are marked with pits. Close ex-

amination under the microscope with both transmitted and reflected

light shows that, in the majority of cases, the pits are over the

center of the chitinous columns and bear no relation to rudimentary

hairs as Dr. W. Horn has suggested. I have seen no pits that

would appear to represent rudimentary hairs though they may

occur.

4. Vewms and thickenings

There are sometimes thickenings running lengthwise of the

elytron as in Domica (fig. 35 and 36). While these thickenings

run parallel with the trachea they are usually between rather than

coincedent with them, except in Caledonica (fig. 25). There are

FIGS. 8-19. ELYTRA OF FRESHLY EMERGED CICINDELAS

Fig. 8. Showing the media, cubitus, and anal (rudimentary in freshly

emerged specimens of C. repanda. Dej.

Fig. 9. The same in a freshly emerged C. punctulata Oliv.

Figs. 10-11. Showing in full detail the tracheation of the right and left

elytra of a freshly emerged C. Leconti Hald. Note the close sim-

ilarity of the main trunks and the marked difference in small

branches, indicating the lack of significance of these. From two

camera lucida drawings of fresh elytra flattened out, one bottom

side up.

Fig. 12. Camera drawing of another specimen of the same species. Note

the fewer cross branches and the longer radius and media.

Fig. 13. Camera drawing of a freshly emerged C. repanda Dej.

Figs. 14-15. Camera drawings of two individuals of C. punctulata Oliv.

Fig. 16. Camera drawing of a freshly emerged C. cuprascens Lec. Note

the degenerate media and heavy cross trachea.

Fig. 17. Camera drawing of a fresh C. hirticollis Say. Note condition simi-

lar to that in 16.

Figs. 18-19. Camera drawings of two different individuals of C. tranque-

barica Herbst.

In these figures note the branching in every case near the curve of the

posterior side of the elytron, and the subcosta ending at the same point with

branches extending inward in every case.

-CICINDELINI

CICINDELIDAE

ns Ee

PLaTe XI

246 V. E. SHELFORD

however some thickenings on the under side of the elytra of most

species which correspond in a general way to veins (particularly

in Mantichora). The outer and inner margins of the elytra are

always thickened and resemble veins almost invariably containing

trachee. The subcosta usually follows the costa very closely at

the base of the elytron but just behind the middle it turns inward

away from the margin in a vein like thickening. The radius is in

a distinct thickening of the elytron which proceeds from the base

for a short distance. This is very constantly present. Aside from

this nothing comparable to veins is present but the rows of chiti-

spaces running the length of wing. These are occupied by the

principal trachea. In some cases the spaces appear very clearly on

the under side of the elytron and in Mantichora there are distinct

ridges over them which have every appearance of veins.

Ill. TRACHEATION

I. The main trunks

The six trunks common in insects are represented in but two

genera (Amblychila and Mantichora), (Figs. 4 and 6), which have

rudimentary wings and specialized elytra fastened together in the

FIGURES 20-29. THERATIDA® AND EURYODINI

Fig. 20. Therates basalis Dej. (New Guinea).

Fig. 21. Therates labiatus Fab. (Oriental region).

Fig. 22. Therates fasacitus Fabr.

Fig. 23. Eurosopus Chaudoiri, Thom. (South America).

Fig. 24. Eucallia Boussingaulti Cuer. (South America).

Fig. 25. Caledonica acentra Chd. with thickenings in the: lines of the trachea.

(New Caledonia).

Fig. 26. Distypsidera sp. (New Holland).

Fig. 27. Distypsidera undulata Westw. (New Holland).

Fig.

Prothyma adonis sb. sp. bellula Fit. (Madagascar).

S &

Prothyma versicolor Dej. (N. Africa).

ae wuheapat: traebictiah te nei e9 nk ?

cei MS BT Eee

EUCALLIA.

tA tr = A , ies

ae ba ° > PST <

z ~~

Qa pw

= =

oc = S

J a ma

ud 3

ad a

= =

= ~

Pad fs

x <

aa iS ae <

: lad Fe AeA) tea 2 STD eT J a |

: o A,

= &

= 3

= ‘

= RRS ee -

— bed SRA ce:

—d

fanning

piri reecy n

248 V. E. SHELFORD

adult. These trachea are demonstrated in the adult dried elytra

without any difficulty. In Omus which is closely related to Ambly-

chila the radius and media have disappeared except for rudiments.

The cubitus is the principal trachea (Fig. 5). With the exception

of Omus and Amblychila it is the anal that has degenerated farthest.

Collyris was never very satisfactory for study but it appears that

the cubitus is reduced and the anal wanting (Fig. 6). In figure 7

(Platychila pallida) the commonest type of tracheation of the family

and probably among the most generalized so far as the first four

trachea are concerned, is shown. ‘The anal is much reduced.

2. Details of Tracheation in Cicindela

Figures 8 and 9 show two different arrangements of the anal

rudiment in Cicindela. In figure 8 it is joined with the cubitus.

In figure 9 it is likewise joined in the same place but a small branch

suggests a backward extension. The number of small branches

and cross connections is large and too variable to be correlated with

other specific characters. Figures 10-19 illustrate this fact. Fig-

ures 10 and 11 are the two elytra of an individual and show a

marked difference. It is evident then that only the main trunks

are at all constant. The costal branch at the center the posterior

third of the elytron at the beginning of the curve is very charac-

teristic of Cicindela.

FIGURES 30-33. ODOMTOCHILINI

Fig. 30. Oxygonia moronensis Bat. (South America).

Fig. 31. Odontochila punctum Klg.? (South America).

Fig. 32. Odontochila brasiliensis Dej. (South America).

Fig. 33. Prepusa ventralis Dej. (South America).

FIGURES 34-37. DROMICINI

Fig. 34. Dromica Kolbei W. Horn. (South Africa).

Fig. 35. Dromica sculpturata Boh. Thickenings shown as dark areas, not

related to the trachea. (South Africa).

Fig. 36. Dromica tuberculata Dej. (South Africa).

Fig. 37. Dromica limbata Bert. (South Africa).

CICINDELIDAE

ODONTOCHILINI

PREPUSA

ey ear

ODONTOCHILA

SSS D9: DEAE epee tee) ap eet

: DRAPER SN ce CBS

Part eres)

PLATE XIII

CICINDELIDAE

DROMICINI

DROMICA

ODONTOCHILA

= Nae wr AARNE. Sete

nea rena Ee

OXYGONIA

250 V. E. SHELFORD

3. Peculiarities of the different Taxonomic Groups

The peculiarities or distinguishing features of Cicindela and of

the Palzomantichoride, Neomantichoride, Pognostomide, Platy-

chilidee and Collyride have been noted. The remaining families or

subfamilies still require mention.

a. Theratide. (Figs. 20-21) The radius and media unite

in the center of the posterior third of the elytron and are united

with the costa and subcosta by three or four branches.

b. Cicindelide-Euryodini. (Figs. 23-29) The main trunks

are similar to those of Cicindela but differ characteristically in the

absence of a costal branch or of its presence near the tip of the

elytron than in any other subfamilies. Compare figs. 26-27 with

10-19. In fig. 24 are shown two backward branchings (such as

occur in Cicindela). The media appears well developed and joined

to the subcosta as a rule.

c.. Odontochilini. These differ only slightly from the Euryo-

dini but as a rule the media is reduced and the costa joined to the

cubitus.

d. The Dromicini appear to have an unbranched and some-

times reduced costa.

e. Ihe Megacephalide (Figs. 38-45) are characterized by a

well developed media which is as a rule the most prominent trachea

of the elytron. The costal branches are somewhat farther posterior

than in Cicindela.

ce OL IS MEIER SP eA LOE cae SNM A AEE ae hc A SMORAM A MOD 5 3.5 Aaa bs S

FIGURES 38-45. MEGACEPALIDZ

Fig. 38. Pseudoxychila bipustulata Latr. (South America).

Fig. 39. Oxychila tristis Fabr. (South America).

Fig. 40. Tetracha carolina (Georgia).

Fig. 41. Tetracha carolina Linn. (N. Carolina).

Fig. 42. Styphloderma asperta Wat. (Africa).

Fig. 43. Megacephala (Phzoxantha) limata Perty (South America).

Fig. 44. Megacephala (Phzoxantha) equinotialis Dej. (South America).

Fig. 45. Megacephala (Pheoxantha) Klugi Chd. (South America).

ches SOAP Mp LS 2: e

AA RNR ODES TERA ras

Tetrache Ho

MEGACEPHALA

OXYCHILA

PS EUDOXYCHILA

at:-

rma W

Slyp hiode

MEGACEPHALA

PLATE XIV

252 V. E. SHELFORD

In the set of figures facing this page the obvious and presum-

ably more characteristic features of elytral tracheation, brought out

in the preceding camera drawings are shown. The figures show

characteristic differences between the different groups correlated

with the general taxonomic differences. The notable characteristics

of the family as a whole is the tendency to reduce the anal. The

exception falls in the Mantichorid groups which are wingless and

have the most specialized elytra. The figures show conclusively

that tracheation is far from haphazard, that it is of a fairly constant

taxonomic significance in this group of Coleoptera.

It has been shown that veins are formed in the development of

elytra before the trachea enter. Accordingly no claim of exact

homology with the corresponding veins of other orders is made.

BIBLIOGRAPHY

Comstock, J. H. and Neepuam, ph t2

1899. American Naturalist, pp. 43; 81, 231, 335, 413, 561, 769, 903.

Horn, WAtTER.

1905. Systematischer Index der Cicindeliden, Deut. Ent. Zeit. Feb. 1905.

Supplement pp. 1-56.

SHELForD, V. E. .

1913. Noteworthy Variations in the Elytral Tracheation of Cicindela,

Ento. News. Mar. ’13 pp. 124-125.

University of Illinois,

June 11, 1915.

CTENOSTOMIDAE ‘COLLYRIDAE THERATIDAE

iN (

POGNOSTOMA CTENOSTOMA COLLYRIS TRYCONDYLA. THERATES

MEGACEPHALIDAE

CICINDELIDAE

EURYODIN} ODONTOCHILIN| CICINDELIN| DROMICINI MEGACEPHALA

NEOMANTICHORIDAE ALAEO PLATYCHILIDAE

th A . fy A

MANTICHORIDAE

AMBLYCHILA OMUS MANTICHORA PLATYCHILA

PLATE XV

eer er i

Bae ae eae

Se oe is

ar ee

mae tee ge

bile ateatac,

Seine >

A STUDY OF THE SCALES OF SOME OF THE FISHES

OF THE DOUGLAS LAKE REGION*

By Artuur T. Evans

During the summer of 1913 while in attendance at the Uni-

versity of Michigan Biological Station on Douglas Lake in Che-

boygan County, Michigan, an effort was made to find out if possi-

ble whether the variation in the scales of different fishes is such

that the scale characters are of any taxonomic value. The objects

of the study were to determine, (1) whether there is a marked

difference in the scales from different individuals of the same

species; (2) whether the scales of an adult and a younger speci-

men of the same species varied sufficiently to be mistaken for scales

of another species; and (3) to determine in just what way the

scales of various families differ. As the length of time for con-

ducting the work was limited only ten of the more abundant species

were studied in detail.

The writer is indebted to Professor Max M. Ellis for help

and suggestions in the course of the work.

Since the examination of all the scales of a number of fishes

would be a big task and quite likely unnecessary it was decided to

examine scales from what appeared to be the three most typical

regions of the body of the fish. Before deciding upon these three

typical regions an examination was made of the scales from various

other parts of the body of the fish to determine just where the most

variation occurred and of what this variation consisted. Through-

out the course of the work whenever a fish of a different species

was studied a complete examination was made of scales selected

from various parts of the body to make sure that variation did not

occur in parts not included in the typical bands. The three regions

selected were, (1) just anterior to the dorsal fin (Band 1); (2)

just anterior to the soft dorsal (Band 2); and (3) just anterior to

the caudal fin. In each of these regions a band of scales was taken

the entire distance around the body of the fish. Each scale was

*Contributed from the University of Michigan Biological Station Number 16.

256 ARTHUR T. EVANS

examined under the low power of the compound microscope and

the following data recorded: (1) number of teeth, if any; (2)

number of radii (this includes both entire radii and radii of which

only parts were visible in Table 1); (3) number of basal lobes,

(4) whether the circuli extended through the apical area or not,

and (5) the form of the circuli.

It was supposed that there might be a variation in the number

of teeth, radii, and lobes correlated with the age of the individual

so in order to find out the exact extent of such variation three

Fgare J.

Right scde

--— Left side

Blank space represents regenerdiion sm Iéoles.

different sizes of the several species of fish were examined when-

ever it was possible to obtain them. In selecting individuals for

study it was assumed that the larger fish were the older.

Perca flavescens (Mitchill)

The first fish selected for a detailed study was Perca flavescens,

because of its abundance in Douglas Lake and the ease with which

it might be obtained, since numbers of individuals might be found

in almost any sizes desired along the beach after a high wind.

SCALES OF FISHES 257

Fishes of these seemingly desirable sizes, that is, 150 mm. or more

in length; 100 to 125 mm. in length; and less than 100 mm. in

length were collected along the beach and brought to the laboratory.

All were preserved in 5% formalin.

All the scales from the three bands on the three different indi-

viduals, each representing one of the three different lengths, were

mounted and the data taken. From these data curves were plotted

and it was found that the resultant curve for Band 1 and Band -

were very similar in shape, but that Band 3 was somewhat differ-

ent. The curves for the three bands of one of these three perch

are shown in figures 1, 2, and 3, and the entire data is given in

Figure ke

fight side

eee eft 5 2188

Table 1. If a detailed study of this data is made it will be found

that the lowest points of the curves are represented by the extreme

scales ; that is, the scales from the median dorsal and median ven-

tral lines. In view of the fact that these points seemed so con-

stant for both sides of the fish and for all the fish examined, data

was recorded only from the extreme dorsal and ventral scales and

the scales from either the second or third row above and below

2a,

258 ARTHUR T. EVANS

the lateral line in subsequent tables. In comparing the curves rep-

resenting the various bands with curves for the corresponding bands

on the opposite side of the fish they were found to be similar, which

indicates that corresponding scales from opposite sides of a fish

are rather uniform but not identical.

Fig ure JF

[fi 9 Ail Side

Wate ae Left side

The similarity between Band 1 and Band 2 seemed sufficient

to warrant the use of only one of these bands. Band 2 was dis-

carded. This should not be taken to indicate that no scales were

examined from the vicinity of Band 2 as scales were examined

SCALES OF FISHES 259

from all parts of the body but data was only recorded for Bands 1

and 3.

Table 1 contains the data collected in the detailed study of the

three different sized Perca flavescens. It includes averaged data

for every scale in the three typical bands. The data for each

scale was collected separately and then the corresponding data for

each of the several scales of the various series were averaged. The

lengths of the three sizes of fishes were, Series 1, 242 mm.; Series 2,

200 mm.; Series 3,90 mm. A detailed study of this table shows a

very great similarity in scales from similar parts of the body, In

studying scales from the various fishes it was always found that

the scales in the row along the base of the various fins are not well

developed and may even fail to show signs of teeth, radii, or lobes.

This is very likely due to the position which they occupy and it

appears that they are entirely normal for the region which they

represent. Many of these scales are covered with skin. Ctenoid

scales are in general not heavily covered with skin. This fact may

account for the presence of cycloid scales along the base of the fins.

It might be contended that the dorsal and ventral scales examined

in this work were such scales. This is probably not the case, how-

ever, as almost without exception the scales from Band 1 showed

radii and lobes. Also a thorough examination was made of all

the scales in front of the dorsal fin and they were found to be very

much alike. In the scales from the tail region or from Band 3,

with very few exceptions, all were highly ctenoid and had both

radii and lobes. This brings out a point in regard to the scales of

the caudal region. In every Perca flavescens examined the scales

of that region were found to be highly ctenoid. This was also true

of most of the other fishes examined. It shows very well in

Micropterus salmoides (Lacepede) and Mucropterus dolomieu

Lacepede which have scales that are cycloid or very weakly ctenoid

on the anterior part of the body while the posterior part of the

body is covered with scales that are heavily ctenoid. In Perca

flavescens it may be said that, in general, the caudal region is more

‘heavily ctenoid than the anterior region. The biological signifi-

cance of this fact was not ascertained. Additional protection from

rear attacks or for use in connection with spawning (like the pearl |

260 ARTHUR T. EVANS

organs in the Cyprinids) might be suggested. Again it may have

no significance whatever.

If Table 2 is examined it will be found that there are many

differences between the scales of the larger fish and those of the

smaller fish; also that there are many similarities. It is very no-

ticeable that the number of teeth increase with the size of the fish.

Scales from the fish of the larger size have the greater number of

teeth, these being twice as numerous as the maximum number in

the scales from the smaller fish, in some cases. The teeth on the

smaller scales are very pronounced, however, even though some of

the scales are very small. This variation in the number of teeth on

the scales of the different sized Perca flavescens seems to be one of

the most certain characters by which the scales from a large fish

may be separated from those of a smaller fish. The radii and lobes

are much more constant. However, there is a tendency for the

smaller scales to have a greater number of lobes than the larger

ones. This is correlated with the fact that in nearly all the scales

from the smaller fish the radii are usually all complete, while in the

scales from the larger fish many of the radii are incomplete. This

is also a very sure way to distinguish a small fish from a larger

one. The more complete and more numerous radii of the scales of

the smaller fish is interesting in view of the fact that the radii in

the scales of Osteoglosside and other more primitive fish are very

numerous, complete, and often cross the entire scale, while there is

a tendency toward the reduction of the radii in the scales of the

higher fish. The scales from the various parts of the body of the

fish are variously shaped. Those from the sides are rather broad

and the teeth are stout in appearance. Scales from the dorsal and

ventral parts of the body vary considerably in shape. Most of them

are more or less lop-sided. The scales along the sides near the

lateral line decrease in size from the anterior end of the body back

toward the caudal end. On the cheek of Perca flavescens the scales

are not toothed. From a smaller fish the scales do not appear so

coarse and the teeth are somewhat more frail. The lobes in the

older scales are much more pronounced, the basal end being deeply

notched and the individual lobes being more elongated. In the

smaller fish the lobes of the scales resemble a series of small semi-

SCALES OF FISHES 261

circles. In other features the scales appear much alike. Scales

from the side of the fish appear more or less rounded and somewhat

flattened on the apical and basal ends. A description of a typical

Perca flavescens scale covering scales from all parts of the body

would include the following characteristics: The shape of the

scales are from oblong to ovoid with from three (some scales along

the base of the various fins without teeth) to seventy-seven teeth on

the outer margin of the apical area, varying in size with the size

of the individual fish the scales are taken from. The apical area

upon which these teeth are set is built up of columnar-like struc-

tures. The radii are all basal and extend out from the nucleus,

which is situated about one-third of the distance from the top of

the apical area to the base of the basal lobes, through the basal

area in the form of spokes varying in number from one to twelve.

Many circuli extend as semi-circles through the outer part of the

basal area of the scale. In the outer part of the scale these cir-

culi are interrupted by the apical area while in the central area

they are not but extend around the nucleus as small circles. This

is much more pronounced in the scales from the smaller fish than

those from the larger fish. In the smaller fish the circuli are all

beaded. In the larger fish the circuli in the center are not beaded,

evidently having lost them in the course of development. The cir-

culi are beaded in the outer part of the older scales. These points

concerning beading are what might be expected since primitive

fish have beaded circuli.

Stizostedion vitreum (Mitchill)

The only examination of the scales of Stizostedion vitreum

was made on the scales of large specimens taken from Carp Creek.

A comparison of the scales of Perca flavescens and Stizostedion

vitreum show dissimilarities in a great many respects. In Stizos-

tedion vitreum the scales are more heavily toothed in all parts of

the body. The dorsal and ventral scales are also heavily toothed

which is not the case in Perca flavescens. Also the scales of Stizos-

tedion vitreum usually have a decided flange between the apical and

basal areas.

In a detailed study of the scales of Stizostedion vitreum all the

circuli are found to be heavily beaded looking in part as if the

262 ARTHUR T. EVANS

circuli were broken in pieces. Also the circuli do not extend

through the apical area even in the central part. Whether this

would hold for smaller specimens is not known. An examination

of scales from all parts of the body showed no regional differences

from Perca flavescens other than the dorsal and ventral regions

being heavily toothed.

Table 3 gives the complete data for the only two specimens of

Stizostedion vitreum examined.

Boleosma nigrum (Rafinesque)

As Boleosoma nigrum is a very common fish in the shoals and

along the rocky shore of Douglas Lake, large numbers were avail-

able for study.

One of the points in which it differs from the rest of the

Percide is in the fact that the breast and the dorsal parts in front

of the dorsal fin are scaleless. Forbes and Richardson* hint that

probably more northward the specimens of this fish are more scaly

than southward. None of the specimens taken from Douglas

Lake, however, were found to have scales on the breast and cheeks,

and only a few were found that had scales on the back. In tabu-

lating the data for Table 4 the absence of scales on the breast and

dorsal parts of the fish made a slight change in the scales taken

necessary. In taking the dorsal scale the data was recorded for the

very top scale near the front of the dorsal fin. The ventral scales

were taken from the ventral line just back of the ventral fins. This

plan was followed throughout regardless of whether the breast

was scaled or not.

Boleosoma nigrum being one of the more highly specialized

Percide it would be expected to possess scales of a higher type.

This is exactly what is found when the scales are studied. Table

4 shows that all the scales are distinctly ctenoid. The teeth are

very prominent and although the scale is rather fragile in appear-

ance it is well developed and the teeth look very formidable.

The data also show that the radii are somewhat more numerous

than in either Perca flavescens or Stizostedion vitreum. When

compared with the scales from very small Perca flavescens they

*“The Fishes of Illinois,” by the Natural History Survey of Illinois. Volume III

pp. 297, paragraph 2.

SCALES OF FISHES 263

seem to be somewhat alike although the young Perca flavescens

scales are much more immature in looks. The lobes appear quite

alike. The circuli in Boleosoma nigrum as would be expected in

a highly specialized fish are plain, appearing as a number of plain

line semi-circles and not extending through the apical area.

Centrarchide

In a further study of scale characteristics the Centrarchide

were selected as a group to be studied. They are very common in

Douglas Lake being represented by five different species.

Fifteen different specimens were examined in every case pos-

sible. Some, however, are represented by fewer specimens as they

were unable to be obtained in greater numbers. Unlike Perca

flavescens most of the specimens were taken in nets. A few, how- ~

ever, were collected along the beach. In the study of the Centrar-

chide the same plan was followed as in the study of the Percide.

Although only data for eight scales were recorded a careful study

was made of scales from all parts of the body. The species studied

were: Ambloplites rupestris (Rafinesque), Eupomotis gibbosus

(Linneus), Lepomis pallidus (Mitchill), Micropterus dolomieu

(Lacépéde), and Micropterus salmoides (Lacépéde).

The study of the scales of the Centrarchide is a varied and

interesting one. A typical scale of one of these fish is very much

like one of Perca flavescens in appearance. The circuli are beaded

and do not extend through the apical area except in the very young

specimens. In such specimens the circuli passing through the

apical area are not beaded. Also in scales from a young Centrar-

chide the apical area is entirely covered with circuli. One of the

marks that will help to distinguish a Percide scale from one of a

Centrarchide is that the apical area in a Percide scale forms a

more obtuse angle than in the Centrarchide@ scales.

In the Centrarchide the dorsal and ventral scales do not have

the same characteristics as the ventral and dorsal scales of Perca

flavescens. In Ambloplites rupestris the shoulder including the

parts of the fish in front of a line drawn from about the fourth

dorsal spine to the tip of the pectoral fin and on the ventral surface

of the body in front of a line drawn from the base of the ventral

fins to the tip of the pectorals, is without ctenoid scales. The scales

264 ARTHUR T. EVANS

from these parts of the fishes body are either entirely without teeth

or with only traces of teeth. The boundry of these regions vary

more or less but in general the above statement will about cover it.

Occasionally a ctenoid scale will be found to occur in these regions

but it is rather uncommon. In Eupomotis gibbosus and Lepomis

pallidus the dorsal regions correspond with those in Ambloplites

rupestris as may be seen by the data. The ventral regions are,

however, entirely different. In both Eupomotis gibbosus and Lep-

omis pallidus the scales from the very lowest ventral parts are all

ctenoid. Further up near the opercular flap only traces of teeth

are found. In Ambloplites rupestris the ventral part of the fish

near Band 1 is found to be covered with scales that are distinctly

cycloid or almost always so.

In all the Centrarchide studied the scales from the caudal

region were found to be highly ctenoid. The scales along the base

of the fins are usually long, irregular in shape, and without teeth,

radii, and lobes.

Ambloplites rupestris

The specimens examined varied in length from 17 mm. to

215 mm. The data (Table 7) show how distinctly cycloid the

scales from the region of Band 1 are. The data for the scales from

Band 3 show that the scales from that region are very decidedly

ctenoid. In shape, the scales of Ambloplites rupestris are much

like a scale from any of the other Centrarchide.

Lepomis pallidus

The specimens examined were from 52 mm. to 125 mm. in

length. Table 9 shows that Lepomis pallidus differs from Amblop-

lites rupestris in that the scales from the ventral part of the fish

in front of the ventral fins, are ctenoid while in Ambloplites rupestris

they are cycloid. The scales from the caudal regions of these two

fish are quite similar.

Eupomotis gibbosus

The specimens examined varied from 62 mm. to 135 mm. in

length. Table 8 shows that the scales are ctenoid except-in the

region of the shoulder. The ventral scales on Band 1 are ctenoid

but farther up the sides near the operculum only traces of teeth are

SCALES OF FISHES 265

found. The scales from the ventral region show more or less of

a tendency to be flanged, that is, the region in which the apical and

basal areas meet is wider than the parts of the scale on either side

of it.

Micropterus salmoides and Micropterus dolomieu

The scales from Micropterus salmoides and Micropterus dol-

omieu will be discussed together as they are both typical Centrar-

chide scales and resemble the other Centrarchide discussed very

much. Further, their own relations are better discussed when they

are considered together. The cycloid regions in these fish are quite

similar. It is also very much more extensive than in any of the

other Centrarchide studied. These scales from all the anterior

part of the body regardless of their position are cycloid or at most

very weakly ctenoid. The posterior part of the fish is covered

with scales that are highly ctenoid. If scales are selected and

examined from the posterior end of the fish forward they are

found to gradually pass from ctenoid to cycloid. Only a few fish

were available for the work so that a more detailed study is lack-

ing. From the data at hand it appears that the ctenoid scales ex-

tend as far forward as about the third or fourth ray of the dorsal

fin. In the study of a large number of scales from the different

specimens it was found that Micropterus salmoides shows a ten-

dency to be more heavily ctenoid than does Micropterus dolomieu.

A description of a typical scale will suffice for either of these two

fish. In the younger specimens the circuli extend around the

nucleus, passing through the apical area, and are beaded, while in

scales from the adults the circuli extending through the apical

area are not so numerous and also the circuli in the central part

of the scale are not beaded. The outer circuli in the adult scales

are very much broken and show very distinctly a tendency to form

teeth.

Percopsis guttatus (Agassiz)

The study of the Percopside was made upon Percopsis gutta-

tus. They varied in length from 75 mm. to 87 mm. This fish has

a scale which is very different from either the Percide or Centrar-

chide. It has no radii or lobes. The nuclear area is located far up

266 ARTHUR T. EVANS

near the apical end. Also the plain circuli in the central area near —

the nucleus extends through both the apical and basal areas. These

circuli appear to be broken in places. Farther out they are inter-

rupted by the very small apical area.

The teeth are small but very pronounced. They are very dif-

ferent from those on the scales of Percide or Centrarchide in that

the apical area is not composed of columnar-like structures. Al-

though the teeth are well formed they appear rather weak due to

the fact that the apical area lacks the columnar-like structures. The

number of teeth on each side scale varies from 2 to 22 and as in

both the Percide and the Centrarchide the posterior end of the

body is more heavily ctenoid than the anterior end. The nuclear

area is also nearer the top of the apical area than in the Percide

and Centrarchide.

In Percopsis guttatus the dorsal part of the body in front of

the dorsal fin is scaleless. The table does not include this region.

The first data recorded represents a scale from the second row

above the lateral line in every case. Table 5 contains the data

collected on Percopsis guttatus.

Esox Lucius (Linnzus)

The scales of Esox lucius show the characters of a more primi-

tive fish when compared with those of one of the more highly

specialized fish examined. The scales are entirely without teeth.

The number of radii found were from one to four and the number

of lobes varied from one to three. In many of the scales the lobes

were found to be separated from each other and the rest of the

scale, in many cases, over half way to the nuclear area.

The circuli are plain and somewhat irregular. They extend

as circles through both the apical and basal areas. In the center

near the nucleus they become irregular in shape. In many of the

scales examined there were irregularities in the upper portion of

the apical area which may be an indication of teeth forming. Table

6 gives the data collected on the two sizes of Esox lucius.

SUMMARY

It has been very recently that scale characters have been con-

sidered seriously as a method of distinguishing between different

SCALES OF FISHES 267

fishes. Undoubtedly external features are a quicker method by

which to identify a fish, but if only a small portion of the fish is

available then the method of identification by scale characteristics

becomes very valuable, if these characteristics are stable. That

they are stable seems almost conclusively proven from the data

at hand. A few of the essential differences in the scale character-

istics of the fish studied may be enumerated.

In Perca flavescens the dorsal row of scales in front of the

dorsal fin, and the ventral row of scales in front of the ventral

fins, are not ctenoid. The rest of the dorsal patch which may be

designated as all the scales in front of a line drawn from the an-

terior end of the dorsal fin to the corner of the operculum, and

all of the ventral patch which may be designated as all the scales

included in the region in front of a line drawn from the base of

the ventral fins to the corner of the operculum, is covered with

scales which are toothed very heavily. This is not true of any other

fsh studied. The scales of Boleosoma nigrum are ctenoid on all

parts of the body. If a scale of a small Perca flavescens is com-

pared with a scale from Boleosoma nigrum they can be readily

distinguished from each other regardless of the sizes of the fishes

that they are selected from. The scale from a Boleosoma mgrum

is a mature looking scale. It is well formed and looks as formid-

able as-a very mature scale from a Perca flavescens. The scales

from a small Perca flavescens have a rather immature appearance

and are easily distinguished as scales from an immature fish. In

such scales the columnar structures are not well formed and al-

together the scale looks frail in comparison with one from Boleo-

soma nigrum. Also Boleosoma nigrum lack the scales on the breast.

In the Centrarchids studied the whole shoulder patch is without

ctenoid scales. The ventral patch is different. In Lepomis pall-

dus and Eupomotis gibbosus the ventral patch is covered with

ctenoid scales, the teeth being more or less lacking up near the oper-

culum. The scales of Ambloplites rupestris are cycloid on all the

ventral patch distinguishing it at once from either Lepomis pallidus

or Eupomotis gibbosus.

Micropterus dolomieu and Micropterus salmoides are distin-

guished from the Percide and C entrarchide studied by the absence

268 ARTHUR T. EVANS

of ctenoid scales on the anterior part of the body. This region with

only cycloid scales extends back to about the third or fourth ray of

the dorsal fin.

Esox lucius is easily distinguished from the rest of the fish

studied by the body being entirely covered with cycloid scales.

Also the lobes are distinctly separated from one another by the

radii, almost one-half of the distance to the nucleus.

Percopsis guttatus scales may be identified by the absence of

any columnar-like structures in the apical area.

The work so far as it has been carried out and so far as the

data can be interpreted seems very satisfactory. Many striking

similarities are seen between the scales of different fishes from the

same families. Also many differences are evident in the scales

from fishes of the various families. In every case, however, the

scales of the primitive fish have shown characteristics of primitive

scales, and the scales of the specialized fishes have shown charac-

teristics of highly developed scales. .

Scale characters as a means of identification would probably

be very little affected by regeneration as the percentage of regen-

erated scales is small. The percentage of regeneration in all the

scales of a single specimen of Amblophtes rupestris about 200 mm.

long was 12%.

TaB_e I, : Perca flavescens. 15 specimens. Length—150-250 mm. Table 2

3 Length 242 mm.

Ba Right Side Left Side

gs Band 2 Band 1 Band 2 Band 3 Location

$2 2 2g 5 3 of Min. Ay. Max.| Min.

gE é 2_|f ; 3 |i 3 | ae

az rire #3 | 83) g]2| 23| 23/s |4|23|23|s|sldzl esl. 0 a

* 8 Bf) Se) 213) 88) 82\3 13] ee|82| 2) Zee] se

E Sle is [21 3"i2*13 [21 8*] 2°] 4 | 2/2*| g#/2 9 2A | 19) 38) 55

8 above 0} Oo 0 Pa 7 2B | 31) 42) 58

no “s 5| 0} O}.. “4 3] ol 2 o} 1 6H]-V 0} 16] 23

: “ : : . : = oa ee lie 4 4 > f| 2 Perca flavescens. 15 specimens. Length 100-125 mm. Table 2 Cont.

— Pha oa ahaleiea sy : 3] 0 2 D | oO 24 ia] 9g 5) 9] 2, 4) sD] o 7 iq) 0 4) 9 0] 3] 8

3 « 5 s| 3| 6laal sl 2 1 2 | 2 2A 17 21 | S28, G9 121s Ai 7a eA: 8) 22): 301 -7|. 19 | 12 Gas ad

—— ne on Se Ms I : ie 2B | 22| 32} 37] 4 7| 10} 5| 6| 9] 2B] 13] 21] 31] 4! 7] 10) 3] 6| 9

rs 5 6 2 6lsa sl 2 7 mal g V Oj aesta2t 6.209] 2 2a PSs, 0} 9} -15)) Tp 5 | 240) 60) Aas

1 below | 81 4 O} 2/10]50} 5) 3 6 Ole B77. Perca flavescens. 15 specimens. Length 35-100 mm. Table 2 Cont.

ae 4 0} 2} 9/50; 6 1 7 11] 3| 7 0 2; 4] Fok ae

at 6 7| 4] 6165] 7] 1 6 11) 1] 9 4 3) 6] “3b o2 gas

Beh 5 4) 5] 4Jos} 6] 1 5 10} 2) 7 5 4.6" 9) a Se

; : 4 7| 5] 6163] 6] 1 5 6| 0} 5 0 2 4|- 71 ae

c 4 Dhani Ae }40|. 27) el 5 AiO 2

7 ; 6 4 odoo si 1 7 6 42 Length 420 = = —

: : 5 ANON 6 Ne IE 6 V4 ers mE s| 6| 6 4

“ Co he 6

Tie. 2\. 6 Gilberts iy ec!

Ot eg 4}. 5 MEFs) | it)

+ ae ae 5 Boleosoma nigrum. 15 specimens Length 35-47 mm. Table 4

hoa PAW 4

14 21 be 2

7 above| 3| 2) 1| 2{/13/ 2| of 1 os Ee eee Oe es noe ee pe ee) = a

6 “ |{3l) 6 1) 5)22; 5 1) 4]12; 3 Of 2IR] 3] of 3] oO 1] 1} ollie i} 2-0

5 “ 137, 4| 3) 3|33) 4} 2| 3]26 5 2! Si33| 4! of 3132| 2] 2] 3lie 4 3} 4 0

4 “ 137) 2 4) 3/47) 7| Of 6}42) 9 1] 7/381 2| 4] 3}4i} 6 11 6f2il si 44 0

3 “ {41)- 1] 6 3/39 5) O} 4435) O 3]10}42) 4] 3{ 1)44 7] 41 6127] 3] S| 4 0

2 “ 133 3 Of 4]44| 6 of 5]50| 5} 4] 440i 31 of 2l4d of al staal 4) al 3 0

1" {46 4 0} 3)56 5) 2 6)53, 4 1) 3)28) 5} a} 5} 32] 5} Of 4/38] 5] 4] 5 Esox lucius. 4 specimens Length 300-410

1 below] 48) 4) 0} 3/31) 5} O} 4]46 S| 1} 4151! 7] o| 6/33 7] Of 6146 4] 21 4 0

2 “ 144 5 1 S}48 5} 1 5742) 4 2) 3i47i 7) of 6145) 6 | 5/47] 3] a2 2

3“ 157) 5S} 2 5152) 5} O} 4745) 4 1 3f62| 7} of 6l48i 5] of 4446 5s} 4) 4 2

4 “ 165} 6 O} 6]51; 6 0} 5/32) 6& OF} Sioo| 6| of 6]53| 5} 1] 4}52) 4] 2] 3 0

ats 50} 6} =O} S}50) 6 Of 5/25) 4 1) 3f51} 7) 1] 6153] 5! of 4}3il 5) 4) 4

Pee OA) tl 715655) PO) 4) 151-2) 80) <1154i=— 5) 3|54 (571— S/S 1=5 1 20|=a| =

Tae SOAS leer 157) G. -O. 5]: .-157] 6} 3) 6148] 5} O} 4 =a] fee

ee ee PN OM 31041 GLA) 21° 31. AR| 5} 2-4} 40) 6. _1|-6

ee ope ll 5157) 5} <0) 4 he = [09 - 3|-—5|- 41 541= Sieetl=4|=

ear Bete Se 2 Of Spl 20 sf Relse DlSeZioo | Doles oe Ole 4A a

eee 140 A O23 157) 6. OS F. ELLS(2 26) = 0/55 | 65/61 |= i Length 140-215

eee 4 a} 0. 24 521-6) 1] 51. sO 4- = 2} 41 O0l =n) — 154s

13“ JR} 3} 3| 3}2s| 5| of 4]. 31} 2) | 2}31] 4) a} 3]. sol sel eyl ot a eel ee

FE 1p eae 22114 = 4 Al 3b. 130} 1} 1) OF 14) OF 2] OF. 42| ool 7

eee re Ore (= Ole Opss| = b= oc) Ol= 41-01 33] 13-1] = 0-04.21 10 181 0

OO .

Fish 3. Length 90 mm..

6 above|12| “7/ 0| 7] 6 5! o| 4] 4 3! of 2lol Z| of of..) ..| ..J..].1..

See os 13} 8 O} 8/19) 8 410] 6) 5) O| 4,0} 7; OF 7]-8 4 OF 3) OF 8

Ae ete OO Pig] 8) 1-84 R12) 0) E115] 6 0; 54 15|- 7. 11 677

: as 13} 7) O} 8) 25) 9 1) 9)12) 7) OF 611) 7 OF 6}25 8 OF 7] 8 8 Eupomotis gibbosus. 15 specimens Length 100-135 mm. Table 8

2 “ #20! 10) O;}10]14; 9 OF 8}18) 8 OF 7/10) 8 .1} 8}22| 9 oO} 825) 9

1 “ 117} 7| of 6}22| 8} of 7}21} 9] 2 slisi 6 1) 5]21] 8} o| 7{15) 10 Band 1 Band 3

1 below} 22} 9} O| 8/29) 7 1) 6119) 7 O} 6/28} 8 1) 8}31) 9} 1) 8]24 7 Teeth Radii Lobes Teeth Radii Lobes

ee coe OT 71241. -7| 0), Of 7) 10| 0) 9730) —8|— 0|=7 | 24| 60/25 }.12)- 5 eee

eee) el Or Azo 7) O61 9} 01-8137 8-01-91. 24)— 7 01-6 | -15| 38 eatiorats so = Min. Av. Max.|Min. Av. Max.|Min, Av. Max.

eee sie ol OF 7 Pes a) OL Sie L 1). OF 10/37). 10-0) 6127) 6|— 0) 5 | -18|=7 —

5 “ |31) 7] O| 6/29) 6 OF 5] 6 8 Of 7/34 7] Of 7/31; 6 1) 5} 7 D o| of} 3[ d 4] 7] 0] 3] 6D 0} 4) 18) 0; 3) 7) OO; 2] 6

eee SOT Or 0 = 7.1.20). 01. 0-51; .|25} 8] O| 6]28) 7] O} 6 2A 0; O; OF 10 11} 14) 9) 10} 13}) 2A 20) 31) 45] -—5| “8 |-11)- -71- 421-10

fe 038) 10)" i 9129) 6) -0).. 5]. -|27; 10} 0} 9}28] 8} O| 7]. 2B 0} 27} 45] 7 9} 11] 6} 8} 11}] 2B (71235556) 5 S211 Se 7a 0

eee ese or LO 7.20 or 0) 7 ps {24 7] 0} 6]29) 6) O} 5S]. V 06231 544 Si 7510: 4o 5 Oey: 0) 2) =: 9) =-0) 3.180 eZ aes

a7 12. 7 1 6126 8 7). 24, 6} 1) 5) 28) 6 = 5S}. Lepomis Pallidus. 7 specimens Length 45-100 mm. Table 9

ee Ph oF 5.130] =. 8) 0 7}. 22| = 8} -0|\-7 | 271 8) 0-716 D 0} 24) 212) sie 25) 4 S30 eae

ie eee oO} OL FIM) 5) 0. 4]. ali —7}==0|- 6 26), 27-01-61 2A 0)=26|SAlb- 26)=10} 213 ieee oes

ee or 7) 61 ce? 7) 026] . 1244 6} OF 5]. 2B o| 311 49] 6| 91 12) 51 glu

re eo GO Mo PAZ) O11}. ae. : Niele ee Ol -6| sigh 1). 23 Slo one

eee AS | OF. 61. a : . - -

15). 121) “S). 0 6]. 16} 5} (O} 4Y.. Lepomis pallidus. 7 specimens = —- ar st ae “) on .

1, 2, and 3, etc., ab =Ist., 2nd., 3rd., etc., scales above lateral line.

ne 2, ‘sd 3, a pion iat aod: 3rd., etc., scales below lateral line. 2A 0} 24) 56 4)59'- 10 1}: 7% 9

R=regenerated scales. 2B 25| 34) 55} 8] 10} 15} 8] 8] 14

We Ole 7] 214) = Ol 2 Alo s isles

Microterus salmoides. 6 specimens Length 35-62 mm. Table 10

D 0) | a6) FS ee ESS ea eae 0 0}, 2) 4 [0 ess

2A OO b=23 aed 810 \ee8 eee 0 4), .6)|' + Seed eoolew

2B & jr (ei [eels |i SAS = 25] fete: Ger cf in #7 0 5) 66.) 7 ee Sol

V OSE le 0 = 413861" Oe 4a 0 Ol 3) 2S) 0 eee

Micropterus salmoides. 6 specimens Length 55-63 mm. Table 11

D 0) 0) a) Fe eT a aS my | ie: Ofs > 0) OFS OF 1S Siero ieee

2A OVO) (0) 6: 7A Si 5] One aac 0} 3)0 5] Ol aeG') Poy eo eo ee

2B Oh OF 0 es 721 Bit Or ear caeall ees O° 4| 91+ 5 6). 71) ete

Vv OWSO Of Ae ede 21s ew el ee Dies 0}: 0} Oly OF Aa S21 Ol eae

Micropterus dolomieu. 3 specimens Length 305 mm. Table 11 cont.

D OPO Ol eA a2 a2 | Sa ee Q)) 0) 0] &-0) =O) Ore sor Onan

2A O}-Ols O]) Bs 8s Bae eee 13). 13] 131) “Sik Sie Stas Sache

2B OSLO OF SaaeSci 5 Poulos onisae 30) 30} 30] -6) 64 6) So) oaies

V Ob DS Ohe He Sale Gt et an Sale QO} 0}... OF 0) 0) Ole

Tasces 8-11

Sewn

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DEPARTMENT OF NOTES, REVIEWS, ETC.

It is the purpose. in this department, to present from time to time brief original

notes, both of methods of work and of resuits, by members of the Society. All members

are invited to submit such items. In the absence of these there will be given a few brief

abstracts of recent work of more generai interest to students and teachers. There will be

no attempt to make these abstracts exhaustive. They will illustrate progress without at-

tempting to define it, and will thus give to the teacher current illustrations, and to the

isolated student suggestions of suitable fields of investigation.—[Editor.]

A RELIABLE METHOD FOR OBTAINING AMCEBA FOR CLASS USE

By CuHar.es A. Kororp

It is a common experience of the teacher in elementary biology

to find that his supply of Ameba for class use is uncertain in source,

variable in amount, and not infrequently fails at the appointed time,

or is so scanty that pupil and teacher both find more experience in

searching for the elusive creatures than in profitable observation.

Those who have available an Oscillaria rich margin at some outlet

of sewer or of a drain where a fairly permanent greenish-black felt

of this alga is present, can often secure an abundant supply of large

Ameba proteus. Decaying Ceratophyllum from pond or lake waters

also yields this Ameba in abundance for a brief period in the pro-

cess of decomposition in the laboratory aquarium. A permanent

Ameba culture of this species might be arranged and regularly

kept up in a culture house with supply of spring water suitably en-

riched. A culture of this sort of an apparently as yet undescribed

Amoeba was maintained at Lincoln, Nebraska, by Miss Powers and

supplied widely to the laboratories of the country from Atlantic

to Pacific for several years, but was unfortunately destroyed by

purification treatment of the city water supply.

In our laboratories at the University of California we have

for several years applied the principle of pure mixed cultures in

a crude way to the production of amcebas in quantity for class use

with such success that we offer it as one solution of the problem

of supply for large classes where the elimination of loss of time

in searching for the animals on the slide is desirable. The amceba

272 NOTES AND REVIEWS

we have cultivated in a small soil amceba, Negleria grubert

(Schardinger) originally described by Schardinger (1899) from .

the human intestine in a case of diarrhea. It is improbable, in view

of the use of pure culture methods in his work, that his culture was

contaminated from dust or water; though it is not impossible. The

amoeba in reality may have been recovered from the’ feces after

transit in an encysted condition through the intestine of the pa-

tient having been introduced in food or water. This is apparently

the same amoeba which Wherry (1913) later isolated from the

water supply of Oakland, California.

There are certain disadvantages in its use. It is very small,

about 9 to 30 microns, is not very mobile at room temperatures, lacks

prominent pseudopodia in most cases, and perhaps worst of all,

enflagellates on slight provocation, often at a very inopportune

time, and quite without regard to class schedules. The compen-

sating advantages are (1) the great numbers easily obtained, ten

or a dozen in a single high-power field, and thus the entire elimin-

ation of the necessity of search for the animals; (2) the occurrence

of cysts, amceboid, and flagellate stages and of both exogenous and

endogenous budding; (3) ease and certainty (in our experience)

of securing the cultures; and (4) ease with which slides of the

various stages may be prepared. A full account of its life-history,

as far as known, is now in press in the “University of California

Publications in Zoology” by Dr. Charlie W. Wilson (1915) now of

Mills College, Oakland, California.

The methods of culture which we employ for laboratory sup-

ply are as follows: We use as culture dishes enameled instru-

‘ment trays 6 by 10 inches in size which fit readily in the horizontal

autoclave, and accomodate each over fifty 22mm. floating cover,

glasses. As nutrient culture medium we use the filtered fluid from

a mixture of 50 grams each of lettuce leaves, horse manure, soda

cracker, and garden soil boiled for half an hour in a liter of creek

water or untreated tap water. This fluid is sterilized for 30 min-

utes in the autoclave and when thoroughly cool is shaken up with

a small quantity of soil and the water poured in the previously

sterilized pan to the depth of 2 centimeters and covered with a

glass plate. The pans should not be placed in the direct sunlight.

AMERICAN MICROSCOPICAL SOCIETY 273

Clean cover glasses are floated carefully by dropping them on the

surface of the culture fluid and are best removed by bent cover

glass forceps. Soil bacteria and the soil amcebas appear within a

few hours, and multiply rapidly, and are at a maximum in about

four days after the culture is started, the period varying accord-

ing to a number of factors such as temperature, richness of the

medium, and certain unknown chemical conditions. The medium

should not be too rich for best results and may be diluted to one-

half or less the strength above indicated by use of a larger pro-

portion of water. The numbers of ameebas are less in the poorer

cultures, but there is danger of excessive bacterial growth and fer-

mentation if the medium is too rich.

The amcebas rise to the surface and adhere in both ameeboid

and encysted stages to the under surface of the cover glass in as-

tounding numbers. For class use it is only necessary to make cer-

tain of infected soil by a trial culture, and of the time inter-

val between seeding the culture and the period of maximum

abundance of amcebas under the conditions of temperature, stock

of culture medium in use, etc. It is desirable to use a uniform,

or the same stock of culture medium for both the preliminary test

and final culture. When these factors are determined it is safe

to proceed and to rely upon the culture to yield the amcebas as

planned. It is possible to plant new cover glasses in the culture

after removing the first supply and within 24 hours to have a new

lot available. The cultures run down rapidly at first and then

slowly for weeks or months after their brief maximum.

We have found it advisable to keep a stock in culture and to

seed from it by adding a cubic centimeter of old culture fluid to

the unseeded new medium instead of using the original soil material.

Such a culture has now been continued in our laboratory for over

thirty months.

The conditions which stimulate enflagellation are apparently

access of oxygen and new food supply. For this reason cover

glasses should be used promptly on removal. They not infrequently

yield enflagellating amcebas within an hour and usually the whole

culture on the cover glass follows suit. Encystment is very common

and occurs both with abundant and with scanty food supply. Stained

274 NOTES AND REVIEWS

preparations are readily made of the amceboid and encysted stages

by floating the cover glass with its attached amcebas on warm

Schaudinn’s fluid amoeba side downwards for a few moments, and

then staining by the customary procedure in Heidenhain’s iron

hematoxylin. Ameeboid, budding and encysted stages are abun-

dant in such preparations and enflagellating, exflagellating stages

and the flagellate stage itself are all to be found by careful search

but are obviously more difficult to obtain in this way since Ry, do

not adhere habitually to the substrate.

Many of the amcebas are in stages of cell division, and many

cysts are binucleate simulating sexual phases. Care is essential

in interpreting these stages as sexual phenomena, which are as yet

not proven to occur in this amoeba. Chromidial extrusion simulat-

ing maturation phenomena also occurs in the cysts and many puzzl-

ing nuclear conditions, probably degenerative or involution phen-

omena, will be found in stained preparations from cultures, espe-

cially in the period of the decline from the maximum numbers.

Cultures easily become contaminated and ciliates from the soil

are usually inoculated with the amcebas. For this reason it is wise,

if one wishes to keep a permanent culture going, to isolate a ciliate-_

free cover glass for seeding the permanent culture.

Our experience leads us to believe this culture method adds a

valuable resource to our biological laboratory and increases our -

means of control of an abundant supply of this very important type

essential both for instruction and research.

PAPERS CITED

SCHARDINGER, F.

1899. Entwicklungkreis einer Amoeba lobosa (Gymnameeba): Ameba

Gruberi. S. B. Akad. Wiss. Wien, Math.-natwiss. Cl., 108, 713-73¢

pls. 1-2.

Wuerry, W. B.

1913. Studies on the biology of an ameba of the Limayx group.

Arch. Prot., 3z, 77-93, pls. 8-9, 8 figs. in text.

Witson, C. W.

1915. On the life-history of a soil amceba. Univ. Calif. Publ. Zool., 15.

(In press).

Zoological Laboratory, University of California.

October 22, 1915.

AMERICAN MICROSCOPICAL SOCIETY 275

NOTES ON METHODS OF LABORATORY TECHNIQUE FROM THE ZOOLOG-

ICAL LABORATORY, UNIVERSITY OF MICHIGAN

The writer presents the following methods of laboratory tech-

nique knowing full well that little originality can be claimed for

them but with the feeling that they have been sufficiently tried to

prove their worth.

Neutral red (Neutral Roth) as an indicator of the reaction of

the digestive fluids of Protozoa.—That neutral red may be used as

an indicator of the reactions of the digestive fluids of Protozoa has

been known for a considerable time but its use as such is not as

general as it should be. For this purpose freshly made solutions of

the dye in the proportion of 1: 10000 to 1: 100000 in distilled water

should be used. A few drops of the solution should be drawn

under the cover-glass or one or two drops of it should be added to

the water on the slide containing Protozoa before placing the cover-

glass. Examination should be made at once because the reaction

is secured within a few minutes. In the presence of alkalies neutral

red becomes yellow while in the presence of acids it becomes red.

Used in this manner the stain does not injure the Protozoa ap-

preciably. This test is readily made even by a beginning student

and it is easily interpreted by him. In this laboratory it has been

used in the study of Paramecium in order to introduce a little idea

of the physiology of Protozoa. It should be further stated that

after the digestive fluid in the food vacuoles becomes well stained

the mouth and gullet are more readily seen than in the untreated

specimens.

To demonstrate the difference in function of the skin glands

of the frog—lIt has been the usual experience that ordinary meth-

ods of fixation and staining of the skin of the frog do not yield

preparations which readily show differences in function of the

poison and mucus glands. Hematoxylin and eosin usually reveal

only structural differences not differences in secretions elaborated.

After numerous trials the following method was found to give best

results: Pieces of skin including the dorso-lateral dermal plice

are removed from a pithed frog, tied to a slide flesh side out and

fixed over night in Bouin’s picro-aceto-formol mixture. Ten micron

sections from this material are stained for a few minutes in a 0.1%

2/6 NOTES AND REVIEWS

solution of thionin in distilled water, washed for a few seconds to

a minute in distilled water, and briefly stained in a 0.5% solution

of erythrosin. The section must then be dehydrated rapidly,

cleared in xylol and mounted in balsam. By this method all nuclei

are stained blue, the contents of the small glands and the granules

within the cells of these glands are stained blue to reddish-purple.

Since thionin is a specific mucin stain these glands are shown to be

mucus glands. While the nuclei of the cells of the large glands are

stained blue the cells are either unstained or are pink; the secretion

which appears in the lumen of the gland as globules of varying sizes

is stained a brilliant reddish pink or red tinged with yellow. Thus

these glands are shown at once to be different in function from the

mucus glands, and the inference is that they are poison glands.

With such striking colorations the two kinds of glands are distin-

guished at a glance. By this method the muscles surrounding the

glands are stained pink, the stratum compactum another shade of

pink, blood cells pink or coppery red with blue nuclei. Boundaries

of epidermal cells are clearly shown.

Fixation in Gilson’s fluid followed by these stains gives fair

results but not as good as by the method outlined above. Unfor-

tunately sections stained by this method are not colored uniformly

from end to end, nor are different slides stained alike, nor is it

possible to turn out slides by wholesale when using these methods be-

cause each slide must have individual treatment. Slides so stained,

however, show many features not well differentiated by hematoxy-

lin stains in combination with other stains.

In the absence of thionin toluidin blue (Toluidin-Blau) used in

the same fashion gives almost the same results. Kresylectviolett

has not been tried on this tissue but it should be pointed out that it

is an excellent mucin stain.

A method. for staining the cephalic glands of immature trema-

todes—In an attempt to work out the relations of the cephalic

glands to their ducts in some minute immature trematodes a number

of aniline dyes, carmine, hematoxylin stains were tried. The ma-

terial had been fixed in formalin of unknown strength. Hzma-

toxylin which was first tried for toto preparations and sections gave

altogether erroneous ideas of relationships of the glands to the

AMERICAN MICROSCOPICAL SOCIETY 2/7

ducts and wrong ideas of the ducts because of over-staining and too

diffuse staining. Carmines were hardly more successful. Out of

a number of aniline dyes solutions of thionin and of toluidin blue,

ca 1% in distilled water, gave excellent results either for toto mounts

or for sections. Whole specimens or sections were stained about

ten minutes in either of these stains, differentiated by washing in

distilled water, dehydrated and finally cleared in xylol and mounted

in balsam. The length of time required for differentiation of the

stain in the whole worms was several minutes while a few seconds

or at most a minute sufficed for the sections. Sections were some-

times given a second stain with erythrosin which served to stain

muscles but did not aid in the study of the glands and the ducts.

The contents of the glands and of their ducts when stained with

thionin or toluidin blue are blue or purple in color and are seen to

consist of great numbers of small globules. In this case the indi-

cations are that this material is mucin. The walls of the glands

and ducts remain unstained. As a result of this fact two ducts

which lie very closely together and which appear as one when

stained with hematoxylin or carmine are shown as separate struc-

tures. Certain other structures are shown fairly well. The method

seems to be inapplicable to toto preparations of large trematodes.

To fasten the wax to the bottoms of dissecting pans.—The us-

ual method of fastening the wax in a dissecting pan is to have strips

of tin or other metal soldered or riveted to the ends or to the bot-

‘tom of the pan and so projecting that the melted wax will run under

the strips and hardening thus the mass is held in place. This meth-

od does very well for tin or copper pans but if it is desired to use

enameled pans it is not satisfactory. A method that is applicable

to any kind of a pan is to use ‘BB’ shot or bits of granulated lead

or zinc to weight down the wax. At the time of pouring in the

melted wax the shot or the bits of granulated metal are placed in

the corners of the pan. When cooled the weight of the metal holds

the wax in place. In case the wax becomes loosened it can be

readily removed from the pan, melted and repoured. When metal

strips are used in the old method the wax must be melted in the pan

and there is danger of opening up the seams in soldered pans.

278 NOTES AND REVIEWS

Celluloid-acetone cement.—A very useful laboratory cement

may be made by adding scraps of celluloid to acetone in which it —

quickly dissolves. Experience will soon teach the proper propor-

tions. Do not make it too thick. Since acetone evaporates quickly

the cement must be kept covered and the work must be rapidly done.

In our laboratory it finds many uses, e. g., in mending small broken

bones, cementing glass to glass or to many other substances, where

a waterproof cement is desirable. In mending small broken bones

with this cement it is advisable to run some of the cement into the

cavity, put pieces together, cover with a thin layer of the cement on

the outside. If a strong joint is necessary chop up some cotton

into short bits and work it into the cement as it is used. For the

mending of bones the transparent celluloid should be used to make

the cement but for other purposes it may be desirable to use colored

celluloid. The writer has seen this cement so skillfully used on

broken bones that very close scrutiny was required to discover the

fracture. Since this cement is dissolved or at least softened by

strong alcohols it cannot be used to mend things that are to be stored

in alcohol.

To render visible lines milled on laboratory apparatus —The

millings on apparatus that has been in use for some time usually

becomes indistinct. To render it again visible polish the surface

with a good metal polish and then fill the milled lines and figures

with ‘Shinola’ shoe polish, allow to dry and then rub off that por-

tion which remains on the plain surface. The lines will be filled

with the blacking. This material being neutral in reaction will not

injure the metal.

To revive or to keep going cultures of Ameba.—Ameba cultures

made after the method of Jennings, i. e., by allowing plant materials

collected from ponds or streams to decay in pond or stream water

usually begin to fail soon after they have begun to yield Ameba in

abundance. Such cultures may be kept going or, even after all

amebz have apparently disappeared, they may be made to yield in

abundance if a small quantity of sterile hay be added at intervals

of a few days. Probably any clean hay would do as well but if

sterile hay is used there is no danger of introducing other encysted

Protozoa. GeorcE R. LARveE.

University of Michigan. |

AMERICAN MICROSCOPICAL SOCIETY 279

ENTOMOLOGICAL NOTES

Bombycine Moths.—The National Academy of Sciences (1914)

has recently published, as First Memoir of Volume XII, Part III of

the “Monograph of the Bombycine Moths of North America”, by

A. S. Packard. The manuscript, left unfinished at the death of the

author, was edited by Prof. T. D. A. Cockerell, assisted by a num-

ber of other workers. This part of the Monograph includes the

families Ceratocampide (exclusive of Ceratocampine) Saturniude,

Hemileucide and Brahmeide. This large volume contains a sys-

tematic discussion of each of the North American species and im-

portant data are given for immature stages. Tables for the separ-

ation of genera and species are included. One hundred thirteen

plates accompany the text matter, of which thirty-four are wholly

or in part in color, representing primarily the larval stages of many

species. Students of Lepidopera will find this work of great in-

terest. .

Air Stores of Aquatic Insects—Ege (’15, Zeit. f. Allgemeine

Physiol., 17:81-124) has investigated the respiratory function of

the air stores carried by Corixide, Dystiscide, and Notonectide.

These air stores are found to have two distinct functions: (1)

They have a hydrostatic function, thus playing a very important

role in connection with the equilibrium of the animal and its relation

to the surface of the water. (2) They are of respiratory import-

ance and related to respiration in two ways: (a) they function in

ventilating the tracheal system when the animal is submerged, and

(b) they constitute a mechanism by means of which the insect is

able to utilize the dissolved oxygen of the water.

Trichopterous Larve—Krafka (15, Can. Ent., 47: 217-225)

has made a study of trichopterous larve and presents a key to the

families which will, no doubt, prove of great service in the identifi-

cation of the immature forms. The key also leads to the identifi-

cation of some of the principal subfamilies. Thirty-seven figures of

structural detail of larve are included in the paper.

Germ Cells in Hymenoptera—Hegner (’15, Journ. Morph.,

26: 495-535) reports the results of studies on the protoplasmic

differentiation in the oocytes of some of the Hymenoptera. The

ovariole of Apis mellifica has four regions: terminal filament, rosette

280 NOTES AND REVIEWS

region, differentiation zone, and posterior linear series of oocytes

separated by nurse cells. Each rosette group arises from a single

oogonium and the component cells are held together by persistent

spindle fibers from preceding mitoses. Oocytes and nurse cells

arise from oogonia, the descendants of which can be determined by

the presence of certain deeply staining rings between the cells.

Granules, apparently mitochondrial in nature, appear near the nuclei

of oocytes, later becoming distributed throughout the egg cytoplasm.

In Camponotus herculeanus var. pennsylvanica, each ovariole con-

sists of a terminal filament, terminal chamber, growth zone devoid

of “bacteria-like rods” and posterior, linear series of oocytes ac-

companied by nurse cells, the former being surrounded and subse-

quently invaded by the bacteria-like bodies. Secondary nuclei, the

origin and fate of which are in doubt, appear near the oocyte nu-

cleus, increase in number, ultimately surround the germinal vesicle,

and finally become distributed throughout the oocyte, particularly

in the vicinity of the follicular epithelium. In Copidosoma gele-

chie, Apanteles glomeratus, and Andricus punctatus the changes in

the oocyte nuclei are very similar, consisting essentially of early

chromosome formation, paired fusion of chromosomes, arrange-

ment on an asterless spindle, and condensation. The oocytes of

Diastrophus nebulosus contain a chromatin body which probably

results from chromosome condensation. In certain growth stages

of Rhodites ignota large numbers of secondary nuclei appear, orig-

inating, apparently, from certain peripheral granules which stain

like chromatin. ;

New Order of Insects—Crampton (715, Ent. News, 26: 337-

350) has studied the morphology and systematic position of the

remarkable annectent form, Grylloblatta campodetformis, which was

recently described by Walker from specimens found in Canada and

placed by him in a new orthopteran family, Grylloblattide. Cramp-

ton finds that the structural features of this species include a com-

bination of characters found in Dermaptera, Isoptera and Gryllide

and that there are sufficient grounds for considering Grylloblattide

a distinct order, the Notoptera. This new order occupies a position

intermediate between the Dermaptera and Isoptera and seems to

AMERICAN MICROSCOPICAL SOCIETY 281

include the ‘“‘nearest living representatives of the common ancestors

of the Gryllide and ‘Locustide’ (Tettigonidz). ”

Luminous organs —Wheeler and Williams (715, Psyche,

22: 36-43) find that in the New Zealand glow-worm, Bolitophila

luminosa, the four elongated Malpighian tubules, which extend to

the posterior region of the body, are differentiated into two parts:

(1) the long, proximal portions, which retain the primitive excre-

tory function of these organs; (2) the short, distal, dilated tips,

closely applied to the intestine, which appear as four curved, lumi-

nous rods in the living larva and constitute the photogenetic organ.

Distinct structural differences occur in the two differentiated parts.

In no other insect are the Malpighian tubules known to have a

photogenetic function, although they sometimes acquire functions

other than that of excretion, as for example, the production of silk

in certain Neuroptera and Coleoptera.

Wilt Disease —Glaser (’15, Journ. Agr. Research, 4: 101-128)

finds that the wilt of gypsy-moth caterpillars is a true infectious

disease which occurs all over the gypsy-moth territory. Epidemics

occur only in regions heavily infested with gypsy-moth and climatic

conditions appear to have an important relation to the disease in the

field. Certain imported parasites may be important factors in the

distribution of the wilt. There is no direct evidence that the dis-

ease is transmitted from one generation to another. Infection

naturally occurs through the mouth by means of the food. The

disease is more prevalent among the older larve.

Olfactory Sense in Coleoptera——MclIndoo (15, Biol. Bull.,

28: 407-460) reports the results of an extended morphological and

experimental study of olfaction in beetles. His results show that

the antennz do not bear any of the olfactory organs but certain

pores, found on the peduncles of the elytra, on the dorsal surfaces

of the wings, on the trochanters, tibiz, sometimes on the femora

and tarsi, and perhaps on the mouth appendages, are the true ol-

factory organs. .

Sex recognition—Sturtevant (715, Journ. Animal Behavior,

5: 351-366) finds that, in Drosophila, the olfactory and tactile senses

are probably concerned with sex recognition and that sight is not

essential. The wings of the male function in the production of

sexual excitement in the female. The characters of certain mu-

282 NOTES AND REVIEWS ~—

tants—white eyes, vermilion eyes, yellow body color, and curved

wings—seem to have no selective value. Evidence is presented in

support of the view that neither sex exercises any “choice” in the

selection of a mate but mating will occur when members of the two

sexes, ready to mate, chance to find each other.

Notonectide.—Essenberg (715, Journ. Animal Behavior,

5: 381-390) reports results of a study on the natural history and

habits of Notonectide. The food is chiefly living or dead insects,

and animals many times larger may be successfully attacked.

Chemicals in solution have very little influence on backswimmers.

They have a strong, positive phototaxis which increases with rise

of temperature and with greater light intensity. They are positively

rheotactic. The young resemble the adults in their behavior and

instincts.

Habits of Water-striders—Essenberg (715, Journ. Animal Be-

havior, 5: 397-402) finds that Gerris remiges is positively thigmo-

tactic, positively phototactic, positively rheotactic, and negatively

geotactic. Sense of smell is apparently present; hearing is not well

developed; and sight is very keen and efficient. No special choice

is shown in the selection of food, any dead or living animal matter

being consumed. Individuals may live for weeks or months with-

out food. They are actively predaceous but never attack animals

below the surface of the water. They possess the cleaning habit

and may be engaged in this activity for considerable periods of time.

Death feigning is common and may be produced by artificial stimu-

lation.

Middle Membrane in Wings.—Marshall (’15, Annals Ent. Soc.

Am, 8:201-216) finds that in Platyphylax designatus (Trichop-

tera) the structure, usually known as the middle membrane, is not

a true membrane but is a thin layer of protoplasm which occupies a

median position within the wing. During the development of the

wing this median layer is not continuous but disappears and is

reformed in the same position. The resulting layer is more mem-

brane like than the one first formed.

Preservative for Insects—Schulze, [’15, Entomological News,

26: 361 (abstract from Deut. ent Zeitschr., 1915, p. 204) ] recom-

AMERICAN MICROSCOPICAL SOCIETY 283

mends the following fluid for the preservation of galls, coccids on

plants, and larve for dissection: 200 cc. glycerine, 200 cc. dis-

tilled water, 1 gram crystallized carbolic acid.

This mixture is said to be serviceable for preservation in the

tropics.

Kansas State Agricultural College. Paut S. WELCH.

NOTES ON OLIGOCHATA

Hemonais—Stephenson (’15, Trans. Roy. Soc. Edinburgh,

50: 769-781) reports the discovery of a new species of Hemonais

in India. Heretofore, this genus was represented only by a single

species from Switzerland. A complete account of the anatomy is

given. In the sexually mature condition, the alimentary canal un-

dergoes atrophy. The mouth remains but the lumen of the canal

becomes indistinct and disappears in V, reappearing only posterior

to XI. The posterior region is nearly normal in appearance but,

in the zone of degeneration, the canal is merely band-like and its

transverse dimensions are less than those of the ventral nerve cord.

Dero and Slavina—Stephenson (’15, Trans. Roy. Soc. Edin-

burgh, 50:789-795) describes the anatomy of sexual individuals

of Dero limosa and finds, in these animals, a curious degeneration

of the digestive tract which has not been described in the Oligo-

cheta except for Hemonais, although a similar condition is known

to occur in certain Polycheta. The anterior opening disappears,

and the mouth cavity, pharynx, cesophagus, and intestine become so

profoundly changed that they are represented only by a narrow

cord for some distance. The lumen is absent anterior to XII.

Apparently, the development of sexuality marks the end of the life

of the individual. In this same paper, the hitherto unknown sexual

organs of Slavina are described in the species punjabensis.

Bifurcation in Lumbricus——Benham (’15, Trans. New Zealand

Inst., 47: 185-188) reports an interesting case of bifurcation in

Lumbricus rubellus. The right side of the third clitellar somite

gives rise to a posteriorly directed branch, about one-fifth as long

as the body and containing fifteen somites. The clitellum is con-

tinued on the three basal somites of the branch and the tubercula

pubertatis is continued along their outer side. The nerve cord and

284 | NOTES AND REVIEWS

the nephridia extend to the end of the branch but the intestine is

absent. Sete are apparently absent on all somites of the branch

except one. Bifurcations are not uncommon among earthworms

but this record is unique because of the unusual anterior position

of the branch, the great inequality of the two corresponding body

parts, and the absence of the intestine in the branch.

Regeneration—Hunt (715, Am. Nat., 49: 495-503) has found

that, in Enchytreus albidus, posterior regeneration occurs when the

animal is cut at any level between “eight segments from the poster-

ior end of the body and eight segments from the anterior end.”

From the caudal end up to the middle of the body, the rate of

regeneration increases almost in direct proportion to the number of

segments removed. Removal of the eight most posterior somites

may cause regeneration of double tails. Regeneration occurs either

in fresh-water or salt-water conditions and salinity apparently has

little or no influence on the rate.

Earthworms—Smith (715, Bull. Ill. State Lab. Nat. Hist,

10: 551-559) describes two new varieties of earthworms from Illi-

nois and presents a tabular key to the described species of earth-

worms known to occur in that state. The key is accompanied by

additional data on the distribution and habits of the different species.

The key will be very useful in the identification of the earthworms,

not only of Illinois but also of the adjacent states.

Kansas State Agricultural College. PauL S. WELCH.

THE OLFACTORY SENSE IN INSECTS

This problem has been a favorite one for students for one

hundred and fifty years, and very diverse conclusions have been

reached. N.B. McIndoo has published a summary of these con-

clusions under the title “The Olfactory Sense of Insects” in the

Smithsonian Miscellaneous Collections, Volume 63, No. 9.

The two principal views relative to the possible seat of the

olfactory organs of insects are :—first, that they are located largely

upon the antenne and palpi; and secondly, that the vesicles dis-

covered by Hicks and situated on the legs, wings, and other parts

of the body are the true olfactory sense organs.

AMERICAN MICROSCOPICAL SOCIETY 285

While admitting that the removal of certain portions and ex-

perimenting with insects may aid in settling this question, the pres-

ent writer is firmly convinced that observation of the normal ac-

tivities of insects, and comparative study of the cytology of the

various cells on the surface of the body of arthropods are more

fundamental and important in determining how the function is dis-

tributed. In the opinion of the writer many arbitrary and mis-

leading statements about the functions of surface structures of the

insects are contained in the literature, largely because the authors

were innocent of first-hand knowledge of the cytology of these

structures. It is desired in this note to present a series of diagram-

matic illustrations of the finer structure of some typical ectodermal

cells taken from thousands of sections of insects.

Apparently all ectodermal cells are potentially sensory cells.

The abundant branching of the nerve fibrils to the bases of these

cells shown in Fig. 1 (Plate XVI) suggests this. The section here

is of the pupa of the Tussock Moth and is perpendicular to the

surface.

Fig. 2. Structure of typical sensory :

scale from Tussock Moth pupa. S.C.—sen- ig. 3. Sensory Peg, Vespa Antenna.

sory cell; N.—nucleus; N.F.—nerve fibril; P.C.—peg cell; N.—nucleus; N.C.—nerve

C.P.—cyto-plastids; C.—centrosphere; S.F. connection; W.—peg cell wall; C.—centro-

—sensory fibrils; T.F.—terminals of sen- sphere; M.T.—membranous tip; F.T.—ex-

sory fibrils. posed fibril tips.

The typical internal structure of such a generalized sensory

cell is shown diagrammatically in Fig. 2. In this type of cell there

are two distinct types of fibrils having different origin. The fibrils

marked (H) arise directly from small bodies in the outer end of

286 NOTES AND REVIEWS

the cell which in their turn arise from division of the centrosphere.

These fibrils and their origin from the centrosphere suggest that

they are homologous with the motile and nervous fibrils of cells.

The origin of the other set of fibrils, which always lie in and

form the walls of all types of cells, is very obscure and discussion

of them is reserved for another paper. These fibrils are always

developed and in position before the cell wall is exposed to the air

and chitinized thereby. Being of a different chemical composition

these fibres are never chitinized but remain in position in the chitin

matrix. Their terminals are always naked and exposed, no matter

how heavy the chitin may be around them.

These fibril openings through the chitinous wall can be and are

modified along with the fibril products in various functional ways.

These modifications of the fibrillations of the epidermal cells form

a most interesting chapter in the structure of insects. The writer

holds that this structure is universal to the hypodermal cells of

insects. In thousands of instances studied we have yet to find any

type of such cells in which these fibrils do not perforate the chitin

walls and establish connection with the outside world.

To observe these structures to best advantage the material

must be well preserved and should undergo thorough staining,—

preferably in Iron-alum-hematoxylin. The tissue should lie in

the mordant two days and in the stain an equal time. This allows

the stain to penetrate the fibrils through the chitin matrix which is

very resistant to the stain.

The sensory cells of the epidermis may be modified from the

type in various ways. They may be sunk below the surface as in

Hicks’ vesicles, or compounded into pore-plates beneath (or flush

with) the surface, or raised into variously formed spines, scales,

hairs, pegs, etc. No matter, however, what the form or elevation

or degree of complexity the main structures are similar to those

diagrammed in the typical cell (Fig. 2). In the later figures are

given a few of the more characteristic variations from the typical

epidermal cell.

Fig. 3, is a peg from the antenna of a wasp, Vespa. It has a

membraneous tip through which the tip of a bundle of nerve fibrils

AMERICAN MICROSCOPICAL SOCIETY 287

are protruding. They extend quite a distance beyond the mem-

brane and stain nicely.

Fig. 4. Pore Plate, Vespa antenna. S.C. Fig. 5, Hick’s Vesicle, Honey Bee. S.C.

—sense cell; N.—nucleus; H.—chitin hypo- —sense cell; N.—nucleus; N.C.—nerve

erm; C.—centrosphere; B.—blood space; connection; C.—centrosphere; C.W.—chitin

S.F.—sense fibrils; F.SP.—fibril spheres; wall of cell; H.—hypoderm; S.F.—sense fi-

C.P.—chitin pore-plate. brils. -

Fig. 4, is a diagrammatic view of a compound pore-plate from

antenna of Vespa. This pore plate has a slit lengthwise of it in

a surface view; many Ichneumon flies have this form with very

enlongate slits. The pore plate is penetrated by the fibrils from the

group of cells which compose it. The nuclear end of the cell may

be extended by growth to a remote position from what it origin-

ally occupied.

The space (G) is filled with blood fluid and may by excitation

and flow of blood to the antenna slightly protrude the pore plate

which is suspended by a thin membranous ring. They might prop-

erly be termed erectile pore plates. At (E) on the diagram is a

row of darker staining spheres which we will call for want of

better name—fibril centrospheres. The ontogenetic origin of this

body from the parent centrosphere and its subsequent functional

history, have never been worked out, so far as we know. These

pore plates, wherever found in insects and related animals, always

have exposed fibrils extending through them. They are not solid

chitin disks as assumed by many writers.

Fig. 5, is a diagram of Hicks’ vesicles, which are a form of

sunken peg with a membranous top through which the nerve fibrils

288 NOTES AND REVIEWS

are exposed. The chitin body (D) is the wall of the sunken peg

cell.

Some writers consider this form of cell alone capable of re-

ceiving olfactory stimuli, denying that all homologous pegs which

are raised varying degrees above the surface are capable of such

function. The writer desires to stress the fact that these vesicles

of Hicks’ (1868) are but sunken pegs; and are not different in

main details from those raised above the surface.

Fig. 6, is a diagram of the sense scale from the antenna of the

crustacean, Oniscus. This shows that the nerve fibrils are exposed

in a similar manner in one of the related families of the Arthropods.

The writer has seen extensive areas around the mouth parts of

spiders where these fibrils are exposed on flat membranes.

Fig. 7. Sense Scale, Pyrameis antenna.

Fig. 6. Sense Scale, Oniscus antenna. §.C.—sense cell; N.—nucleus; N.C.—nerve

S.C.—sense cell; N.—nucleus; N.C.—nerve connections; H.—hypoderm; W.F.—wall fi-

connection; W.—wall of scale; C.—centro- brils; F.S.—flat part of scale; C.—centro

sphere; S.F.—sense fibrils at tip. sphere; S.F.—sense fibrils.

Fig. 7, is a diagram of a scale from the antenna of a butterfly—

Pyrameis,

From observation of many thousands of these various scale

types the writer is convinced that the evidence shows many forms

of epidermal scales which have all the cytological details necessary

to receive in some degree olfactory stimuli.

AMERICAN MICROSCOPICAL SOCIETY 289

As to what extent any particular type or set of cells actually

do receive such stimuli we deem it impossible at present to state.

Experiments which show that insects can smell both with and with-

out the antenne are of little value.

Some authors contend that the Hicks’ vesicles on the legs are

olfactory in function, while they absolutely deny that apparently

homologous cells on the antennz and palpi are capable of receiving

such stimuli. The antennz and palpi are but leg rudiments, a pair

from each of the five segments of which the head is composed,

being modified for various uses. In some forms of insects such as

some Chironomide the antenne are becoming rudimentary, while

the first pair of true legs are greatly enlongated and used as sense

organs, being extended toward and waved toward objective points.

In other insects which do not feed in the adult state, such as some

Ephermeride, the antennz are very rudimentary.

Again some insects which do not feed in adult stages, such as

some families of moths, have very large and much branched an-

tenn. In this case they seem to be connected with sexual func-

tions. An almost endless list could thus be given of the modifica-

tions of the various forms of legs in the different insect families.

These legs are homologous. There is nothing to indicate that their

epidermal cells may not be homologous also. The minute structure

of these cells as detailed above further reinforces the conviction

of their homology. But the fact remains that all insects respond

to odors, no matter what the modificactions of the epidermal appen-

dages. | |

Many experiments have been devised by various students to

try and show by mutilation which organs are olfactory and which

not. Antennz, palpi, tongties, legs, wings, styles, halters, stings,

etc., have been removed in an endeavor to locate the olfactory

sense. While the results are equivocal, most observers agree that

the sense is not confined to any one set of appendages. We believe

that the antenne can and do act as receptors of olfactory stimul1

under normal conditions, both for the reasons given above and

because of observations upon the animals. Extensive observations

were made this summer on normal insects engaged in feeding and

other duties, to try and determine this point. For many days the

290 NOTES AND REVIEWS

various bees which visited a patch of columbine were observed. As

the nectary tubes of these flowers are too long for the tongue of the

bees, they invariably went to the rear of the flower and ripped the

nectary open with the tongue. The bee working on the outside

of the flower was in good position to show the normal use of the

antenne. The antennz were invariably waved toward, then placed

in direct contact with the nectary, and the presence or absence of

food determined thereby. Out of thousands of bees which visited

the flowers not one was observed to feed without first making an

examination with the antenne.

Many ants were studied in their travels, and the antenne were

found to be constantly in use. When near enough, making contact

with the object; when further away, waving them towards the

object.

Many species of Ichneumon flies were observed. Their anten-

nz are almost constantly in motion, apparently receiving most of

their stimuli without contact.

For these reasons we believe that there are many types of epi-

dermal cells widely scattered over the body which are normally

capable of and do receive olfactory stimuli.

The arbitrary assumption of this function as belonging to any

one type of cells without adequate cytological investigation can but

lead to scientific mistakes. The call is for more and better cytologi-

cal work.

Battle Creek, Mich. E. W. RoBeErts.

ARACHNOIDISCUS IN MARYLAND DIATOMACEOUS EARTH

About a year ago a friend in Maryland sent me a piece of

diatomaceous earth which was uncovered by the excavation for a

State road. It did not look very promising, being very sandy.

Some time later, however, I cleaned a part of it and during the

process was rather disgusted to find fragments of Arachnoidiscus

mixed with it. It never seemed possible that the diatom might

belong there, so I cast the stuff away and started anew only to find

them in the new batch. I then became suspicious, but remember-

ing an article I had read by Kitton in an old number of Science

Fig. 1. Nerve Fibrils to Ectodermal Cells in Tussock Moth.

* ie

eee

Thy

in va

Fig. 2. Arachnoidiscus ornatus, Marylandica.

PLATE XVI

AMERICAN MICROSCOPICAL SOCIETY 291

Gossip in which he attributed the finding of a frustule of Arach-

noidiscus in British waters to the use of dirty cleaning vessels, |

secured an entirely new set of glasses and started again. The result

was conclusive. Arachnoidiscus, apparently ornatus, was there,

plentifully, together with Actinoptychus calicinus, a form I have

never before seen in the Maryland deposit.

Repeated cleanings have verified the find. I have since visited

the locality and made careful examination of the bank from which

it came. |

The Arachnoidiscus apparently occurs only in the topmost part

of the deposit, and a few feet underneath the level at which it occurs

most plentifully it disappears entirely when we reach the typical

rich bed of diatomaceous earth. Another outcrop about five hun-

dred yards away also shows it; but elsewhere I have been entirely

unable to find it.

The deposit, a part of the Maryland Miocene, of which the

Richmond beds form a part, is at a point known as Bird’s Hill on

the State Road about nine miles south of Annapolis. At this place

is about one hundred feet above sea level and is the highest point

I know of at which this earth shows. The deposit is only a few feet

above sea level at Ferry Landing opposite Nottingham and at the

Calvert Cliffs on the Chesapeake Bay, both these latter places being

about twenty-fivé miles south of Bird’s Hill.

The Arachnoidiscus here mentioned closely resembles Arach-

noidiscus ornatus of the Pacific and I have taken the liberty of

naming it Arachnoidiscus ornatus, variety Marylandica.

I may be mistaken in thinking I have really made a discovery

and regret that the great authorities are no longer alive to pass on

it but I can find nowhere any reference to such a find either in

Atlantic waters or in the fossil deposits adjacent thereto.

I am sending with this mounted specimens which I hope may

be reproduced for the benefit of your readers who may be interested.

(See Pl. XVI, Fig. 2).

BLANTON C. WELSH,

U. S. Army, Retired.

Montclair, New Jersey,

July 8, 1915.

292 NOTES AND REVIEWS

A SATISFACTORY DISSECTING BOARD

The dissecting of the cat, dogfish and some of the smaller ani-

mals caused in my laboratory, as I presume it has in others, a de-

mand for an adequate dissecting board. In taking charge of a

course in cat anatomy seven years ago there came into my posses-

sion also some dissecting boards supplied with plumb-bobs and

B G

Details of Dissecting Board.

strings for spreading and holding the specimens in place. Such

trays are little if any better than a plain board with nails driven in

the sides to which the strings may be tied. Both of these types

were replaced by the simple piece of apparatus herein described

which has given complete satisfaction.

The principle part of the device consists of a board 70 cm.

long, 40 cm. wide and 12 mm. thick screwed to two cross pieces B

30 mm. thick, one of which is 55 mm. high and the other 75 mm.

high. ‘These pieces are cut in such a way as to give a dish to the

board 12 mm. deep. The difference in the heighth of the two pieces

gives a sufficient slope to carry away any liquid that might come

from the specimen. To either side of the board are fastened five

iron cleats as shown in the figure (A). Each cleat C is bent at an

angle of 120° and the free end, 15 mm. long, carries a V-shaped

slot for its full length. The slot at its open end is 4 mm. wide

The specimen is held in place and spread by means of pieces of

Jack-chain (No. 16 or 18) 20 cm. long provided with fish-hooks

AMERICAN MICROSCOPICAL SOCIETY 293

with the barbs removed. After the hooks have been attached to

the animal the chains are drawn into the slots of the cleats where

they are held firmly.

University of Utah. NEwTon MILLER.

MECHANISM OF MENDELIAN HERIDITY

In a book bearing this title T. H. Morgan and co-workers have

undertaken to bring to the general biologist the significant steps

that have been made by the geneticists in the study of inheritance,

and the correlation of these results with the discoveries made by the

students of the minute structure of the germ cells. It is perfectly

apparent that these cells, the egg and the sperm, must contain the

mechanism by which hereditary qualities are handled from gener-

ation to generation. It is manifest that the wonderful results

which the Mendeélians have been able to get by breeding experiments

will be explained, if ever, only through the study of the minute

units of cytoptasmic and nuclear structure, which must be their

material basis.

As is well known the author strongly supports the view that

the chromosomes are the particular part of the protoplasm which de-

termines such hereditary phenomena as follow the Mendelian prin-

ciples. No cytological work has done more to discover the facts

which tend to support this view than the brilliant investigations of

Professors Wilson and Morea and their students in Columbia

University.

This book brings together the wonderful series of parallels be-

tween the observed conditions of the chromosomes and the statis-

tical behavior of unit characters in breeding, which we describe

collectively as Mendelian. For example, the observed segregation

of chromosomes in the genesis of the germ cells coincides remark-

ably with the theoretical demands of the Mendelian principles of

segregation resulting in the “purity of the gametes.” Similarly there

has been established in some organisms a parallelism between the

nuinber of chromosomes and the number of hereditary groups of

characters ; also in some degree between the size of the chromosomes

and the size of these groups of characters.

294 NOTES AND REVIEWS

A more difficult task outlined and undertaken in this book is

to show that there are parallels in the behavior of the chromosomes

to those more complex dissolving of groups of characters which

allows more or less freedom (and more or less linkage) of one unit

quality to other unit qualities. If chromosomes carry the unit char-

acters it is clear that each chromosome must carry factors relating

to numerous unit characters, since there are many more characters

than chromosomes. From the way that different unit characters

behave from generation to generation, it is clear also that there

must be machinery for the separation and crossing over of the

factors of the unit characters from one chromosome to another in

differing degrees. The authors find this possibility in the twist-

ing, the partial unions, and the separation along new lines that

occur in the period of conjugation of the chromosomes in the ma-

turation of gametes.

Brief statements are made of other offered explanations of the

Mendelian phenomena of inheritance.

The discussion is given under the following Chapter headings:

~ Mendelian Segregation and the Chromosomes; Types of Mende-

lian Heredity; Linkage; Sex Inheritance; Chromosomes as Bearers

of Hereditary Material; Correspondence between the Distribution

of the Chromosomes and of the Genetic Factors; Multiple Allelo-

morphism; Multiple Factors; The Factorial Hypothesis. The Ap-

pendix contains directions for the breeding of Drosophila, the fruit

fly on which much of the investigation of Mendelian phenomena

in animals has been made; a Bibliography; and an index. Me-

chanically the book meets every expectation.

Mechanism of Mendelian Heredity, by Morgan, Sturtevant, Muller, and Bridges.

262 pages, 64 illustrations. Henry Holt & Co., New York, 1915. Price $3.00.

CONSTITUTION

ARTICLE I

This Association shall be called the AMERICAN MICROSCOPICAL

Society. Its object shall be the encouragement of microscopical

research.

ArTICLE II

Any person interested in microscopical science may become a

member of the Society upon written application and recommenda-

tion by two members and election by the Executive Committee.

Honorary members may also be elected by the Society on nomina-

tion by the Executive Committee.

ArTIcLeE [IT

The officers of this Society shall consist of a President and two

Vice-Presidents, who shall hold their office for one year, and shall

be ineligible for re-election for two years after the expiration of

their terms of office, together with a Secretary, a Treasurer, and

a Custodian, who shall each be elected for three years, be eligible

for re-election, and whose terms of office shall not be coterminous.

ARTICLE IV

The duties of the officers shall be the same as are usual in simi-

lar organizations; in addition to which it shall be the duty of the

President to deliver an address during the meeting at which he pre-

sides; of the Custodian to receive and manage the property and

permanent funds of the Society under the direction of the Executive

Committee and in conjunction with a permanent committee to be

called the Spenecr-Tolles Fund Committee, and to make a full and

specific annual report of the condition of all the property, funds,

and effects in his charge; and of the Secretary to edit and publish

the Transactions of the Society.

ARTICLE V

There shall be an Executive Committee, consisting of the offi-

cers of the Society, three members elected by the Society, and the

296 CONSTITUTION

past Presidents of the Society and of the American Society of Micro- -

scopists who still retain membership in this Society.

ARTICLE VI

It shall be the duty of the Executive Committee to fix the time

and place of meeting and manage the general affairs of the Society.

ArTIcLE VII

The initiation fee shall be $3, and the dues shall be $2 annually,

payable in advance. But any person duly elected may upon payment

of $50 at one time, or in installments within the same year, become

a life member entitled to all the privileges of membership, but ex-

empt from further dues and fees. All life membership fees shall

become part of the Spencer-Tolles Fund, but during the life of such

member his dues shall be paid out of the income of said fund. A

list of all life members and of all persons or bodies who have made

donations to the Spencer-Tolles Fund in sums of $50 or over, shall

be printed in every issue of the Transactions. The income of said

fund shall be used exclusively for the encouragement and support of

original investigations within the scope and purpose of this Society.

The principal of the fund shall be kept inviolate; ‘Provided, how-

ever, that nothing in this constitution shall prevent the Executive

Committee at any regular meeting from transferring the Spencer-

Tolles Fund to a University, or other incorporated institution for

original research, under such conditions as shall safeguard the per-

manence of the Fund, and its application to the general purpose

for which it was intended; such power to be vested in the Execu-

tive Committee only after securing, and in obedience to, the ex-

pressed will of a majority of the constitutional members of the

American Microscopical Society, or after the constitutional failure

of said society.

ArTIcLeE VIII

The election of officers shall be by ballot.

-ARTICLE IX

Amendments to the Constitution may be made by a two-thirds

vote of all members present at any annual meeting, after having

been proposed at the preceding annual meeting.

AMERICAN MICROSCOPICAL SOCIETY 297

BY-LAWS

ARTICLE [

The Executive Committee shall, before the close of the annual

meeting for which they are elected, examine the papers presented

and decide upon their publication or otherwise dispose of them.

All papers accepted for publication must be completed by the

authors and placed in the hands of the Secretary by October Ist

succeeding the meeting.

ARTICLE II

The Secretary shall edit and publish the papers accepted, with

the necessary illustrations.

ArtTIcLeE ITI

The number of copies of Transactions of any meeting shall be

decided at that meeting. But if not decided, the Secretary shall,

unless otherwise ordered by the Executive Committee, print the

same number as for the preceding year.

ARTICLE IV

No applicant shall be considered a member until he has paid his

dues. Any member failing to pay his dues for two consecutive

years, and after two written notifications from the Treasurer, shall

be dropped from the roll, with the privilege of reinstatement at any

time on payment of all arrears. The Transactions shall not be sent

to any member whose dues are unpaid.

“ARTICLE V

The election of officers shall be held on the morning of the last

day of the annual meeting. Their terms of office shall commence at

the close of the meeting at which they are elected, and shall con-

tinue until their successors are elected and qualified.

ARTICLE VI |

Candidates for office shall be nominated by a committee of five

members of the Society. This committee shall be elected by a

298 CONSTITUTION

plurality vote, by ballot, after free nomination, on the second day

of the annual meeting.

ArTIcLE VII

All motions or resolutions relating to the business of the Society

shall be referred for consideration to the Executive Committee

before discussion and final action by the Society.

ArticLe VIII

Members of this Society shall have the privilege of enrolling

members of their families (except men over twenty-one years of

age) for any meeting upon payment of one-half the annual sub-

scription ($1).

ARTICLE IX

There shall be a standing committee known as the Spencer-

Tolles Fund Committee to take general charge of the fund and to

recommend annually what part of the income shall be expended for

the encouragement of research, but the apportionment of the sum

thus set apart shall be made by the Executive Committee.

The Spencer-Tolles Fund Committee shall also have general

charge of the expenditure of such money as may be apportioned,

under the conditions laid down by the Society for its use.

The Custodian shall be an ex-officio member of this committee.

ARTICLE X

The Executive Committee shall have the power annually to

appoint two members to represent the Society on the Council of the

American Association for the Advancement of Science, in accord-

ance with the regulations of the latter organization.

Revised by the Society, December, 1913.

CHARLES EDWIN BESSEY

PoATe VII

c ; . ; F F be ae ay Ae as

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NECROLOGY

BEssEeY, CHARLES Epwin, Ph.D., L.L.D., ’98........ Lincoln, Nebr.

BODINE, DONALDSON: TOG GO ee Ora oe Crawfordsville, Ind.

GAGE, Mrs. SUSANNE PHELPS, ’87........c00ceee00- Ithaca, N. Y.

Hyatt, JONATHAN DEUELL (Hon. Member)..New Rochelle, N. Y.

OMT ee WAT Le cee ch re Os eaten Po eet eres Portland, Me.

TYRRELL, E. G. Harcourt, 713........ Eshowe, Zululand, S. Africa

A SKETCH OF THE LIFE OF CHARLES EDWIN BESSEY

By Raymonp J. Poo.

Charles Edwin Bessey was born in a log house on a Wayne

County, Ohio, farm May 21, 1845. His parents who were of

French and German descent were Adnah Bessey and Margaret

Ellenberger Bessey. The earlier ancestors emigrated from the Con-

tinent to England during the period of extreme religious persecu-

tion which characterized the Huguenot days. Later some of them

came to America and settled first in Pennsylvania and later migrated

to Ohio about 1832.

Professor Bessey’s education was received in the country

schools and academies of Ohio. The preparatory days were fre-

quently interrupted by periods of teaching, by the uncertainties

accompanying the war and by the death of his father in 1863.

But after several years of preparatory study in the lower schools

Bessey entered the freshman class at the Michigan Agricultural

College in July 1866. He completed the college course and was

given the degree of bachelor of science on November 10, 1869 at

the age of twenty-four.

When he went to the college Bessey was convinced that he

wanted to become a civil engineer or surveyor since he had a

great liking for the out-of-doors and had had some practical ex-

perience in surveying and a natural inclination for that line of work

from his father. His father was a surveyor of note in northern

300 NECROLOGY

Ohio. But young Bessey found that his mind was drifting toward

things botanical. This tendency was discovered by some of his

instructors who encouraged him to enter the field of botany. At

first he spurned that encouragement and advice but as his college

work drew to a close the inclination toward botany became more

and more pronounced until finally he determined to become a bot-

anist. This decision was plainly shown when he accepted an assist-

antship in horticulture and was placed in charge of the greenhouse

at his Alma Mater immediately after his graduation. However

that position was held for a very short time because in December

of that year (1869) he accepted an offer to become instructor in

botany and horticulture in the Iowa State College at Ames. He

began what was to be a fifteen-year service in Iowa in February,

1870.

In 1872 he was promoted to a full professorship at Ames and

during the same year the Michigan Agricultural College conferred

the degree of master of science upon him. Still later he was made

professor of botany and zoology at Ames and in 1874 he went to

the University of California by invitation of the president to deliver

a series of lectures on botany. As one examines the outlines of the

lectures which were delivered in that course it is noted that they

were dominated by systematic botany of that day but they also

contained an excellent summary of the whole field of botany as

it was then known. The sequence in the taxonomic part of the

lectures was from lower to higher forms but what is most signifi-

cant is that with the flowering plants the sequence followed was

the Benthamian reversed.

At Ames Bessey soon became the president’s helper and later

his confidential friend. The president found that his services were

of much value in his office and upon the grounds of the college.

Many of the fine trees and walks and drives that now grace the

campus at Ames College were planted or laid out by Bessey or

under his supervision. Because of this close association with the

president Bessey first secured a near view of the methods and man-

agement of higher educational institutions. As he once remarked:

“This work was all outside my regular duties and I received no

extra money for it but it paid in what it did for me.’’ How true

AMERICAN MICROSCOPICAL SOCIETY 301

that was can be best appreciated by those who were so fortunate

as to be closely associated with him in his later years when all of

the values of the earlier experiences were crystallized into a most

fruitful and inspiring educational climax.

An incident of great importance to Professor Bessey was his

first meeting with Dr. Asa Gray which occurred at the Dubuque,

Iowa sessions of the American Association for the Advancement of

Science in August 1872. Gray was the retiring president of the

Association at that time and Bessey was elected to membership

during those meetings. Professor Bessey also met many other men

who were to become his long-time friends and co-laborers in botany

and in the American Association at that meeting. His initial meet-

ing with Dr. Gray was especially pleasant and resulted in arrange-

ments being made whereby Bessey went to Harvard during the

winter of 1872-1873 where he studied under the great master bot-

anist. The subject of botany had been so richly illumined by Dr.

Gray during those three months that the young western botanist

returned for another period of work at Harvard in the winter of

1875-1876. Those months of enthusiastic study with Gray and the

years of extremely pleasant correspondence that followed were

among the most delightful memories of Professor Bessey’s later

years,

Bessey had served the state of Iowa in a varied educational

and scientific program for a period of fifteen years when in June,

1884 he received notice that he had been elected to a professorship

in the University of Nebraska. The election had been made with-

out his knowledge but he went to Lincoln “‘to look the place over.”

He found that there was no laboratory for botany and that there

was no botanical equipment whatever at Nebraska so naturally he

was loath to leave his well established department at Ames. The

position was accordingly refused. However a second offer was

made to include the deanship of the industrial faculty and in that

form Bessey accepted the call. The inaugural address was delivered

in September 1884 but Professor Bessey did not move to Lincoln

until after the completion of the college year at Ames, November

18, 1884. He began his first class work at the University of Ne-

braska in January 1885.

302 NECROLOGY

The moment that Bessey arrived in Lincoln he became an

enthusiastic citizen of his new city and state and he began at once

to make himself at home in the new position. In doing that he

was but true to the request of his mother expressed many years

before when Bessey as a mere boy was leaving his home in Ohio

to go to certain work in Michigan. Upon that occasion his mother

said to him: “Charles, wherever you are be one of the people of

that community; act as though you intended to live there always,

and make yourself so useful that they cannot spare you.” The

degree to which he fulfilled that request when he went to Michigan,

to Iowa and finally to Nebraska must have been one of the most

gratifying recollections of his aged mother as she reviewed the

career of her boy especially at the times of his visits to the old

Ohio home which were made annually until the mother passed away

at a ripe old age only a few years before the death of her illus-

trious son.

The state of Nebraska loved Dr. Bessey and during his life of

more than thirty years in this state he reciprocated that affection

to the fullest. But that was only one of the many directions toward

which an overflowing measure of devotion and enthusiasm carried

that good man. His broad-mindedness coupled with the many-

sidedness of his personality made Bessey a most valuable citizen

of the state. His intellectual horizon was wide enough and his

sympathies deep enough to include the great and small affairs of

the state and its people and to stimulate the development of the

highest and best attainment in all of those with whom he came

in contact. f

No one was more active in the scientific organizations of the

country than Bessey. From that first meeting of the American

Association at Dubuque he seldom missed one of the annual gather-

ings of the numerous societies to which he belonged. He was al-

ways a conspicuous figure at such meetings where his active co-

operation and his wise counsel were often sought and as often freely

given. As the years wore on and his associates and former students

became more and more numerous and widely scattered there were

times of most delightful reunions especially between the master

and his pupils.

AMERICAN MICROSCOPICAL SOCIETY 303

Many scientific honors were conferred upon him during his

long association with American men of science. In 1880 he was

made a fellow in the American Association for the Advancement

of Science. He was president of the Society for the Promotion

of Agricultural Science and of the Western Society of Naturalists

in 1889. Upon four different occasions he was vice-president of the

American Association and chairman of the botanical section. He

was also a charter member of the Botanical Society of America of

which he was president in 1895. From 1880 to 1879 he was botanical

editor of the American Naturalist and from the latter date until

the time of his death he held a similar position with Science. Dur-

ing this long period scarcely a paper or book appeared and escaped

a review or at least a notice from his pen. As I have looked over

collections of hundreds of such reviews I have been impressed with

the tremendous amount of labor which all that necessitated. If

one had in his possession all of the original books, papers, periodi-

cals, bulletins, monographs, theses, etc., etc. which Professor Bessey

reviewed or noted during that long service to science he would be

equipped with a most excellent and fairly complete library of the

more modern works on botany. That was a service which is prob-

ably not appreciated by one unfamiliar with its magnitude and sig-

nificance. Perhaps the highest scientific honor which came to him

was his election to the presidency of the American Association for

the Advancement of Science at the Minneapolis meeting in 1910-

1911. Dr. Bessey also enjoyed membership in many other scien-

tific clubs, societies and academies in America and abroad. He

secured the degree of Doctor of Philosophy from the University

of Iowa in 1879 and the Doctor of Laws degree was conferred

upon him in 1898 by Grinnell (Iowa) College.

Profesor Bessey was an effective speaker and a voluminous

writer. Many of his earlier addresses and publications had a direct

bearing upon the practical application of scientific botany to the

every day life upon the farm and in the orchard, garden or forest

or upon the wide grassy prairies and plains. Scores of articles

of this sort were published in the farm papers of Iowa and Ne-

braska during his forty-five years’ of labor in those two states.

The well known series of textbooks which he began before he en-

304 NECROLOGY

tered upon his professorship at Nebraska and the numerous techni-

cal papers are too familiar to the members of this society to re-

quire enumeration at this time. For more than a quarter of a cen-

tury he worked upon the “phyletic idea” in taxonomy and the

phylogenetic development of the various groups of the plant world.

Botanists probably know of this phase of his contributions (after

his texts) better than of any others. His last paper was entitled

“The Phylogenetic Taxonomy of the Flowering Plants.”

Of the many services Bessey rendered to science in general

and to botany in particular which are of interest to the members

of the American Microscopical Society perhaps the most interest-

ing was the introduction of the laboratory method of instruction

with the use of compound microscopes at Iowa State College in

1873. Possibly there were one or two other laboratories of that

kind in America at that time but Bessey did not know of their ex-

istence. Later (1881) he gave the first botanical instruction in the

University of Minnesota in which microscopic methods were in-

cluded with the rest of the laboratory work. The microscopes for

that course were borrowed from the college at Ames, since the

University of Minnesota owned no such instruments at that time.

Bessey also introduced that kind of instruction into the Nebraska

schools after he had begun similar work at the University. He

also published several papers dealing with microscopic plants and

their relationships in the Transactions of the American Microscop-

ical Society. Among the more important of those contributions

may be mentioned those upon the phylogeny and classification of

diatoms, desmids and other algz and also upon bacteria and phy-

comycetous fungi. He was president of this society in 1902 and

presided at the Pittsburg meeting in June of that year.

But if everything possible had been said of his numerous and

varied contributions to science and of the many delightful and

enviable features of his life among men we must still insist that

we had not touched upon the most powerful and ever-broadening

effects of this man’s work because these were recorded in the class-

room, in the laboratory, and all about the college and university

as a captivating teacher and fatherly guide for the young. Possibly

there have been greater botanists but there has never been a greater

AMERICAN MICROSCOPICAL SOCIETY 305

teacher of botany than Dr. Bessey. Few men ever enjoyed a broad-

er knowledge and a wider point of view of his subject. He was at

home in nearly every phase of the great study of plants. And he

insisted that his students secure the very broadest training possible

in the subject. The stimulating methods of the man and the esprit

de corps that were constant delights about his department attracted

hundreds of students from surrounding states and even from dis- |

tant lands. The laboratories increased in number, the library and

the herbarium grew in size and efficiency but never did this accre-

tion catch up with the demands of his department. The herbarium

of about 250,000 specimens was finally crowded out of the building.

He was compelled to take the large classes to some other part of

the campus and even there to divide them into smaller sections be-

cause there was not a room large enough to accomodate the whole

class at one time. He was always crowded for room and it is pe-

culiarly sad that he did not live to enjoy more commodious quar-

ters in the new biological laboratory which is now in process of

construction and which will bear his name. Dr. Bessey regarded

teaching as his greatest work, as his greatest service to mankind

and in that he labored with a joy and a bouyancy which would have

kept him young for many additional years had not the long strain

of the scarcely varied school room duties worn down the physical

body and hastened the approach of the final crisis.

Charles E. Bessey was married to Miss Lucy Athern of West

Tisbury, Martha’s Vineyard, Massachusetts on December 25, 1873.

To this union three sons were born, Edward, Ernst and Carl. The

three boys graduated from the University of Nebraska with high

honors. Edward and Carl specialized in engineering. The for-

mer died on July 12, 1910 while he was assistant professor of

electrical engineering in the Colorado Agricultural College. Carl

is at present assistant chief of a large engineering firm with offices

in Chicago. Ernst followed his father’s tendencies and specialized

in botany. He is at present professor of botany in the Michigan

Agricultural College where his father received his undergraduate

training. Mrs. Bessey, the mother, retains her home at 1507 R

Street Lincoln.

306 NECROLOGY

JONATHAN DEUELL HYATT

Jonathan D. Hyatt, an Honorary member of the American

Microscopical Society, died at New Rochelle, N. Y. Dec. 18, 1912,

in his 88th year. Mr. Hyatt was a charter member of this society

at its formation in 1878, and was elected to Honorary membership

in 1905. He was its President in 1881.

He began his career as a teacher in the county schools of

Dutchess County, N. Y.. After serving as principal in various

high schools in New York State he became principal of the High

School in the Borough of Bronx. He retired from teaching in

1904.

Mr. Hyatt was much interested in the natural sciences, and

was a. diligent worker with the microscope. He was one of the

original members and a President of the New York Microscopical

Society, a member of the Torrey Botanical Club, and Fellow of

the Royal Microscopical Society. His anatomical studies of the

honey bee and the cicada are among his best work in natural history.

His family is notably long-lived. His mother was 100 years

and 614 months old at her death; two brothers each lived to be 86%

years of age; he is survived by a sister now 92 years old.

JONATHAN DEUELL HYATT

PLATE XVIII ;

AMERICAN MICROSCOPICAL SOCIETY 307

LIST OF MEMBERS

HONORARY MEMBERS

Crisp, Frank, LL.B., B.A., F.R.M.S.,

5 Landsdowne Road, Notting Hill, London, England

PREAUM, MAGNUS (icc ccerercdscwecbcssereededaccsseedpueds Meadville, Pa.

Warp, R. Hatstep, A.M., M.D., F.R.M.S.......... 53 Fourth St., Troy, N. i a

LIFE MEMBERS

Brown, J. STANFORD, Ph.B.; A.M......... 299 Madison Ave., New York City.

Cape, Serm: BUNKER: 00 < cesicdavine seep see P. O. Box 2054, Philadelphia, Pa.

DuncaANSON, Pror. Henry B., A.M....... R. F. D. 3, Box 212, Seattle, Wash.

Exuiott, Pror. ARTHUR H...........e0se00- 52 E. gist. St. New York City.

HATELY, JOHN C.......cccececscsccceeres Chicago Beach Hotel, Chicago, Ill.

MEMBERS

The figures denote the year of the member’s election, except 78, which

marks an original member. The TRANSACTIONS are not sent to members in

arrears, and two years arrearage forfeits membership. (See Article 1V of

By-Laws. )

MEMBERS ADMITTED SINCE THE LAST PUBLISHED LIST

Allen, Harrison Sanborn

Allen, Wm. Ray

Bean, A. M.

Binford, Raymond

Bretnoll, G. H.

’ Chester, Wayland Morgan

Conger, Allen C.

Eddy, Samuel A.

Edmondson, Charles H.

Elliott, Frank R.

Esterly, Calvin O.

Hansen, James

Hersh, Amos Henry

Hilton, William A.

Hughes, Sally P.

Jennings, Henry Ralph

Krecker, Frederic H.

Land, William Jesse Goad

MacConnell, John Wilson

May, Henry Gustav

McEwen, A.

McLaughlin, Alvah R.

Mead, Harold Tupper

Metcalf, H. E.

Peery, George Gose

Place, J. A.

Pool, Raymond J.

Riley, C. F. Curtis

Robinson, J. E.

Sheldon, John Lewis

Smith, Gilbert Morgan

Smith, Isabel S.

Stone, George Eathl

Swezy, Olive

Tinsley, Randolph Word

Torrey, Ray Ethan

Waller, C. B.

Waterworth, W. A.

Whiting, William J.

Wilson, Charles Earl

Wodsedalek, Jerry Edward

Wolfe, Jas. J.

308 LIST OF MEMBERS

ACKERT, JAMES Epwarb, ’II............ Kas. State Ag. Col., Manhattan, Kas

PUSON, LALVEED We Ebest Sore eer eee 3935 Pine St., Philadelphia, Pa.

ALLEN, : HArkison: SANBORN,“ MGA gee Trach s ees sole eo ni eA

PPG Pee hehe aac ye cet ME ys Ae 120 Clowes Terrace, Waterbury, Conn.

PRE Wik DACA Y VE a cates han I cena Te ecg oS th RR ah cae

et Nk ie we aisheik Kansas State Agricultural College, Manhattan, Kansas

ALLEN, Wynrrep E., A.M., ’04........ 1345 N. Harrison St., Stockton, Cal.

PXNGAG? J ACU tare ie SP aes ene eee 540 S. Main St., Manchester, III.

PRNOLD,) PRANK! OTS. ca eckiaad eee 408 House Building, Pittsburg, Pa.

ATHERTON, Pror. L. G., A.B., M.S., ’12..State Normal School, Madison, S. D.

APWOOR Tits (2Ot ery lian oy sons hte? 16 Seneca Parkway, Rochester, N. Y

BALDWIN, HERBERT B., ’13.........0200cceee 927 Broad Street, Newark, N. J.

Banger, Prot Howagn (J...) PHD 711. 406d OE ee ee

rae Eugenics Record Office, Cold Spring Harbor, Long Island, N. Y.

BARKER, FRANKLIN D., Ph.D., ’03..... University of Nebraska, Lincoln, Neb

RARER OEE WV LOB. Ca VPA aY Las cc ereleateniec a uk see Clemson College, S. C.

BASS Ct MS oe Sie. tee 741 Carondelet Street, New Orleans, La.

Bauscu, Fywarn, 78... 6. sc va cee ss 179 N. St. Paul St., Rochester, N. Y.

TEAUISCH WALLIAMS Cee, ooh Sad peice ween Rate St. Paul St., Rochester, N. Y.

BEAN Pas vey AGA, ESS bes Gas Sa ee Pete peter one Forrest Grove, Oregon

BELL ALBERT TBS. VAUM Voges hae La. State Univ., Baton Rouge, La

BENREROFK, F059 MoS. Ute eae eae Alfred College, Alfred, N. Y.

BENNETT, Henry C., ’93....Hotel Longacre, 157 W. 47th St., New York City

Bevis; JOUN BG PEN ak at pea ecces hh Saseeu eee 11r Market St., Camden, N. J.

BINFoRD, RAYMOND, Ph.D., ’15............. 226 College Ave., Richmond, Ind.

Birce P Ror. 6a. SCD. LL Is on, ae. ce 744 Langdon St., Madison, Wis.

BLACK; J. ticks Tee ye ee 735 Wilson Bldg., Dallas, Texas

Brame AS Mes Neg ere es a ce Ohio State University, Columbus, Ohio

Bootu, Mary A., F.R.M.S., ’82.......... 60 Dartmouth St., Springfield, Mass

Boye, Prov is MD 82. fee oa University of Nevada, Reno, Nevada

BOYER, ha PO LALM S08) oe elec hie 6140 Columbia Ave., Philadelphia, Pa.

BRETNOLL (Gar ied tot ee eae Ui ot 512 S. 14th Street, LaCrosse, Wis.

BropE, Howarp S., Ph.D., ’13....... 433 E. Alder Street, Walla Walla, Wash.

Brookover, CHAs., A.B., M.S., ’05..Med. Dept. Univ. of Ark., Little Rock, Ark.

BROW AMOS OP NPR DO PTE foo cut rvs Sc 20 E. Penn St., Germantown, Pa.

Reowse, te A tee na a, William Nast College, Kiukiang, China

BROWNING, SIDNEY Howarb, ’11..Royal London Ophthalmic Hospital, London

BrYANT, Pror. Eart R., A.M. ’Io...... Muskingum College, New Concord, O.

BUCKINGHAM, EDWIN W., J8.,......ccceceeecs Va. Med. Col., Richmond, Va.

Buin; JAMES EOGAR, PSO sy 08.4 ote 4328 ws e/a aes 141 Broadway, New York City

BOLEITT, ¥ ROP. ote ae he IA oa bck Oe eee Chapel Hill, N. C.

BURRILL, | PROP WF ik ae tale natn 656 ok cee ene oe ae aad Urbana, III.

CAMPBELL, JOHN PENDLETON, Ph.D. ,13’...University of Georgia, Athens, Ga.

CARLSON OG ON Be eats Ps ee cates a oo he Doane College, Crete, Nebr.

CaAgres,' Prov, CRARLES, (6150, CAGaT Oe suis Sas oe Parsons College, Fairfield, Ia.

AMERICAN MICROSCOPICAL SOCIETY 309

Carter, JOHN E., ’86.......... 5356 Knox St., Germantown, Philadelphia, Pa.

Carvers Prov. G: LS A.M: 12.00. 002.06. 526 W. 123rd St., New York, N. Y.

CHAMBERS, RoserT, Jr., Ph.D., ’13...... Univ. of Cincinnati, Cincinnati, Ohio.

Cuester, WAYLAND Morean, M.A., ’15..Colgate University, Hamilton, N. Y.

Cxiark, GeorcE Epw., M.D., ’06.........-005- Genessee St., Skaneateles, N. Y.

Crark, Howarp W., A.M., 712...... cc cece cece cece cece cence Fairport, lowa

CLEMENTS, Mrs. F. A PH) 805 oes sos Univ. of Minn., Minneapolis, Minn.

CORR, NN RACPRDS 7S arIG Ck Nee ee i cba hee Virb Ota We elas: Falls Church, Va.

CoGHILL, PRror. Faewih re ea LHUPES DI Kas. Univ., Lawrence, Kas.

Cotton, Haroxp S., PhD, (cane Lab., Univ. of Pa., Philadelphia

GONE PAGES Ie See ee as esas Editorial Staff, “fF uinbdeain, Chicago, Ill.

CONES THSSE DL OTE sa s/cwiep sie c abies fae ee es 542 W. Packard St.. Decatur, Il.

ConGER ALLEN GL0MLA., IS diecditea. ole’ P. O. Box 663, East Lansing, Mich

CoNLON, JAMES J., Ph. Ee ARCUATE yeaa ae 717 Hyde St., San Francisco, Cal.

GOeT AVE pe PE. DOV TT. SPER ance lel a ttih cae ee dle olen Veen Os ihe dt oe

REUSE ee Me a Reha Coll., Merrimam Park Sta., St. Paul, Minn.

Cort, GrorcE F., ’11..... si HAR oes Barbee Ree DL aH 1195 Main St., Buffalo, N. Y.

Covey, GEORGE W, oF 8 SCs Oy DEI Sy RO Sk gary cee ag College View, Nebr.

CRAIG 1 HOMAS OF. Gk ok ae sb in.c.ace,e, 000 26 Selkirk Ave., W., Montreal, Canada

Crozier, WILLIAM JOHN, PH.D. 715......cc ce eee cee cecr ees ectceceenes

ip in mes, A Fe PP Bevanida Biological met Agar’s Island, Bermuda

DaRBAKER, LEASURE KLINE, Ph.D., M.D., "IL... 1... seer eee eee cece neces

aN, he ORR ATOR cw Cw eh peo ale 5 415 N. Highland Ave., Pitsbute. Pa.

Dap ETON eRe PMs 1A sso eien Sa euniole «als oes Box 143, Chadwicks, N. Y.

DAUGHERTY, LEW1S So PHD Org, sci ee bein weldeseie » Cameron, Mo.

DAvis 7 3is.2 MAD, OG Gea hi aide nts rein tieeses 209 Locust St., Evansville, Ind.

Davis; Prov: S;, PRD. 12s... 8. University of Florida, Gainesville, Fla.

Deere, Emit Oxar, A.M., S.M., ’13....Bethany College, Lindsborg, Kans.

DeWitt, CHAREES HL.) M.S,;. 711... 6. 0.000% 355 College Ave., Valparaiso, Ind.

Dissrow, WiLLIAM S., M.D., Ph.G., ’ol....... 151 Orchard St., Newark, N. J.

once; ‘CAREOEL SWerid ceo a te Siew ens ss e's Mo. Bot. Garden, St. Louis, Mo.

DOLBEY, EDWARD Pit OOe se has 3613 Woodland Ave., Philadelphia, Pa.

WOLe, J) Wa BUR ie, 259 e Re clic ne a tbe aes Beco eseie Fairfield, Iowa

DRESCHER, Wirt, O76 ob ias. Care Bausch & Lomb Opt. Co., Rochester, N. Y.

Dopckon;* WINFIELD, B.S57 Ils. tons ces cals 6030 Engleside Ave., Chicago, Ill.

Epmonpson, Cuartes H., Ph.D., ’15.....--eee0- 1360 Alder St., Eugene, Ore.

Eppy, MILTON W., Eos. cn ioe ae cll ctnecccscseecess State College, Pa.

PODY) SAMEIER, PUI SEs «chap es srace snes 600 West Main St., Decatur, III.

EGGLESTON: (bis) BAMA, 71365 cate Ox a sae Marietta College, Marietta, Ohio

EIcENMANN, Pror. C. H., ’95..........- 630 Atwater Ave., Bloomington, Ind.

Ex.uiotr, FRANK R., M.A., af DSM RE Rea stdin at nie biplane Wilmington, Ohio

Exuis, Pror. M. M., Ph.D., *12.........cceseeees 1109 13th St., Boulder, Colo.

E.rop, Pro. rian J., M. im BMiSF08)) fi8% beak ee eee ree

PRE ATER Ee ccdhctclera ejale' stains ier University of Montana, Missoula, Mont.

REWELE Tt OO gear ceedetesenees wees 210 The Normandie, Seattle, Wash.

310 LIST OF MEMBERS

PSTeeY CALVIN’ Of SS ds lve aul Occidental College, Los Angeles, Cal.

Evans, ARTHUR THOMPSON, ’I4..........00. 932 Lincoln Place, Boulder, Colo.

Eyre, Joun W. H., M.D., M.S., F.R.M.S., "O0.ai cus He ST ae a ee ee

Pe Uviioaly RUM eee Rie ane urs Guy’s Hospital, London, E. C., England

Paniow) (Peo We Geer sys wep aie cee 24 Quincy St., Cambridge, Mass.

BAttic VE poR Ps Wi Bis, MSS iad ers eee Springfield, Ohio.

Fett, Gro. E., M.D., F.R.M.S., '78....0..05. 1392 Amherst St., Buffalo, N. Y.

Fetiows, Cuas. S., F.R.M.S., ’83...111 Cham. of Comm., Minneapolis, Minn.

FerGuson, Marcaret C., Ph.D., ’11........ Botanical Dept., Wellesley, Mass

Finpiay, Merzin C.,'A.M., "15.0 6.ceedeccs cose Park College, Parkville, Mo.

HISCHU ALY NORV uaa sueeb be burned da briicas Box 1608, Milwaukee, Wis.

Fitz-RaNnpotPpH, RayMmonp B., F.R.M.S., Il... ..c.ccecececccucucccccess

FI teh AA Sekt. State Laboratory of Hygiene, Trenton, N. J.

Fiinr, JAMES IM M.D ore ys Keats Stoneleigh Court, Washington, D. C

BogtR as D4 M.D. Ori kcedicueieaaes 202 S. Thirty-first Ave., Omaha, Neb.

Furniss, H. W., M.D., Ph.D., ’05...... U. S. Consulate, Port au Prince, Haiti

Gace, Pror. Stmon H., B.S., ’82...........0005 4 South Ave., Ithaca, N. Y.

Ga.Loway, Pror. T. W., A.M., Ph.D., ’or...... 1332 West Wood, Decatur, II1.

GARRETSON: o0GENE, 7125.6 .00) 2650 58) 428 Fargo Ave., Buffalo, N. Y.

GESNER, BROWER CLAIR, ’II........ 110 Steadman St., Moncton, N. B., Canada

GotpsmiTH, G. W. B.A., '13........ cu ceeee S. La. Indus. Inst., Lafayette, La.

Gowen, Francis: Hota) oui fhe. R. D. 1. Box 15, Exeter, N. H.

GRAHAM, CHartes W., M.E,, ’I1..........cccecece. Huntington, L. I., N. Y.

GRAHAM, Jon Younc* PhD. "145 0 8 eee University, Alabama

GRA Rg a he oon Seth de et tee 3535 Telegraph Ave., Oakland, Cal.

Gray, WILLIAM CALVIN, ’I4.........2cccececes Lock Box 228, Toledo, Iowa

Gircoay, Esinly Be oPhDi ished he ae Buchtel Col., Akron, O.

GRIFFIN, LAWRENCE E., ’13.......... University of Pittsburg, Pittsburg, Pa.

GUTBERLET, JoHN E., Ph.D., ’11............ Carroll College, Waukesha, Wis.

Guyer, Micuaet F., Ph.D., ’11....... University of Wisconsin, Madison, Wis.

TAGE BoE EMD aU Oy Ree a Hagler Building, Springfield, II.

Hatt, Arice Louise, M.D., ’12......... 730 5th Ave., New Kensington, Pa. .

Hanamay, C, E., F.R.MS., ’79........00. State and Second Sts., Troy, N. Y.

Hance, Ropert T., B.A., ’13........ Zool. Lab., U. of Pa., Philadelphia, Pa.

RIAN RINGON, (EV Aouegae ts 4 Oy skiers tice cheb Meee ocr eom Charleston, III.

TIANSEN, JAMES POI GUNS E Os Chadd acne: St. Johns Univ. Collegeville, Minn.

Harman, Mary T., ’13....... Kansas State Agr. College, Manhattan, Kansas

Havoen, Horace Eowiny JR. 14i 056s eee es Sacks College Station, Texas

Pixar hee Bia ar ioe Wash. State College, Pullman, Wash.

HeErMBuRGER, Harry V., A.B., ’14..... Nat. Hist. Bldg., U. of I., Urbana, II.

FARNDERSON; WWILLIAM. (ATS 27s vol oka k cca ee Millikin Univ., Decatur, II.

Hersu, Amos Henry, A.B., ’I5......0ceecees 561 S. Lime St., Lancaster, Pa.

Hertzoc, MAxMILIAN, M.D., ’or............. 1604 Mallers Bldg., Chicago, III.

Hicks; Arena’ Oy rr s io, ecm ela. 178 Union Ave., Long Branch, N. J.

FULTON; Winkiae OR Ph Dit trey po oe oe 2 nt Be Claremont, Cal.

AMERICAN MICROSCOPICAL SOCIETY 311

Hyorru, Lupvie C., ’12........ Meadowdale, Snohomish County, Washington

RAC eaL ary) NV ij) FDS este Sak VE aS De a oa Ddnldlele' de 49 6th St., LaGrange, III.

Howarp, Rozert Nessit, ’12..Ookiep, Namaqualand, Cape Province, S. Africa

How anp, Henry R., A.M., ’08.............. 217 Summer St., Buffalo, N. Y.

Hupson, Ettis Hernpon, B.A., ’14....3403 Hamilton Ave., Philadelphia, Pa.

BRANCH E COAL OS CISC eee ee ales cur ee Borest Grove, Oregon

Ives, Freperic E., ’02......... Woodeliff-on-Hudson, Weehawken P. O., N. J.

Jackson, Danret Dana, B.S., ’99......... 930 President St., Brooklyn, N. Y.

AMES W ORE MD tra Oss oe lab yee, 1231 Locust St., Philadelphia, Pa.

RRSP R ARON an EY ees pL isd ded enivwiehy 603 S. Fern Ave., Wichita, Kas.

FRMMER, ALAS S MOA Ee Selec a da pola Geshe Science Hall, Indianola, Ia

JUMMINGR FIRWRY KATO ec caccae dnaedeicdude Box 582, Lompoc, Cal.

JOO Toe ace § eC tse so sk Joplin, Mo., R. F. D. 4-147

JoHNSoN, Frank S., M.D., F.R.M.S., ’93...... 2521 Prairie Ave., Chicago, III.

TOMDAR (PROP. Ti Te T8 dk isk cae vos University Place, Charlottesville, Va.

PAIDA TS TEA MOR, | 0005 bio daceid ouhcuciee'yewecds 610 Lake St., Madison, Wis.

Beroce.). Hoa MD 9B. odds Sucre 202 Manchester St., Battle Creek, Mich.

Kincaip, Trevor, A.M., ’12........ University of Wavhingin: Seattle, Wash.

Ry TOES SSIES gO Ry eR rp CC ae BRE TN gO Langdon, N. D.

PNM EY SEM EARTS VT ESs Ota lisinidivwed dos ch 0k P. O. Box 261, New Orleans, La.

TE MS RG a Ose PRPS Aa A eR 1015 Blondeau St., Keokuk, Ia.

Kororp, Cartes A., Ph.D., ’99....... University of California, Berkeley, Cal.

RCS Sieg BAL). OTs. 52 5 ca cden atl ica 32 S. Fourth St., Easton, Pa.

KRECKER, Frepertc H., Ph.D., ’15..... Ohio State University, Columbus, Ohio

eeeew a arya. (MM Gore oer te ee y 520 Elm Street, San Jose, Calif.

Lacy, Frank W., ’14.......... U. S. Naval Hospital, Las Animas, Colorado

i AG CO RRR pees EMEA T Lf SLORRE SOF Om Sat NIRS | oc. QR San By BETS)

CARA Bank of New South Wales, Warwick, Queensland, Australia

LAND. DV UIIAM: JESSE, GOAD PIE DN £85 oil aes cccueoeuk gk dehwneean tia

s ahinae' eS aint dihs Sai cd elt an The University of Chicago, Chicago, III.

Lanpacre, Py L., B.Aig03.2 0... osc0 0 Ohio State University, Columbus, Ohio

LaRue, Grorce R., Ph.D., ’11....University of Michigan, Ann Arbor, Mich.

ATH AM, Messe VAS M.D... D.DiS. AF RIM.S.: 88... eis ek Os

pO atime ee eh TN 1644 Morse Ave., Rogers Park, Chicago, III.

LATIMER, Homer B., M.A., ’11..Neb. Wesleyan Univ., University Place, Neb.

LEHENBAUER, Purp, A.M., ’II.......cecceceee Univ. of Nev., Reno, Nevada

Lewis, Mrs. KatuHerine B., ’89...“Elmstone,” 656 Seventh St., Buffalo, N. Y.

BEBO SE AAAI Uae Sais Wh us ole a's and anh Ce nddacdsous 406 Galena St., Dixon, III.

Uy OEE Rtg Fa ee Re eG Okla. Ag. Exp. Sta., Stillwater, Okla.

renee NVA TAM MID OG 8 kkk a dade e tee tensen Nashville, Tenn.

GM ADOT O98 os ee aiid dss x 289 Westminster Road, Rochester, N. Y.

LoncFELLOow, Rogert Cartes, M.S., M.D., ’11........ 1611 22nd St., Toledo, O.

Lyon, Howarp N., M.D., ’84..........065 828 N. Wheaton Ave., Wheaton, III.

Lyte, Ropert W714... .cceeces 212 Mutual Life Building, Buffalo, N. Y.

312 LIST OF MEMBERS

MacConneLL, JoHN Witson, M.D., ’15........cccecccecceee Davidson, N. C.

MacGiiiivray, ALEXANDER D., ’12....603 W. Michigan Avenue, Urbana, III.

Mack, Marcaret Evizasetu, A.M., ’13........ 210 Maple St., Reno, Nevada

NAGATRY Tin, SME Sig tage eg ak Nat. Hist. Bldg., U. of I., Urbana, Ill.

Marr, Grorce Henry, M.E., ’11............. 94 Silver St., Waterville, Maine

MARSHALL, Co.iins, M.D., ’96........... 2507 Penn. Ave., Washington, D. C.

Marsmats, Rove, PhD. ,fopied ce Ie ka oh oe DeKalb, II.

MaAnsrazi, WoS.\PRD., "320k ck bs pac 139 E. Gilman St., Madison, Wis.

MartLanp, Harrison S., A.B. M.D., ’14...... 1138 Broad St., Newark, N. J.

MASSEY; Pade ADB BSS fs 80 654 Socde) cs abe nab Clemson College, S. C.

Martuer, E., M.D., Ph.D., ’02.......... 228 Gratiot Ave., Mt. Clemens, Mich.

May, iHenay ‘Gusray, B.S, 2x8 ssacesaeceeses 506 W. Oregon St., Urbana, III.

Miverws Rot Jai Star oases aan 1156 W. Decatur St., Decatur, Ill.

MAYWALD, FREDERICK J., ’02........ 1028 Seventy-second St., Brooklyn. N. Y.

McGaiza; Arnert,’ Ph:D., 80.000 dccdc ns 2316 Calumet Ave., Chicago, II.

BEGG Remy, AG EES Wb odd d dh eda Univ. of Nevada, Reno, Nevada

MOBWEN HIG Se RA BK 1118 Marbridge Building, New York

MORAY» Foemnrt Bak: Wks CU ese Cela 259 Eighth St., Troy, N. Y.

McKeever, Frep L., F.R.M.S., ’06......0c0005 P. O. Box 210, Penticton, B. C.

McLaucuHiin, AtvaH R., M.A.,, ’15...... Presbyterian College, Clinton, S. C.

McWitttta ms, Jou; tds: Rion aeuee Lock Box 62, Greenwich, Conn.

MeEap, Harotp Tupper, S.M., °I5........005. 316 McCabe St., Mitchell, S. D.

. Meacer,\A; Cisrroay, ‘M.D. °RR.MS., "Banu cke ci vee eee

Tee TPL Pew Tee tae, Law 1A ts 324 Montgomery St., Syracuse, N. Y.

Miecray WB 5 PRD: Sogisd, Gbbis ecb ceeein 200 E. State St., Athens, Ohio

Mercany, HeeE 1s i 8 University of Minnesota, Minneapolis, Minnesota

Mercas,\Prov..Zeno :P. sB.Acy ase eh adored sede etoed ane

ELLOS PRP . HS Dept. of Zool., Univ. of Minn., Minneapolis, Minn.

Miiier, Cuarzes H, ’11...... Med. School, John Hopkins U., Baltimore, Md.

Miter, Joun A.,Ph.D., F.R.M.S., ’89........ 44 Lewis Block, Buffalo, N. Y.

Miter, Rupotr C., Ph.G., M.D., ’11.........000. 403 Ray St., Seattle, Wash.

Minenart, Pror. VELEAR Leroy, A.B., ’11..2070 Rosedale Ave., Oakland, Cal.

Mockert Jee SeGrieee ss ae 620 First Natl. Bk., Lincoln, Neb.

Morivxn; Hi) MiDi. aps Po ooo ed 341 W. Fifty-seventh St., New York City

Moopy, Rozert O., M.D., ’07...... Hearst Anat. Lab. U. of Cal., Berkeley, Cal.

Morris, CAPEL, ’I2........00 Leafield, Gibsons Hill, Norwood, London, S. E.

Murree, Pror. E. H., A.M., LL.D., ’12...... Brenau College, Gainesville, Ga.

Myers, FRANK. Fy BFR I. a 331 Market Street, Bethlehem, Pa.

NOUe WLR Hy Bib irscca's aarti Univ. of Neb., Lincoln, Nebr.

Norris, Pror. HARRY WALDO, ’II.......cccccecce 816 East St., Grinnell, Iowa

Nomron; (Citances Eo MID sei isccecak 118 Lisbon St., Lewiston, Maine

Oq.ever; C-S2B.S Se Dates). Bex 1006 N. Union St., Lincoln, II.

Qrourr A Wi WEA ies sia ctvo a tek 1495 E. 118 St., Cleveland, O.

Onvera; Dosrieso | Dat 700 J hs oe ack acide. Gijon (Asturias), Spain

Ossorn, Pror. Herpert, M.S., ’05..... Ohio State University, Columbus, Ohio

AMERICAN MICROSCOPICAL SOCIETY 313

Cher PIARVEY DINGY: OSes Oh ee Shu ccicwes Spencer Lens Co., Buffalo, N. Y.

PALMER, THOMAS CHALELEY, B.S., ’II...... ccc ccccesees Media, Pa., R. F. D.

Parker, Horatio N., ’99........ College of Agriculture U. of I., Urbana, III.

PATRICK OP RANK, = PRD: "OTH 7... 421 Bonfils Bldg., Kansas City, Mo.

REUBEN MEIN INI OT Ga EAks Nab dees code Me deleeas P. O. Box 503, Altoona, Pa.

wee ensenoaie (s0SR 7 HOM TS. ci sc tnskewacnceeceteeceueus Salem, Virginia

Pennock; Epwarp) 779). weer dl ates. 3609 Woodland Ave., Philadelphia, Pa.

PeAvAd, THs: WoseVe lly iad s. boosie eied pbs ees Encampment, Wyoming

PETERSON, NIELS FREDERICK, ’II........0csecceeees Box 107, Plainview, Nebr.

Bre et EDWARDS TIVE cee yok 008 Raa On Kine obs tl Gein ea wees oRIREED SOR

...-Madeley House, Bulstrode Way, Gerrard’s Cross, Bucks, England

PEACH. cA Ae Ikan ewe we akw scale 40 Sunnyside Drive, Athens, Ohio

Potiarp, Pror. J. W. H., M.D., ’12. Washington and Lee Univ., Lexington, Va.

Poors RayMone Joe PisD.. 2790 eves oe cds Station A., Lincoln, Nebr.

Pounp, Roscoz, A.M., Ph.D., ’o08..... Harvard Law School, Cambridge, Mass.

PUN GRRS BOARD ALE UIRD Lis iw kas dco ee dba seeee eae w ne Waxahachie, Texas.

Pearcy MoO MCS, Fass bios eS 421 Douglas Ave., Kalamazoo, Mich.

Prien, Pror. Otto L., M.D.V., ’11........ 5 and 6 Fedl. Bldg., Laramie, Wyo.

PCE CAP MED, OFS EOE Os Chat en sd sadaddeead adn Notch, Stone Co., Mo.

Peron) Groece, M.D., 80. vieik cia es ede ddes che 1o1r H St., Sacramento, Cal.

COIREIAN; “MARVIN -C.,"AMB ISA Leese eee Wesleyan Col., Macon, Ga

RANKIN, WALTER M., 713......-.0000: Princeton University, Princeton, N. J.

MUNDUSIEM ORE. £5, I2s nse dicen Oeewe keke cae hes 203 Seneca St., Manlius, N. Y.

PMMMOM EAE TON | T1:, OO ESO RE ile cic o's ca ane hae Pee eee

Pre Wis bas a se. ae U. S. Bureau of Animal Industry, Washington, D. C.

Rector, FRANK Les.iz, M.D., ’I1.......... 36 Forty-first St., Brooklyn, N. Y.

Meese cegor: Arorer: ModPh Db (op. ) OS scales diced his adhe Parloavevaee

Reals ceReks boas seneeedes W. Va. Univ., Morgantown, W. Va.

PERI AE PUUIN I. sichere edie onl dele wa Kuala Lumpur, Selangor, Fed. Malay States

Be VUIKEIAE Taga Mac lte ace a cs ceca 9go1 College Avenue, Wheaton, IIl.

~ wacmanns; Avra PIED lia os. ce vec ess University Station, Austin, Tex.

PEA RNS PLING 5 SOOT is em ck s cies hace Sas 1114 Floyd St., Lynchburg, Va.

Baaky, <r 3e Coens (Mid. 16s ose ce s+. 616 Maryland Ave., Milwaukee, Wis.

PEDERTS EG. WY TALS Bbw arcs vidoe emai ide pin’ 65 Rose St., Battle Creek, Mich.

Roprersyibts Woy s i. sie. State Normal School, Cape Girardeau, Mo.

PRORERTE PW PET CaUGS oid ble vives ceintin ers 345 West Michigan St., Chicago, II.

PORE GOW skoda lag. ES sib wiclereiaiecs'arn glaletwle watsjenvie's ab Box 405, Temple, Texas

Rogers WAL Tee Ptr. Pcie ete Univ. of Iowa, Iowa City, Ia.

Ross, LutHER SHERMAN, S.M., ’II.........0-. 1308 27 St., Des Moines, Iowa

Pee = a Cosh, Pe Edo acs cw ah ne ws AEE & SASS Hyd TR 8. Hudson, Ohio

SAWYER, WILLIAM Hayes, JR., ’13........-- 18 Arch Avenue, Lewiston, Me.

Scott, GeorGe Firmore, A.M., ’13.College City of New York, New York, N. Y.

OEE NM IT ED da SEU ine cc k's hb adis olccnans ase Univ. of Wyo., Laramie, Wyo.

Se ANTz. bi asf PR. DOA LS ale be Bureau Plant Industry, Washington, D. C.

SOUR MMMI EP EOL ONDE hel oG'h) 22% bata w-areresaiein'e erate 809 Adams St., Bay City, Mich.

314 LIST OF MEMBERS

SHELDON, JoHN Lewis, Ph.D., ’15....... W. Va. Univ., Morgantown, W. Va. °

SHIRA, | TAUSUIN ERIN, Ay CER ee ced eect wee eRe eA Homer, Minnesota

SHULTZ CHAS. SiS euiAawe Ls ak Seventh St. Docks, Hoboken, N. J.

SARLEW i Tor they 02. ede ou kien alites Elkins Park, (near Philadelphia), Pa.

sLocum) Caas./ FE.) PHD y'M-D., 78.0062. cs. eked 218 13th St., Toledo, Ohio

SOMA Ls Y SEWARD: 5 Sh Bes visiviatalale noah omccoibidead motaleuk etaee yaa Wyncote, Pa.

SMITH, BERTRAM G., Ph.D., 713.......... 936 Forest Avenue Ypsilanti, Mich.

SmitH, Pror. Frank, A.M., ’12.......... 913 W. California Ave., Urbana, II1.

SMITH, GILBERT MorcAn, Ph.D., ’15............ 1606 Hoyt St., Madison, Wis.

Omiru, Isaner. S$.) M.S.) 7258... ccs eces 1120 W. College St., Jacksonville, Ill.

SMT EO OG LY oN Mike tio ent 131 Carondelet St., New Orleans, La.

SOR CO ERIM. oe tee 08 ond ok ss leh oda bebe GEE LURE

Sa a ae 2, take gpk 37 Dryburgh Road, Putney, London, S. W., England

SPAULDING, M. H., A.M., 713...... 508 W. College Avenue, Bozeman, Mont.

SPuRGEON, CHARLES H., A.M., ’13..... 1330 Washington Ave, Springfield, Mo.

STBOEINS 1). 01,49 f'n. M:D., Or sionk,.asen 50 E. 41st St. New York City

SIEVENS, Ff wor) HE MS ras cbse ea ac Ae ea eee

eC SRN EMC Ts Agricultural Experiment Station, Gainesville, Fla.

STONE, CsEORGE ATHE 715 65 oes cc ea we 1725 LeRoy Ave., Berkeley, Calif.

STUMP MEVER, GEO A.B Si 213. cain cule Sud nn cn le Le Monroe, Mich.

STUNKARD, Horace W., B.S., ’13........ Nat. Hist. Bld. U. of I., Urbana, III.

STURDEVANT, LAZELLE B., A.B., B.S., ’03..... Univ. of Nebraska, Lincoln, Neb.

Sirsaatins)* Pane.) Te Toa B6 ie do as. Oa pee Phe tO eee re Ames, Iowa

Swezy, Ortve, Ph.D., ’15..... East Hall, University of Calif., Berkeley, Calif.

‘SWINGLE, Pror. Leroy D. ,’06.......... Univ. of Utah, Salt Lake City, Utah

Tuomas, ArtHuR H., ’99........ Twelfth and Walnut Sts., Philadelphia, Pa.

TIMMINS, GEORGE, ’06........0ccceeccees 1410 E. Genesee St., Syracuse, N. Y.

TINSLEY, RANDOLPH Worn, B.S., '15.....ccccccccecceese Georgetown, Texas

Zou Fases Covh. Ag MDS trp eds plus oe ee uke in Boulder, Colo.

DOBREV, (RAY PR rraN, BIS Pte sia adc. wc cnendesle eae eithowie Grove City, Pa.

REN NERS IMMEDI AS. Hi ESO. tie on kote 817 Crescent Place, Chicago, III.

Tsou, Yinc-Hsuan Hsuwen, M.S., ’13..P. O. Box 78, Univ. Sta., Urbana, II1.

DURNER, CLATR EG, MAS igo cel, Bid elesve eves Mass. Inst. Tech., Boston, Mass.

SOUTILE PROP ASTLIMD PRD. T1s iL tobe seen cee ees eee

EON Nt Dah at 2a ap ean University of Virginia, Charlottesville, Va.

VALENTING, HERBreT ei PTT EL Aha ec bee odes 141 Milk St., Boston, Mass.

WANT CUBAVE. CEL ARL UM il EE Oe eek oe Bn an 310 N. H. Bldg., Urbana, III.

VARRELMAN, FERDINAND A., 'I3....2+-++-1358B Spring St., Berkeley, Calif.

WAGNER | POWARDETS Tae iid eee wo 124 Willet St., Jamaica, Long Island

Watre.) Pornrgrcr Cee PRD I Lt) ees b saacaco emerald ond Sask san eahe ape

Wey ta Medical Department, Western Reserve Univ., Cleveland, Ohio

Watxen; Fina: R.Ph Dip 7070s oie... 3: University of Nebraska, Lincoln, Neb.

WAEKERWILEVA DRERE CES PUI o's sb hole brane Gunes Station A., Lincoln, Nebr.

WaLtee (Co Bo: PR De itae ol. fog. .--. Wofford College, Spartanburg, S. C.

Wanparck; J. Ci 712 his bein ss Ne aha x acetate Ro ales « 306 E. 43rd St., Chicago, III.

AMERICAN MICROSCOPICAL SOCIETY 315

Warp, Henry B., A.M., Ph.D., ’87......... University of Illinois, Urbana, III.

WaATERWORTH, W. A., "I15....cecceeees 286 Lambton Quay, Wellington, N. Z.

MV Atsow aon) Croyvbe: MIE) 1T. Gi Lites ois nce cedeen tees Kingwood, W. Va.

WME AL Me TA Raabe Cele cate akin ao oes Univ. of N. M., Albuquerque, N. M.

ROLES tarth 463 Wt Oke does nda Nod eeer cues Box 416, Fergus Falls, Minn.

WEIGH TPAUL Ss, PID tn uu kee ces Kas. St. Ag. Col., Manhattan, Kas.

MWrersH iru, B.C ria. sek ssaeds 24 Upper Mountain Ave., Montclair, N. J.

Wr HeLre. PS PDO OOh Nita che ben aneeee 79 Chapel St., Albany, N. Y.

WuHeE ptey, H. M., M.D., Ph.G., F.R.M.S., ’09..2342 Albion Pl., St. Louis, Mo.

WET TR EAD Et NEL, LOS als bg hetdide ates glen vam ve Center Sandwich, N. H.

WHITING, - WILLIAM Ji5- I5s sb vest avescvts 36 Stanley St., New Haven, Conn.

Wireman, Harry L., Ph.D., ’13...... University of Cincinnati, Cincinnati, O.

WreSinsow EDWARD) 140s is hc oo ea bes 219 West sth St., Mansfield, Ohio

WiLiiAMson, Wo., F.R.S.E., ’07..79 Morningside Drive, Edinburg, Scotland

Witson, Cuaries Eart, A.M., '15.......0ccescces Agriculture College, Miss.

Wot cortt, Rosert Henry, A.M., M.D., ’98...Univ. of Nebraska, Lincoln, Neb.

WobDSEDALEK, JERRY EpwarbD, Ph.D., "15.6... ccccccccccecess Moscow, Idaho

WWI ANS PID 7S cols wcunssinace pane Trinity College, Durham, N. C.

Woon, *Antuurm . Kine) 2140. 85208 yu Ardsley-on-Hudson, New York

Yor, Jonn Howe, M.S., '14........2.000- Graduate College, Princeton, N. J.

ZAPFFE, FREDERICK C., M.D., ’05........0008- 3431 Lexington St., Chicago, Ill.

ZeE1ss, CARL (care Dr. H. Boegehold).......... m peeye apt, Jena, Germany

GO ee SAVED: 15.5 DAB sg OS raceciarhiein valve Foie Vow ween 1040 Otis Bldg., Chicago, Ill.

SUBSCRIBERS

ACADEMY OF NATURAL SCIENCES......eeeee0% Logan Square, Philadelphia, Pa.

PERT CULTURAL UA RPi ITA PEIRBARY hc we kc's apie Gane vuln Kenan Knoxville, Tenn,

A.tFreD Dickey BioLoGicAL LIBRARY......... DePauw Univ., Greencastle, Ind

AMERICAN Museum oF NATURAL HISTORY,.....cccccccsccscccecccccece

Si. jue M ee ete 77th St. and Central Park, New York, N. Y.

AML PRS ACOLLEGE LLIBRARY (064 ch oc cis ccc aiceaiseed da diec oddiarets Amherst, Mass.

TRAMCOCH SSCTENTINIC ALIBRARY «o's sain ocse'n p ocbaceie denis oe sib sedivie bot .Plainfield, N. J.

PUUORT CARS TERT DIME ARY 5 os cic cca de acd am a pemibibietcbnadivnde ga’ Beloit, Wisc.

BIBLIOTHECA DE FACULTAD DE MEDICINA.........e0.e00- Montevideo, Uraguay

BICKFORD DIOLOGICAS, « LIBRARY. a. na ois dueied etaleicnca Bates Col., Lewiston, Me.

BOstONs rele BEAR Y . cinc's Aas «ap ace maisid eh cloak Seen 0.0% scien’ bible Boston, Mass.

Boston Society oF NATuRAL HistTory............ aiettheles St., Boston, Mass.

BrRooKLYN INSTITUTE OF ARTS AND SCIENCES..Lafayette Ave., Brooklyn, N. Y.

RURAL OF OCIBN CED MAOBARY | 5c x s.ccndiee ciccuineee scuicicwanee eK a> Manila, P. I.

ES NE hcl aspen pswnoee Coxe een? 126 Summer St., Boston, Mass.

CAVRERAN IR Chics cicthe'na'ccee’ Casilla de Correo-69, Montevideo, Uraguay

CAREC MEM HGTOMARY «oa picicidc dic ccow slp hd aie nre abe eWeeae bende Allegheny, Pa.

CRMMEGUEOLADRAR UR oe cos cies. et tape nas ave hab Bs taRM aN Cae Pittsburg, Pa.

316 LIST OF MEMBERS

Cuemists CLus Liprary, C. A. H. Exriorr..52 East 41st St., New York City

CerCAGO:: WwiPRerry) CSRRARY 10.05% DY iyo.sis)..cocs.epescreroreelen n Pe aioe wih Chicago, Il.

CLARKE, THOS. J......e..ceeeceeeeeeee-251 Willoughby St., Amherst, Mass.

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INDEX

Acanthus vulgaris, ampullze of Loren-

zini, 131

Acidophilous cells in Amphibians, 154

Address of President, 7

Adrenals, acidophilous cells, in Amphib-

ians, 154

Air Stores in Aquatic Insects, 279

American Species of Genus Atractides,

185

Ameeba, Method of Obtaining, 271

Amphibians, Acidophilous cells, 154

Ampulle of Lorenzini, in Acanthus Vul-

garis, 131

Aquatic Insects, Air Stores, 279

Arachnoidiscus in Diatomaceous Earth,

290

Atractides, American species, 185

Badhamia, Cultivation of Plasmodium,

161

Bessey, Chas. E. Life Sketch, 299

Beetles, Tiger, Elytral Tracheation, 241

Behavior of Insects and Spiders, 50

Bifurcation in Lumbricus, 283

Biological Monographs (Illinois), 56

Biology and Social Problems, 61

Blood Sucking Muscid Larve, 193

Bombycine Moths, 279

Botanical, Micro-Technic, Development

of, 71

Bouin’s Fluid, 55

Brookover, Chas., Some points on the

Development of the Nose, 7

Bulky Objects, Clearing, 195

Caddis Fly, Wood Boring, 192

Catocala Moths, 50

Cell Changes in Epidermis of Tadpole’s

Tail, 167

Celloidin, Clearing Fluid for, 55

Celluloid Acetone Cement, 278

Cement, Celluloid Acetone, 278

Cephalic glands in Trematodes, Staining,

276

Cestodes, Revision of Family Proteo-

cephalidze, 56

Cestodes, Technic, 158

Cicindelidz, Elytral Tracheation of, 241

Clay for Modeling, 197

Clearing Bulky and difficult objects, 195

Clearing Fluid for Celloidin, 55

Cobb, Margaret, Some Fresh Water

Nematodes of the Douglas Lake Re-

gion of Michigan, 21

Cobb, N. A., System for locating ob-

jects on Microscope Slides, 189

Coleoptera, Olfactory Senses in, 281

Collecting Diatoms, 53

Constitution and By-Laws, 295

Cort, William Walter, North American

Frog Lung Flukes, 203

Crustacean Mounting, 292

Cultivation of Plasmodium of Badhamia,

161

Cultures of Amceba, How to Revive and

Keep going, 278

Cuticula, Penetration of Foods thru, 196

Damar as a Mounting Medium, 195

Daphnia without Sexual Forms, 161

Dero and Slavina, 284

Development of Botanical Micro-Tech-

nic, 71

Development of Nose, 7

Diatomaceous Earth, containing Arach-

noidiscus, 290

Diatoms, Collecting Methods, 53

Dimorphism in Spermatozoa, 191

Dissecting Pans, Fastening Wax in, 277

Double Eyepiece for demonstration, 198

Douglas Lake, Nematodes, 21

Scales of Some Fishes, 255

Earthworms, 284

Egg Concealment, 192

Elytral Tracheation of the Tiger Beetles,

241

Embryology, Vertebrate, 161

320

Embryos, Stain for, 52

Entomological Notes, 49, 190, 279

Epidermis, Cell Changes of, in Tail of

Tadpole, 167

Euparal, 50

Evans, A. T., A Study of the Scales of

Some of the Fishes of the Douglas

Lake Region, 255

Evolution of Sex in Plants, 59

Examining Stools for Eggs, 54

Eye Piece, double, for demonstration,

198

Fishes of Douglas Lake, Scales, 255

Fixing Flies for Sectioning, 56

Fixing Fluid, Bouin’s, 55

Flies, Solution for Fixing, 56

Flukes, in Lungs of North American

Frogs, 203

Fly’s Tongue, for Object, 52

Fossil Fungus Gnat, 194

Fresh Water Nematodes of Douglas

Lake, 21

Frog, Glands of Skin, Difference in

Function, 275

Frog, Lung Flukes, 203

Fungus Gnat, Fossil, 194

Germ Cells in Hymenoptera, 279

Glands, Cephalic, of Immature Nema-

todes, 276

Glands, of Frog’s Skin, Difference in

Function, 275

Glands, Poison, of Insect Larve, 49

Gnat, Fossil Fungus, 194

Hematoxylin—Orange-G, for Embryos,

Hematoxylin, Ripening quickly, 199

Hezmonais (Oligocheta), 283

Hankinson, T. L., Treasurer’s Report,

Henning’s Solution for Flies, 56

Heredity, Mechanism of, 293

Hyatt, J. D., Necrology, 306

Hydropsychid Larve, proventriculus, 192

Hymenoptera, Germ Cells, 279

Immersion fluid, superior, 199

INDEX

Inland Waters, Life of, 200

Insects, Behavior of, 50

Insects, Key to Families, 194

Insects, Mounts for Class Use, 198

Insects, New Orders of, 194, 280

Insects, Olfactory Senses, 50, 284

Insects, Preservative for, 282

Key to Families of Insects, 194

Kofoid, C. A., Method of Obtaining

Ameeba for Class Use, 271

Laboratory Technic, Michigan, 275

LaRue, G. R., Notes on Laboratory

Technic, 275

Lichtgriin, and Safranin, 51

Lids of Wooden Slide Boxes, Hinges,

196

Life Members, New, 49

Life of Inland Waters, 200

Light-Producing Organs, 281

Locating Objects on Microscope Slides,

189

Lorenzini, Ampulle of, 131

Lumbricus, Bifurcation in, 283

Luminous Organs, 281

Lung Flukes of North American Frogs,

203

Marker, 197

Marshall, Ruth. American Species of the

Genus Atractides, 185

Maryland, Diatomaceous Earth, 290

Material for Laboratory Use, Ameeba,

271, 278

Mechanism of Mendelian Heredity, 293

Members, List of, 307

Membrane, Middle, in Wings, 282

Mendelian Heredity, Mechanism, 293

Metcalf, H. E., Ampullze of Lorenzini in

Acanthus Vulgaris, 131

Metcalf, H. E., Cell Changes in the Epi-

dermis during the Early Stages of Re-

generation in the Tail of the Frog

Tadpole, with Special Reference to the

Nucleus-Plasma Relation, 167

Methods, Biological, University of Illi-

nois, 195

AMERICAN MICROSCOPICAL SOCIETY

Microscopic Technic, 50

Micro-technic, Botanical, 71

Microtome, Table for, 197

Middle Membrane in Wings, 282

Milled Lines, Rendering Visible, 278

Miller, R. T., Dissecting Board, 292

Minutes of Philadelphia Meeting, 63

Mites, Water, Genus Atractides, 185

Modeling Clay, for Beginners, 197

Morphology of Thysanoptera, 193

Moths, Catocala, 50

Mounting Crustacea, 292

Mounting Medium, Damar, 195

Euparol, 50

Mounting Zoophytes and Polyzoa, 55

Muscid Larve, Blood sucking, 193

Museum Jars, seal for, 197

Necrology, 299

Nematodes, Fresh Water, from Douglas

Lake, 21

Neutral Red as an Indicator of diges-

tion reactions in Protozoa, 275

New Orders of Insects, 194, 289

North American Frog Lung Flukes, 203

Nose, Development of, 7

Notes, Reviews, etc., 49, 149, 189, 271

Notonectide, 282

Nucleus-plasma relation, in cells of Tad-

pole’s Tail, 167

Objective, oil immersion, 196

Objects, Locating on Slides, 189

Oil immersion objective, best results, 196

Olfactory Sense in Coleoptera, 281

Olfactory Senses in Insects, 50, 284

Oligocheta, Notes on, 283

Oégenesis, in Philosomia cynthia, 191

Optic projection, 58

Orange-G—Hematoxylin, for Embryos,

Penetration of fluids thru heavy cuticle,

196

Pflaum, M., Custodian’s Report, 65

Philadelphia Meeting, 63

Photomicrography, 200

Plants, preserving colors of, 199

321

Plasma, Relation to Nucleus, 167

Plasmodium of Badhamia, Cultivation,

161

Poison glands of Insect Larvez, 49

Poison of Spiders, 193

Polyzoa, Mounting, 55

Pool, R. T., Memorial of C. E. Bessey,

299

Preservative for Insects, 282

Preserving Plants in Natural Colors, 199

President, Address of 1914, 7

Projection, Optic, 58

Proteocephalidz, Revision of Cestode

Family, 56

Protozoa, Digestive Reaction in, 275

Protozoology, Applied to Soil, 149

Proventriculus of Hydropsychid Larve,

192 |

Psychobiology, 60

Regeneration, 284

Regeneration, Cell

Stages of, 167

Reliable Method of Obtaining Amceba,

271

Removal of Balsam from Slides, 197

Report of Custodian, 65

Report of Treasurer, 66

Ripening Hzmatoxylin, 199

Roberts, E. W., Olfactory Sense in In-

sects, 284

Safety Razor Blades for Microtones, 196

Safranin P. and Lichtgriin, 51

Saturniide, hearing in, 49

Scales of Fishes of Douglas Lake, 255

Seal for Museum Jars, 197

Sex in Plants, Evolution of, 59

Sex Recognition, 281

Sexual Forms, Lack of, in Daphnia, 161

Shelford, V. E., Elytral Tracheation of

the Tiger Beetles, 241

Size Dimorphism in Spermatozoa, 191

Skin Glands of Frog, Difference in

Function, 275

Slavina and Dero, 283

Slide Box, Hinges for Lids, 196

Changes during

322

Slide Marker, 197

Smith, Gilbert M., Development of Bo-

tanical Micro-Technic, 71

Social Problems and Biology, 61

Soil, Application of Protozoology, 149

Spencer-Tolles Fund, Report, 65

Spermatozoa, dimorphism in size, 191

Spiders, Behavior of, 50

Spider, Poison, 193

Staining cephalic glands in immature

Trematodes, 276

Stylopization, 190

Stylops, 190

Subscribers, 307

System of Locating Objects on Slides,

189

Technic for Cestodes, 158

Technic from University of Mich., 275

Termites, 191

Trichopterous Larve, 279

Thysanoptera, Morphology, 193

Tiger Beetles, Elytral Tracheation, 241

- Tongue of Fly, for object, 52

INDEX

Tracheation of Elytra of Tiger Beetles,

241

Treasurer’s Report, 66

Trematodes, Staining Cephalic Glands,

276 ‘

Trichoptera, Wood-Boring, Proventricu-

lus of, 192

University of Illinois, Notes on Methods,

195

University of Michigan,

Technic, 275

Vertebrate Embryology, 161

Vials and Bottles, sealing, 199

Water Mites, Atractides, 185

Water Striders, Habits, 282

Wax in Dissecting Pans, 277

Welch, Paul S., Entomological Notes,

49, 190, 279

Welsh, B. C., Arachnoidiscus, 290

Laboratory

- Wilt Disease, 281

Wood-Boring Trichoptera, 192

Zoophytes, Mounting, 55

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