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No. I.— Contributions to the Morphology and Physiology of the CelJ
By A. B. MACALLUM, M.B., Ph.D.

(Heprintedfrom the Transactions of the CaiMcliati Imtitute, Vol. I., Pt. S.)

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1891. ' • ' '■

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' 1

V' ,>.,,'

,1.;, -r ■:;:■: '.-^[i. ■

[Extract from Transactions of the Cana<lian Institutf, ISUO.'^

-'-;

CONTRIBUTIONS TO THE MORPHOLOGY AND PHYSIO-
LOGY OF THE CELL.

By A. B. Macaixum, H.A., M.B., Ph.D.,

Lecturer in Physiology, University of Toronto.

(Read r^th November, iHgo.)

In the interior of the epithelial cells of the alimentary canal, anil in
the glandular cells of the pancreas in amphibia, are usually found struc-
tures which are of great mterest to both the morphologist anil the physio-
logist. Typical examples of these occurring in the gastric mucosa of the
salamander have been described and illustrated by Lukjanow,* and one
has but to glance over the figures he has given in order to gain an idea
of the number and variety of these bodies. They are much mure abun-
dant in the intestinal than in the gastric epithelium of a well-nourished
animal, and, so far as my observations go, they present, on the whole, a
greater complexity of form than those described by Lukjanow. What is
the significance of these bodies? With the exception of some of the
intranuclear forms, they can, I believe, be arranged in the three follow-
ing divisions :

1. Parasites.

2. The remains of broken-down cells and nuclei swallowed by the

healthy adjoining cells.

3. Material swallowed by the epithelial cell from the food passing

over its free surface (in the case of the intestinal epithelium).

4. Plasmosomata migrated or extruded from the nucleus (only in

the glandular cells of the pancreas).

It is, no doubt, impossible, in many cases, to determine to which of
these classes this or that particular body belongs since intracellular
parasites simulate plasmosomata and kindred structures in some stages
of their existence, and I propose, therefore, to treat of the structures in
a general way, pointing out, wherever possible, their relationship to one
or other of the classes given above excepting, however, those connected

* Beitrage zur Morphologic der Zelle — Arch, fur Anat. und Pliys., Siippl. Hd. zur Phys, Abth.,
18J7. P- 66.

.;;•>.■•■,

TRAN8ACTIO.NH OK THE CANADIAN INSTITITE,

[Vol. I.

with division 3, the treatment of which I postpone until I have finished
my experiments on the methods of the resorption of chromatins ( Nu-
deins).

To illustrate the parasitic nature of some of these forms, I will now
describe undoubted examples of intracellular parasites from the intestines
of the spotted newt and the lake lizard (Necturm).

I. A Cellular Parasite fko.m the Intestinal Epithelium of

DlEMYCTYLUS ViRIUESCENS.

In April of this year I obtained from the neighborhood of Toronto a
number of spotted newts for the purpose of studying the phenomena of
secretion in the pancreas and in making preparations of this organ I
found it frequently convenient, on account of the small size of the animal
and its organs, to include the anterior portion of the intestine. In the in-
testinal epithelium of one of the newts was found a large number of
forms like those shown in Figures 3 and 4, and I immediately endeavored
to work out their history. Before detailing the results of this work it
may be well to state that the particular object from which the sections
studied were made was hardened in F"lemming's Fluid and alcohol, stained
in toto with hjematoxylin, imbedded by the chloroform process in paraffin,
the sections therefrom placed in scries on the slide and stained with eosin
and safranin, before being permanently mounted in balsam.

The structures in question are so numerous that every second or third
epithelial cell.for long stretches of the section, contained one of them. They
are always placed in the outer half of the cell between the nucleus and the
free border, and have a nearly uniform diameter (9-1 l/i, averaging lO/x)
and an approximately spherical shape. They do not appear to have a
definite or distinct membrane, and what takes its place appears to be a
zone of homogeneous or faintly granular protoplasm which, in many
cases, is denser and thicker at one side of the body than at any other.
From this zone trabeculae of granular protoplasm pass inwards to ter-
minate in a more or less centrally placed protoplasmic mass. In a
number of these bodies sufficient to render the peculiarity prominent, the
bulk of the protoplasm is collected at one side (Fig. 3), while the thicker
portion of the protoplasmic rim occupies the opposite side with a large
crescentic, oval, or round cavity intervening. The protoplasmic mass
stains lightly but readily with eosin and contains a round homo-
geneous nuclear body, which stains deeply with safranin and measures less
than 2/A (i.S/^)- Sometimes the nuclear body is placed in a cavity in
the protoplasmic mass and connected with the latter by a few fine
strands. In a few instances, the nucleus was surrounded at a distance by

1HK<J-90.J

MOKPHOLOOY AND PIIVSKlLOdY OK THE CtLL.

a distinctly marked membrane which, however, may have been only a
thickening of the protoplasm bordering tiio cavity in which the nucleus
was situated.

Not so common, but still quite readily seen, are forms like that repre-
sented in Fig. 5, in which, in place of a single nucleus, there arc a large
number (over twenty) of safranophilous spherules, each surrounded by a
small quantity of finely granular protoplasm and marked off fioni the
rest of the mass by a delicate membrane (Fig. .S</.) These spherules
are homogeneous and measure much less than J/x. Fig. 6 apijarently
represents a later .stage of the same body, and in this one sees that
the homogeneous spherules have become transformed in such a way,
that the stained material in each is arranged in a horseshoe or crescen-
tic form, according to the specimen examined (Fig. 6, 7, He). The com-
parative .scarcity of these forms, the very small size of the objects and
the absence of a sharply defined contour to the stained material, render
it extremely difficult to determine this arrangement satisfactorily in many
cases, but in thin and well stained sections, and with good objectives
(2mm. immersion apochromatic, Zeiss), forms like those figured appear
now and then.

There can, I think, be no doubt about the parasitic nature of these
intracellular bodies, and we may, therefore, regard the stage described
in the last paragraph as that of sporulation.

I endeavored to determine the mode of transition from the stage in
which there is a single nucleus to that of sporulation. It was not an
easy subject for study, because, for every hundred that one observes
belonging to both stages, th>,re are not more than one or two forms that
can be ranked as transitional. Two of such are represented in Fig.
Sd and c. I have been led to consider them as stages in the formation of
spores, because they present structures which resemble somewhat kary-
okinetic figures. For example, in the form represented in Fig. 8d, the
centrally placed stained body may be regarded as belonging to the
dyaster stage and seen from one of the poles ; in it also structures, bear-
ing a resemblance to individual chromatin loops, can be made out. This
arrangement comes out well sometimes in preparations stained with
ha;matoxylin and safranin, but oftener the safranophilous substance is
collected in a ring form resembling, to a certain extent, the equatorial
plate of nuclear division. Probably the explanation of Fig. 8^ is
that it represents a multiple form of karyokinesis. The difficulty of de-
termining the nature of such conditions will be readily understood,
when it is remembered that the safranophilous bodies are usually not 2/a
in diameter, and that, consequently, its metamorphic elements must be
very small.

i I

TRANSACTIONS Of THE CANADIAN INSTITUTK.

[Vou I.

If the determination of the division of the nucleus is difficult, much
more so is that of the full history of the spores. They are so small at
first that, apart from the mother organism, they cannot be distinguished
from other cellular contents, such as the swallowed portions of the
debris of neighboring cells and the spore stages of other parasites. It is
only in a few cases that circumstances favor the determination of some
of the forms after they have escaped. In Fig. I, for example, is
shown a cavity in the interior of a cell, evidently once occupied by the
parasite in question, and in the neighborhood of the cavity is a number of
bodies like plasmosomata, of similar, or nearly similar size. These are
evidently the spores derived from the organisnr. which occupied the
cavity. In a few instances, with the best conditions for observation, forms,,
like those shown in Fig. ga, are seen. Here the structures are comma-
shaped, and their resemblance to other forms in the same Figure, to that
of Fig. \oa and to those in Fig. 2, is such as to suggest a developmental
relationship. The probability, however, that very young forms of Sporo-
zoan parasites arc similar to those represented in Fig. ga, is sufificicnt to
invalidate any conclusion that might be drawn from this resemblance.*

There is more certainty in regard to the larger comma-shaped forms,
such as are shown in Figs. 2 and lo^n These are intensely safranophilous
bodies, and measure from 3 to 6/j.. Their outlines are sometimes distinct,
sometimes not, this depending on the way in which the organism is dis-
posed in the field of the microscope. If the tail should happen to be above
or below the head of the comma the organism may be recognised with
difficulty. The connection between these and the spherulating forms can
be seen by glancing at Fig. 10 a-h. In further development the head of
the comma enlarges, the safranophilous substance collects into a small
round mass, leaving the protoplasm which contained it more or less
coarsely reticulated or finelj' granular, and with feeble staining capacity.
The tail still retains its safranophilous character and remains distinct
for several stages. The space between it and the head tends to increase
when its point becomes applied to the head (Fig. loc). At the same
time it becomes somewhat elongated {d), and the safranophilous sub-
stance in it condenses into a thin band bounding the convex side of the
crescentic cavity. The head also undergoes further changes {e). The
protoplasm becomes collected at its periphery as a rim to which the small
round safranophilous mas.s, the nucleus, is attached by delicate proto-
plasmic strands. In the next stage protoplasmic strands may stretch
across the crescentic cavity, to the remains of the tail or the point of the

•Compare with Steinhaus' Figures of the intracellular parasites in the pancreas of the Sala-
mander, Ziegler's Beitrage Zur Path. Anat., Bd. VII., Taf. XI.

1889.y(».]

MOHI'lIULOUV AM) I'll YsKlI.iiilY OK TilK CKLL.

tail may fuse with llii; head ; in the latter case tin; crcsccntic cavity
persists (/), The safranophilous substance ^railually disappears from the
thin band representing the remains of the tail, till finally its staining capa-
city is scarcely marked in some of the forms, althoujjh its density is notice-
able. This sketch of the orj^anism developctl out of the comina-shapcil
body explains thus the occurrence of a denser, frequently more tler|)ly
stainintj zone at one side, the presence of a cresccntic cavity, or of a cavity
next the zone, and the frequently excentric position of the nucleus in the
adult orjfanism {h'igs. 3 and 4). In individual cases, in which these pecu-
liarities are apparently wanting, it may be that they cannot be observed,
because the organisms are not favorably placed in the microscopic field.

We can, I think, now account for many of the forms shown in Fig. 9,
especially those in which a deeply stained crescent occurs with a sphere
in its cavity — they are merely comma-shaped parasites in the process of
transformation into that stage in which sporulation takes place. In the
same way we may explain some of the forms illustrated by Luk-
janow,* especially his Figs. 14, 15, 16, 6ia and /;, 66, 72, 74 and 75, and
probably al.so Figs. 7, 11, 13, 6.S, 69, jy and 94. His F"ig. 48 would seem
to indicate that he saw the sporulating phase of the same organism.
All his studies were made on the gastric mucosa of the salamander. I
have found in the gastric mucosa of Diemyctiiliis very few abnormal
structures of this character. If they are parasitic, their comparative
absence from the stomach may be attributed to the digestive and
resistent action of the gastric mucosa, and it is probable that the irregu-
larity and atypical character of many of the structures drawn by Lukja-
now may be due to the physiological action, during life, of the glandular
elements in which they occurred.

It is interesting to note the structure of the cytoplasm around the
full-sized organisms (Figs. 3-7). It is constituted of very fine rodlets,
each with a thick end directed towards the organism and passing in a
radiating manner peripherally into a zone of what appears to be finely
granular protoplasm, but which is, probably, a portion of the cytoplasmic
reticulum condensed. The border of thickened points in many cases
clcsely resembles a membrane. It depends, apparently, on the vitality of
the cell whether the radiating arrangement of the cytoplasm occurs or
not. It may be absent, as in Fig. 2, when the cell shows signs of
degeneration. It is difficult to understand the function of this mechanism,
but we may suppose it to act as a filtering apparatus.

'Op. cit.

TRANHACTIO.NH OF THE CANADIAN ISHTITUTE.

[Vol,. I.

II. On CUkOMATOI'MAdOUS AND OTIIKK I NTKACI'.LLUKAR PAKASITKS
IN THK InTKSTINK OF NlX'TURUS LATKKALIS.

Ill the intestinal epithelium of Necturus arc often found forms which,
from their peculiarities, must be ret,'arcled as parasitic. When I observed
tiicm first, I considered them to belong in a general way to that class of
intracellular structures which Lukjanow* has described as occurring in
the gastric mucosa of the .salamander, and of which there are not a few
examples in the intestine of Necturus. They are well shown in prepara-
tions made from recently captured animals, and their characters are pre-
served well in the tissues fi.xed with Flemming's I'^luid, or corrosive sub-
limate, and stained with alum cochineal, or haematoxylin or eosin.

The chromatophagous forms have usually an irregular outline and the
protoplasm extended in one or more long pseudopodial processes, which
taper often to fine threads In some ca.ses the whole organism is thread-
like (Fig. IS/). They are easily distinguishable in alum-cochineal pre-
parations in the unstained, epithelial cytoplasm, in which they may be
found, and by their stain being in every respect similar to, and as deep
as, that of the chromatin bodies of the epithelial nuclei. With high-
powered objectives the stain is seen confined to the fine granules which
densely crowd the cytoplasm of these organisms. There is sometimes
a quantity of unstained protoplasm at the thicker end (Fig. 14/), or
a more or less curiously shaped mas.s may lie in its neighborhood
(Fig. \6 pr). Sometimes the bodies are found in the interior of nuclei,
but, as a rule, they are not easily recognizable in this position, unless they
show amoeboid outlines or are fixed in the act of migrating from the
nucleus. One is shown in the latter condition (Fig. 15/}. The nucleus
is in this case partially deprived of its chromatin by the parasite, which
owes its staining capacity to the chromatin it absorbs or invaginates.

An explanation of the relations of such structures as are shown in Fig.
13 (/) can be at best only problematical. Here two parasites, each in a
separate cavity in the cytoplasm, have their prolongations hooked
around one another. This is only one of several instances observed of
such a condition, but the preparation drawn shows the process most
distinctly. It may be a case of conjugation.

There are a number of forms which are either wholly unstained by the
coloring reagent, or which possess one or more stained spherules or
granules (Fig. 1 3 p). These may, in some cases at least, represent young
stages of the chromatophagous forms.

•L. c.

lf*8l)-90.

MORPIIOLOOY AND PIIY8I0L0RY OF TIIR CKLI,.

In Fig. 12 is shown a cell from the base of the tpiihclial layer,
which has certain peculiarities worthy of note. In one of its two nuclei
is a cavity containing an cosinophilous, dumb-bell-shaped structure. The
chromatin of this nucleus is very much condensed, but a portion of it is
extended into the cavity in the form of doubly-beaded ro<llets. The
structure here reminds one strongly of that of the cytoplasm about the
parasites in the intestine of Dicmyctylns as described above, and I am
inclined, therefore, to regard the dumb-bell structure as a parasite. The ele-
ments in the neighborhood of the second nucleus may be parasitic also.
Such a case as this illustrates fairly well upon what slender grounds one
has to judge of the parasitic or non-parasitic nature of some intracellular
bodies.

III. On Certain Structukks in the Pancreatic Cells of

AMI'IHUIA.

In the pancreatic cells of Amphibia are structures which, since their
discovery by Nussbaum,* in 1883, have excited attention amoni^st a
number of cytologists, on account of their supposed participation in the
processes of secretion. From the fact that they presented resemblances in
position and form to structures described by v. la Valette St. George and
BUtschli, as occurring in the testicular cells of some invertebrates, Nuss-
baum gave them for temporary use the name nebenkerne. It will be
seen from the description given below that these elements are not normal
portions of the gland cell at all, and, therefore, do not merit the title, which
has, since Nu.ssbaum's paper was published, maintained its place in nearly
all the publications on the subject. I do not intend to discard the term,
however, because the full history of the structures have not been worked
out, and they may really belong to a stage of a Sporozoan parasite, whose
adult form may already be described and named. In that case the con-
tinued use of the term nebenkern applied to these elements is preferable
to the coining of a new word for temporary service probably, and I will,
therefore, not offer any further excuse for adopting it in this work.

If the elements in question were normal, it might be advisable to give
them an English name equivalent to the word nebenkern, in which case
the words " paranucleus," or " accessory nucleus " might suffice. The
word cyiozoon, on the other hand, is precluded, since it has been adopted
by Gaule and his pupils to denote, according to their views, the elements
in certain stages of cell metamorphosis or cell rejuvenescence.

According to Nussbaum's description, the nebenkerne are placed in the

• Uber den Bau und die Thatigkeit der Driisen. Arch, fiir Mikr. Anat.. Bd. XXL, p. 296.

TRANSACTIONS OF THE CANADIAN INSTITUTE.

[Vol. I.

protoplasmic portion of tae gland cell, between the nucleus and the mem-
brana propria ; they are oval in outline, and either solid or more or less
spirally twisted. There may be one or more in each cell, and when one
only is present it is usually larger than the several taken together, which
may happen to be in another cell. On the fourth to the fifth day after
feeding the animal (salamander), tney are present in every gland cell,
while they may be found with difficulty, or not at all, in animals recently
fed, and they are rare in animals which have fasted for a long time.

Nussbaum also found solid nebenkerne in the oesophageal glands of
the frog, and in the exhausted, unicellular glands in Argulus, and thread-
like ones in the pancreas of Triton.

As to the nature of these bodies, Nussbaum came to no conclusion.
Ogata*, on the other hand, put forward a view which connected them with
processes of secretion and cell renewal. According to his account, they
are the plasmosomata of the nucleus, which have wandered into the cell
protoplasm. The small nebenkerne are homogeneous, spherical, or ellip-
tical in outline, often elongated and do not stain with haematoxylin, but
they readily imbibe eosin, which, consequently, obscures their presence
amongst the similarly stainefl zymogen granules. In the larger neben-
kerne the chromatin substance is pr 'nt, consequently they are either
colored homogeneously violet or have one or more corpuscles colored
deep violet to pure blue. The large nebenkern can, on the one hand, in
old and exhausted cells, develop into a new cell, which, situated immedi-
ately adjacent to the membrana propria, pushes the disintegrating
nucleus and remains of the old cell towards the lumen, and increases its
own cytoplasm, in which zymogen granules appear ; on the other hand,
it may, in ordinary cells, break up into zymogen granules. It depends
on the general condition of the gland, whether the nebenkern breaks up
into zymogen granules, or developes '"nto a new cell. The production of
zymogen :s not, however, limited to the nebenkern, for the granules were
seen in the process of formation in the nucleus.

Ogata found in the moderately large, as well as in the full-sized
nebenkern, cavities and fissures which gave them various appearances.
Sometimes the structures were seen to sit cap-like on the nucleus.

Ogata stimulated the pancreas either by pilocarpin or by electrical irri-
tation of the medulla, and found the number of nebenkerne greatly
increased. When two or more doses of pilocarpin were given at intervals
of twenty-four hours, the resulting number of nebenkerne was smaller

•Die Veranderung der Pankreaszellen bei der Secretion. Arch. fUr Anat. and Phys., Phy*-
Ahth., 1883, p. 405.

1889-90.]

MORPHOLOGY AND PHYSIOLOGY OF THE CELL.

than when only one dose was given. He explains this on the ground
that the first dose has greatly increased the number of nebenkerne, and
thereby weakened the cells, which now respond to the second dose less
readily.

Ogata also traced a relation between the disappearance of the neben-
kerne and the appearance of new nuclei.

Platner's* first published views coincided to a certain extent with those
of Ogata. His description substantially is this: The large round
nucleolus of the pancreatic cell elongates, and moves towards the peri-
phery of the nucleus, often pushing out its membrane. The long axis of
the nucleolus corresponds to the radius of the nucleus. A portion of
the nucleus becoming constricted off, this part contains the nucleolus and
is separated from the main portion by the formation of a homogeneous,
septal wall. The nucleolus and the separated portion of the nucleus con-
stitute together the nebenkern, which, when the main portion of the
nucleus regains its usual size, sits on it like a demilune. The nebenkern
becomes homogeneous, separates from the nucleus and breaks up into
granules which are probably zymogen. These observations were made
on the pancreas of Anguis fragilis, and were corroborated in that of the
frog.

Platner's second studyf led to somewhat different results. He used
for this purpose the pancreas of a number of Reptilian and Amphi^ m
forms, but he obtained the most decided results from that of the sala-
mander. In the latter the irregularly contoured nuclei of exhausted gland
cells stain deeply with safranin, so that the nuclear franicvvork becomes
indistinct. Of the many or several prominences on each nucleus one
only remains finally. Into this the chromatin, distributed throughout the
nucleus, wanders, with the result that the prominence appears as a dark
red bud on the remaining portion of the nucleus, which now gradually
returns to the normal condition, namely, that in which the nucleus shows
an unstainable caryoplasma (Kernsaft). These buds are variously shaped,
large or small, round or irregular. The nuclear membrane in most of
the cases still covers it. Often it has vanished and the contents, still
colored deeply, lie as fibrillar or coiled elements, or as partially granulated
material, in the protoplasm of the cell. The constriction between* the
nucleus and the bud deepens, till finally they separate, the bud now losing
its uniformly staining capacity. At the same time the protoplasm of

•l^)er die Entstehung der Nebenkerne und seine lieziehung zur Kerntheilung. Arch, fiir
Ni»r. Anat. Bd., XXVI., p. 343.

tBeitrage zur Kentniss der Zelle und ihren Theilung. Arch, fiir Mikr. Anat. Bfl. XXXIII.
p. 180.

TRANSACTIONS OF THE CANADIAN INSTITUTE.

[Vol. I.

the cell increases, till it attains its normal maximum volume. The
retrogressive metamorphosis of the nebenkern, as Plainer now terms the
separated bud, goes hand in hand with the vigorous formation of zymo-
gen granules in the cell. The nebenkern stains less readily with hsema-
toxylin, and its volume decreases gradually, till either only fibrillar
remains of the same are visible among the zymogen granules, or it is
indistinguishable.

It is seen that these observations raise the question how far " partial "
chromatolysis, as Platner terms the formation and degeneration of the
nebenkern, thus described, enters into the processes of secretion, but
Platner leaves the matter undecided,

Platner accounts for the discrepancies in the two descriptions of the
mode of formation of the nebenkern by stating that in the pancreas of
Anura, which formed the basis of his earlier observations, the determina-
tion of the various points is difficult, because of the small size of the cells
in which the nebenkern sits cap-like on the nucleus.

Steinhaus* solves the question of the nature of these bodies differ-
ently. He denies their normal occurrence in Amphibia. They were
not present in the pancreas of six axolotls which he examined and they
were also absent from the pancreas of frogs obtained from one locality,
though present in those of another. Even in the pancreas of some sala-
manders they are absent. He states that they have no connection with
the processes of secretion, as the formation of zymogen granules goes on
as well in the cells deprived of these bodies, as in those possessing them.
They lie unchanged and, so far as the formation of zymogen granules is
concerned, inert in the cell protoplasm. He never saw any structures
which proved either the origin of these bodies out of the constituents of
the cell, or their convi rsion into zymogen granules, or their connection
with cell renewal. Steinhaus studied the condition of the nucleus in all
the phases of secretion, but could observe nothing which would be
considered as nuclear budding, according to Platner's description.

Steinhaus gives no verbal description of the nebenkerne, but in his
figures he represents them as varying in size and nut.^ber in each cell
and as thread or worm-like forms more or less coiled, some of the larger
onrs of which have one end of the thread thickened to resemble a head.

Steinhaus considers these bodies as parasites whose relationship to
the Haematozoa is unmistakable, but so long as we know only this stage

*Ueber parasit&re Einschlusse in den Pnncreaszellen der Amphibien — Ziegler's Beitrage zur
Path. Anat. und zur AUgem. Path,, Bd. vii., p. 367.

1889-90.]

MORPHOLOOY AND PHYSIOLOGY OF THE CEIX.

in their life-history, it is impossible to say anythin;^ more definite than
that everything at present determined points out their kinship to the
Sporozoa

It is of interest here to note the occurrence of supposed ncbenkcrne in
the pancreas of the dog* Melissinos and Nicolaides found that
these are intra- as well as cxtranuclear forms, sometimes of
curious shape and composition. The intranuclear ones, the
plasmosomata, may wander from the nucleus into the cell sub-
stance, where, as these observers arc led to believe from the
results of experiments with pilocarpin, they break up into zymogen
granules. They deny the correctness of I'latncr's view, that the appear-
ances, from which Ogata was led to believe that nuclear plasmosomata
migrate into the cell, are artificially produced, and, in support of their
position, they mention that in a quarter of an hour a^^ter the adminis-
tration of pilocarpin the plasmosomata show all the stages of migration
from the nucleus, the extranuclear forms are numerous and zymogen
granules arc present, while in half an hour after the administration,
neither plasmosomata, cxtranuclear forms, nor zymogen granules are
visible. The extranuclear forms, which arise by migration from the
nucleus, of the pla:!mosomata, they call nebenkernc and these they dis-
tinguish from others, which are more or less complicated in their
structure and composition and which lie in distinct cavities in the cell
protoplasm. These latter they think are: (i) excretions of the cell
protoplasm ; (2) the remains of leucocytes ; (3) chromatolysed nuclei.

METHODS OF STUDY.

I used several methods at the outset of this research but finally gave
the preference to one mode of preparation which included either Flcm-
ming's Fluid or corrosive sublimate as the hardening reagent. This
mode of preparation was as follows :

The animal {Diemyctylus viridescens, Amblystoma punctatiim, Pletlio-
don glutinosiis) was decapitated, the abdominal cavi' 't opened, the
pancreas snipped away and immediately dropped into a saturated solu-
tion of corrosive .sublimate, where it remained ten to fifteen minutes, or
into a quantity of Flemming's Fluid, wnere it was left from one to twenty-
four hours, according to the need. The operation of removal was usually
done within twenty seconds, this interval including the decapitation
process al.so The object of this was to prevent any post-mortem

*Untersuchuni;cn Uber einige intra- and cxtranucleare Gebilde im I'ankreas der Siliigethiere auf
ihre Dezieliung zu der Secretion. Von C. Melissinos. Mitgetheilt von K. Nicolaides. Arch.
fUr Anat., und Phys., Phys. Abth., 1889, p. 317.

ii\

TRANSACTIONS OF TIIK CANADIAN IN8TITUTK.

[Vol. 1

changes in the pancreatic cells and I believe that it was attained in
every case. The piece of tissue was after removal from either of these
fluids, vva.shed for a few seconds in distilled water, then transferred to 70%
alcohol for three hours, in the case of the corrosive sublimate prepara-
tion, and for twenty-four hours, when the Flemming's Fluid was used.
When the latter was allowed to act longer than one hour, the alcohol
was changed as often as it presented a trace of chromic acid coloration.
The harden ng was completed by a stay of twenty-four hours in 95 per
cent alcohol. The organ was now transferred to the staining fluid, alum
hiumatoxylin, (a few drops of a saturated solution of ha;matoxylin in ab-
solute alcohol to a saturated .solution of pure ammonia alum in distilled
water: allowed to stand one month in summer sunlight before using, and
kept from deterioration by crystals of thymol), for ten to fifteen hours. In
order to prevent overstaining, I found it advi.sable to dilute the original
ha;matoxylin solution with twice its volume of distilled water, in which
dilution, after the time allowed, there is only a pure chromatin .stain in
the nuclei of the pancreatic cells and a faint shade of purplish blue in
the nebenkerna. The objects are now washed in distilled water to
remove the alum and the excess of the staining fluid, and are then put
in a quantity of a i per cent solution of eosin in 30 per cent alcohol for
from two to three hours. Washed in 95 per cent alcohol, till the latter
■was but faintly colored with the eosin after one hour's action, the
object was placed in absolute alcohol for five minutes, then in pure
chloroform for fifteen hours on the average, after which it was kept
in a saturated solution of paraffin in chloroform at 3S°C. for about
eight hours, and finally placed for a like period in melted paraffin
{melting point 52°C). The sections were made of a thickness not ex-
ceeding 5m with the Thoma-Yung microtome and fixed by the ribbon
method in series to the slide with a diluted Sch'allibaum's clove oil-
collodion mixture, (clove oil i volume, collodion 3, equal parts of
absolute alcohol and ether 3). I used, sometimes in the case of the
corrosive sublimate preparations, the Gaule method oi' fastening the
paraflfin sections to slide, but, as the process of staining on the slide was
not employed, except when the action of saffranin was required, it did
not present any points of advantage over the other, which was the
quicker. The paraffin was removed with benzole and '^'le sections
mounted in benzole balsam.

The staining of the object as a whole with haematoxylin and eosin has
the advantages of giving a regular and uniform depth of reaction in the
various sections and the different parts of each, and of preventing the
loss of important elements entailed by the process of staining on the
slide. I found that a little practice enabled one to judpe of the length

188J

[Vol. 1

1889-90.1

MORPHOLOOY AND PUYSIOLOOY OF THE CKLU

of time necessary to give the tissue its proper depth of stain, and I
determined that a stay of eight or ten hours longer than usual in the
diluted hematoxylin solution, did not seem to increase the depth of the
stain, or to make it more diffuse. Probably the explanation of this is
that the equilibrium between the coloring matter in the diluted solution
and that deposited in the tissue is reached when the chromatin is
saturated. This, of course, is merely an application of the principle,
that length of time and degree of concentration are elements in the
right employment of staining methods and that these are, roughly
speaking, in inverse proportion to one another.

In order to determine if the nebenkerne contribute in any way to the
elaboration of the secreted elements of the pancreas I resorted to the use
of pilocarpin. I had a large number of Diemyctyli at my disposal, and
on these I studied the action of the drug, so far as the nebenkerne are con-
cerned. Batches of ten, twenty and thirty were taken, and into the ab-
dominal cavities of each of these less than 2 mgrm. of pilocarpin was
injected. Three of these were, at certain periods after the injection, decapi-
tated, the pancreas of each removed, hardened with corrosive sublimate,
and treated as described above. These periods were usually : i, 2, 3, 4,
5, 7, 9, [2, 17, 22, 36,44, 52 and 60 hours, and these were chosen in some
cases for convenience. I took three at each period, because, if I depended
on one, misleading results might be obtained. It was found that the
averages of the results obtained from each three agreed with each other
in presenting an unbroken outline of the history of the nebenkerne.

I treated very young forms of Amblystotna punctattim also with pilo-
carpin, the method of employment of the latter in this case being to dis-
solve twenty to fifty milligrams in about half a litre of water and placing
the animals therein for a period of five to twelve hours. As they mea-
sured between thirty and thirty-five millimetres in length, it is obvious
that an intra-abdominal injection of a solution of the drug was out of the
question.

The specimens of Necturus kept in the laboratory aquarium were not
used for this investigation, since, owing to their not having been fed for a
long time, the pancreas presented a more or less atrophied condition. It
was found impossible to stimulate the gland in these to activity, or even to
make it secrete at all.

There is a great advantage to be obtained from the concurrent use of
the two hardening reagents, corrosive sublimate and Flemming's Fluid.
The former fixes thoroughly and quickly the zymogen granules as well
as the cellular and nuclear structures in the pancreas, while A^ith Flem-

THANHACTIONS OF THE CANADIAN INSTITUTK.

[Vol. I.

mine's Fluid, though the cell structure and nucleus arc well preserved, the
zymogen is dissolved out of all the cells except those at the immediate
periphery of the organ. This removal of the zymogen is due to the acetic
av 'd in the fluid, which penetrates '"here another constituent of the same
mi.xturc, osmic acid, is unable to ditl'use. The action of acetic acid in this
reagent enables us to distinguish between zymogen and other granules
which have the same staining capacity with cosin. The osmic acid, fur-
thermore, gives a dark tinge to the nebenkerne and unusual bodies in those
cells near the periphery and thus brings them out in clear contrast to the
other cytoplasmic structures.

OnSKKVATION.S.

In sections made from the pancreas of Dieinyctyliis, which has been
hardened with P'iemming's Fluid and stained with hematoxylin and eosin,
one observes in addition to the nucleus and cell protoplasm and, some-
times, zymogen granules, other structures which can be ranged in two
groups at least. One of these groups comprise forms whose fundamen-
tal structure elements are thick or thin fibrillar, either in sheaf shape, or
wound in a ball fashion (Fig. i). Sometimes the fibrillae may be so thick
as to merit the designation threads (Fig. 8). These forms are usually
but not always, placed between the nucleus and the membrana propria,
and they frequently sit, cap-like, on the nucleus, or the latter may be
indented by them. In the second group, which are, at the outset, unlike
the first, in that they are placed in cavities of the cell, are structures which
present a varied form and composition. They are sometimes eosinophil-
ous, sometimes chromophilous, and at times they present both characteis,
They are numerous in the pancreas of a freshly captured animal, but are
not so much so as the members of the first group.

The members of these two groups of intracellular elements have been
confused by other observers, and Ogata describes them as derived from
the plasmosomata migrated from the nucleus, while Steinhaus appears to
believe they are all parasites. In order to show that the views of these
observers are hasty generalizations from a limited number of results, I
propose to go fully into the description of the structure origin, mode of
production, and history of each group. As plasmosomata, migrated, or
extruded from the nucleus, are sometimes present, and as they have a
different history, they merit special attention as a third group. These
three groups may then stand in the order of description as follows :

1. Parasites.

2. The remains of broken down cells and nuclei swallowed by

healthy adjoining cells.

1889-90]

MOKPHOLOOY AND Pll YSIOKOOV OF TIIK I'KLt.

3. Plasmosomata, migrated, or extruded from the nucleus' into tlio
cell protoplasm.

I. Parasites.

These arc, as already said, usually, but not always, placed between the
membrana propria and the nucleus of the cell. They vary in size, measur-
ing in their extreme limits i/x and 9/i, and their shape, usually oval, may
also be oblong, spherical, elongated, club-like, or crescentic in section.
They are not very sharply .separated from the protoplasm of the cell and
if the latter is dense, their outlines arc distinguished with difficulty.
Their structure varies also, but there are certain features in this
respect which arc tolerably constant for the great m<ajority of the.sc
forms. These are the central cavity and the fibrillatcd appearance,
the fibrillre, as a rule, appearing as if wound around the central
cavity. The central cavity may contain from one to several /ymogen-
like granules. The fibrillar do not appear as if wound tightly, but are
more or less tortuous in their course and the outermost ones may appear
ragged, or project loosely into the surrounding protoplasm. This fibril-
lated arrangement is best seen in Flemming's Fluid preparations from
freshly captured Diemyctyli^ and, especially, in those on which the reagent
has been allowed to act for twenty-four hours. The osmic acid and the
hematoxylin in such give these bodies a dark brown stain, which deeply
contrasts with the lightly or non-stained, surrounding protoplasm. In
corrosive sublimate preparations, on the other hand, the fibrillation usually
does not appear so distinct except under high powers when it readily be-
comes manifest, and hrimatoxylin gives it a faint reddish violet stain.
Zymogen granules are entangled in the peripheral fibrillac, often so abun-
dantly, that they obscure the presence of the organism in question.

This stage is the most common, but in order to understand its na-
ture, it will be necessary to consider the characters of the other forms
found even in the same .sections. These present more the appearance
of pla.smodia, are usually much smaller, and they take a deeper and more
uniform stain with eosin. In the protoplasm of these, one can, at times,
see concentric laminated slits, which are apparently an indication of a ten-
dency to form fibrill.'c, but which may al.so indicate that these plasmodia-
like masses are derived by the fusion of the protoplasm of a coiled
thread. Such coiled threads are rarely seen in ordinary preparations,
but very frequently in sections from the pancreas of some Diemyctyli,
which have fasted for about two months (Fig. 8). These coils have
been, now and again, found to be dense in sections from the pancreas
removed fifty to sixty hours from the animal after the injection of pile-

TKAN8ACTI )N8 OF THE CANADIAN INHTITUTK.

[Vol. I.

carpin. When these bodies are very small, the number of turns in the
coil is not more than two or three, whereas in the largest forms the
number of turns cannot usually be made out.

All the forms, then, are either plasmodia-likc masses, or are composed
of Pbrilla; or threads. Whether the plasmodia arc elements of a separate
stage in the metamorphosis of the bodics,or whether they are merely formed
by the fusion of the protoplasm of the threads, cannot be decided defi-
nitely. It can certainly be determined that the fibrillated stage is one
of degeneration, for one can find the fibrillated forms in all conditions up
to disappearance. Figs, i, 2, 3, 4, and 6nb show this. The first step in
this consists in a more or less parallel straightening of the fibrillar and a
consequent flattening of the whole mass, then the cell protoplasm pushes
it towards the periphery where it lies, usually, directly under the cell
membrane (Figs. 2, 3, and \nb). Here the fibrillae disintegrate one by
one, till finally, owing to their fineness and small number, they can not
be distinguished from the cell protoplasm. Platner has described the
occurrence of such fibrillated remains in the cell protoplasm, and he con-
siders them derived from the nebenkerne.

I am inclined to believe that the coiled thread is the intact form of the
parasite, and that the plasmodium-like mass may be either an earlier or
a subsequent stage in the life history of the parasite. In the case of the
latter form, the fact, that it is usually smaller than those in which the
fibrous or fibrillated structure is manifest, tends to show that it is a
younger stage, but not conclusively, since even small fibrillated masses
occur sometimes.

I have withheld the proofs that these forms are parasitic till now. Of
course each fact adduced is not of itself sufficient to prove the correctness
of my view, but all taken together are conclusive in this respect. These
facts may be summarized in the following items : —

They are not present in the pancreas of the great majority of young
forms oi Amblystoma pmictatum. I sectioned the whole of the pancreas
of seven of these and found these bodies in only two of t' em. Of these
two, one contained only eleven of the structures, while the rest possessed
hundreds, and in both these cases, as well as in the other five, the cells
exhibited all stages in secretion. I treated five other larvae with pilocarpin
and examined the pancreas at intervals of four, seven, eleven, thirteen,
and twenty-two hours after, without finding a single specimen of this
nebenkern. The larva, in which the greatest number of such were found,
measured in total leng»^h a little over thirty millimetres, while the others
were of the same length or some what longer, and we may conclude, there-
fore, that the occurrence of these bodies does not depend on the stage of

[Vol. I.

lB8y-!iO.]

MOKPIIOLOaV AND PHY8I0L00Y OF THE CELL.

ns in the
)rms the

oni posed

separate

y formed

ded defi-

ge is one

itioiis up

t step in

X and a

n pushes

the cell

; one by

r can not

ribed the

\ he con-

rm of the
earlier or
ise of the
vhich the
it it is a
d masses

now. Of
)rrectness
t. These

of young
pancreas
Of these
possessed
the cells
•ilocarpin^
, thirteen,
:n of this
2re found,
;he others
de, there-
z stage of

developmen; although it may depend on the change in the food, or
habitat, which the increased development entails.

2. They are present in all the cells of the actively secreting pancreas
of Diemyctylus, as well as in that of an animal fasting for two months or
more. When two or more are present in a cell, they are, usually, but not
always, small. I have found them present in the cells apparently without
diminution in number at every indicated interval, after the injection of
pilocarpin. In corrosive sublimate preparations of the gland cells distendeil
with zymogen granules, these bodies are, in many cases, not seen. If one
relied wholly on corrosive sublimate as a hardening reagent, one might
conclude that this is a stage in which the nebenkerne are absent, having
been used up in the formation of zymogen, and such a conclusion has been
ativanced by Ogata. That the bodies are not absent, but merely obscured
by the granules, is shown in preparations made with Flemming's Fluid
from a pancreas in the same condition. This reagent dissolves out the
zymogen in the centrally placed tubules, and, if allowed to act for twentj-
four hours, blackens the structures in question, thereby showing them to
be as numerous in this phase of cellular activity as in any other. I
have, however, found that they, as a rule, stain somewhat more readilj'
with eosin at certain intervals after injections of pilocarpin, and this
condition is concurrent with the filling up of the exhausted cell with
zymogen, and with a subsequent exhaustion of the same. The deeper
stain during the formation of zymogen is due to absorption of the latter
diffused from the nucleus, its seat of formation, while, in the other case,
the cells, having their energy exhausted, cannot destroy c vi ; *^^egrate
the organisms, which absorb tlie cell juices and thereby I ii, greater
readiness for eosin. I think this latter condition is in some .onnected

with the vitality of the animal, for it is less apt to appear in vigorous
animals, and I found it best exemplified in sluggish ones, while in sonic
cases, again, it appeared in forty-five to fifty-five hours after the adminis-
tration of one dose of pilocarpin.

3. They are not derived from the nucleus by constriction and partial
chromatolysis, as Plainer describes, although other structures described
farther on, with which these have been confused, may be so derived. I
have examined series of sections made from the pancreas of over seventy
Diemyctyli, exhibiting all the phases of glandular activity and yet I
have never in a single instance seen the bodies in question, in any way,
derived from the nucleus, nor are they plasmosomata which have migrated
from the nucleus and have undergone a certain amount of extranuclear
development, a thesis which Ogata adopts and defends. I have found
extranuclear plasmosomata, and, as will be seen from the description

TKANHA(.'TIOVH OF TIIK CANADIAN INHTITUTB.

[Vol. I.

further on, traced their history, which is totally unlike that of the
structures in question. Given, then, that they are derived neither from
the cell protoplasm nor from the nucleus, the only remaining conclu-
sion possible is that they come from without — in otiicr words, they arc
p.irasitic.

4. The parasitic nature of these bodies is best shown by their form in
the two young Amblystomata referred to above. Fig. 10 rt, b, c, e,f{nh),
represent the commoner types of these and a resemblance to a " wur li-
chen " typo is readily seen in the.se.

5. The fibrillation and gradual di.sappearance of these bodies occur
without any participation whatever in the processes of cell activity and
secretion. There can be no doubt about the correctness of this, and
moreover, IMatner's description practically admits it, although he thinks
tliat the desintegration of these bodies furnishes material for an increase
in the amount of the cell protoplasm and, possibly, of its zymogen. It
is not to be denied that the desintegration and possible assimilation of
these bodies increase the cell protoplasm and may, therefore, very
indirectly assist in the formation of zymogen.

The statements made by Steinhaus that these structures are not derived
from the cell or nucleus, that they have no functional relation to secretion,
nor have anything to do with cell renewal, I can, therefore, fully con-
firm. His observation that they are inconstant even in the same species,
agrees with mine as to the young Amblystomata. His figures, however,
of these bodies resemble but few of mine, and show the " wurmchen "
form to be more common than I have been permitted to see in my pre-
parations. If Platner's statement is correct, that the fibrillar remains of
these bodies can be observed in the pancreas of the .salamander, it is evi-
dent that Steinhaus has overlooked the full history of the structures.
Steinhaus is also in error in concluding that the parasites alone are the
nebenkerne of Ogata or Platner, for bodies have evidently been included
in this class by the two observers, which are not parasitic at all.

What are these parasites "i Steinhaus believes that they are similar to,
not to say identical with, those described under the names H.tmatozoa
and Cytozoa. There are several facts which speak for the correctness of
this view. The forms of some of them correspond with that found in the
blood cells of the frog, the " wurmchen " of Gaule and known as Drepan-
idium ranarum of Lankester. The latter is also to be found in the blood of
Dtemyctylus. Kruse* states, however, that it is not present in the blood
of the tadpole and this fact is to be taken in connection with the absence,

*Virchow's Arch., Bd. 120, p. 553,

1 889-90.]

MOHPIIULOliV AND I'HVHIOl.UliV OK TlIK < Kl.l,.

!'.»

pencrally, of the pancreatic parasites in youn^j Amhtystouuxta, if an expla-
nation is desired of the latter phenomenon. Furthermore, the dct;enera-
tion and disintc[,'ration of the pancreatic parasites antl the complete
absence of the reproductive processes show that some otiier tissue is the
breodin}^ tjround of the parasite, and their presence in every pancreatic
cell points to the blood as their source.

The destruction of such lar^e numbers of the parasites in the pancrea-
tic cells seems to indicate that the pancreas of Amphibia is a pro-
tective as well as a .secretive oryan, and that it plays this part specially,
since the parasites have not been found in any other w^i\\\ after the most
careful search,

2. Karyolytic and Cytolytic Pkoducts

These elements are few in some Diemyctyli, abundant in others, the
latter especially in freshly captured animals. They are found only in
groups of the cells at certain spots in the sections and they present cha-
racters which definitely distinguish them from the elements described
in the foregoing section. Probably the best representation of these forms
is given by a glance at Figs. 6 chm, 4 rchc, pvieg, 5 pvt.

Their fcrm is usually spherical or approximately so, and their size, as
well as their structure, varies. They often consist of chromatin and eos-
inophilous substance, or simply of protoplasm which has a special affin-
ity for staining reagents. Less commonly, they may contain cosinophi-
loiis granules like the zymogen granules, or these may be present with
the chromiitin masses. Apart from the occurrence of eosinophilous
granules ard the slightly stained protoplasm, the structure of these bodifes
is mostly varied by the quantity of chromatin present and the form vhich
it takes. £ ometimes the whole of the structure seems composed of chro-
matin (Figs;. 3 and 6 chm), but more frequently the latter forms a small
o-ldly shapi:d mass irregularly placed in the structure. One may sec
rings, rods, crescents, hooks, and spirals formed of this substance and
variously disposed in the protoplasmic mass carrying them. These bodies
usually lie in the cavities in the protoplasm of the containing cell, a pecu-
liarity which readily brings them to view when their affinity for staining
reagents is very slight. These elements are sharply distinguished from
the parasitic bodies in that they never fibriliate and they, moreover, have
a different fate. The latter can only be studied in the pancreas of freshly
caught animals, and in those in which the various phases of the resting
cells are being developed. In the active gland they may be numerous
but as the resting phase of the gland cell is step by step being estab-
lished they are found to become correspondingly smaller, the staining

TRANSACTIOSB OP TUB CANADIAN INSTITUTB.

[Vol. I.

with liitmatoxylin less vivid, while tlie lar^jcr bmlics disintegrate and the
fiaj,Miients become scattered through the cell. The disappearance of these
clenicnts, the concurrent increaHe in the cell protoplasm and the appear-
ance of zymogen granules are not matters of physiological relation.
The removal, or rather the disappearance of chromatin, is on the other
hand in some way connected with the abundance of chromatin in the
greatly enlarged nucleus of the containing cell (Figs. 3, 4, 5, and 6).
The nucleus may be somewhat distorted in its shape, and this is without
doubt due to the abundance of the chromatin which it has absorbed
from the elements in the cell. The processes of disintegration and
absorption go on till finally in the resting gland cell a few protoplasmic
masses, scarcely larger than zymogen granules, may remain.

The origin of the.sc bodies is to be sought for in the broken down
gland cell. Indeed one can see them so derived in the sections. In
Figs. 3 and 5 are some of the remains [cdi and r^//t') of such disintegrated
cells lying in the intercellular spaces, while the surrounding cells contain
masses, which, from their position, are evidently swallowed portions of the
same. The farther a cell is removed from these intercellular masses the
freer it is from the intracellular elements in question and at a distance not
greater than the diameter of a cell the.se may be ab.sent altogether. In
other word.s, wherever one finds the intracellular bodies numerous one can
also in the same or in the ne.xt section find intercellular elements to in-
dicate the place of origin of the former. It is quite possible that disin-
tegrated leucocytes may give rise to the same, but I have seen no evi-
dence of such, except, perhaps, in such forms as that represented in
Fig. 6a.

These bodies are also present more or less in the pancreas of all the
young /I /;//'/j'i-/o;/w/rt examined and they exhibit here also the same vary-
ing compo.sition and structure.

These bodies do not participate in the processes of secretion. The
presence of eosinophilous granules, like those constituting the zymogen,
led Ogata to consider them as breaking up into zymogen and from the
fact that the parasites may appear to contain zymogen granules more or less
imbedded in them, he concluded that the latter are earlier phases in this
formation of zymogen. These eosinophilous granules are not formed of
zymogen, however, because in the more centrally placed cells in a sec-
tion of the pancreas prepared with Flemming's Fluid, the zymogen gran-
ules are dissolved out by the acetic acid in this reagent, but the eosinop-
hilous granules are not affected. This phenomenon has a bearing on the
mode of secretion and I will, therefore, forego an explanation of it till I
come to this subject farther on.

[Vol. I.

lt(!S'J9(l.]

MOI(l'l|l)I.()<iY AM) HIIVHlDUXiV OK TIIK CKLI..

Nothing can probably demonstrate more effectually the non-secretory
nature of these elements tiian tlie fact that they are present in the cul)ical
cells lining the ducts and ductletsof theglaml (Figs, ii />,<•//;;/ and \2ri/n).
Nor arc these bodies confined to the pancreas, for I have found them in
the cphithelial cells of the intestine, in the liver, the kidney and cutaneous
epithelium of Diciiiyctylus and Nictiiriis. , They indicate, however, how
little of a tissue is normally lost to itself and how it husbands its waste
material. It is, of course, on first view, surprising that the pancreatic cells
.should exhibit am'i'boid properties, but it is less so when we remember
that the hcp;itic cells, wiiich in sections have a definite and apparently
fi.ved form, manifest in the teased out scrapings from the cut surface of
the fresh liver annnboid movements.

3. Mi(;i<ATi:i) OR E.XTkUDKi) Pi,asmosom.\ta.

Plainer denies that the nuclear plasmosomata migrate, and, at first, I
was inclined to this view. It is easy to sec in well hardened sections of
the pancreas plasmosomata driven by the knife from the periphery of the
nucleus into the cell, the nuclear membrane torn, and the Ccivi'y pre-
viously occupied by the plasmosoma empty. This occurs chiefly when
the plasmosomata are large and placed next to he nuclear membrane.
The apparent protrusion of the nuclear membrane, in some cases, is really
due to a shrinking of the same at every part, except opposite the plas-
mosoma, which offers a resistance. I found, however, as the investigation
proceeded that there were phenomena which could not be so explained.
For example, in the pancreas of a young Amblystovia, about one-fourth
of the nuclei showed plasmosomata which were fixed in the act of passing
from the nucleus to the cell. I saw plasmosomata of dumb-bell form half
outside and half within the nucleus and some were embedded in the cell
protoplasm. I saw this condition, moreover, but less marked, in the pan-
creas of a specimenof Dicmyctyliis removed twenty hours after the injection
of pilocarpin. Though the evidence was unmistakeable, I cannot but think
that if the phenomena are constant or normal, they should be observed
oftener. In any case the migration or extrusion has, from all that I see,
no connection with the processes of secretion. If it is a case of extrusion,
one might imagine it to occur readily in the pancreas of any specimen of
Diemyctylus, unless one were to suppos*^ that in certain stages of cell ac-
tivity the nucleus is more contractile. My attempts to establish the
correctness of such a supposition resulted unsuccessfully.

That the extrusion or migration is not a normal phenomenon appears
to be borne out in the history of the extranuclear plasmosomata. They
either disintegrate and form granules like that of zymogen in size and

'! \

i I'

TRANSACTIONS OF THE CANADIAN INSTITUTE.

[Vol. I.

sLainin^ reaction, or persi't for a time in a cavity of the cell protoplasm
and gradually loose their eosinophilous character. Forms of the latter
are rare but they can be distinguished from the cytolyscd products of
other cells by the fact that they are more or less eosinophilous, and by
the fact further, that one only is to be found in a cell, while similar bodies,
protoplasmic or otherwise, are absent from the adjoining cells. For the
purposes of the diagnosis of course serial sections are necessary. But
with these aids even, the process of determining whether a slighth'
eosinophilous, extranuclcar mass is a plasmosoma derived from the
nucleus is a difficult one. The disintegration into zymogen-like gran-
ules is easily distinguishable on account of the fact that the resulting gran-
ules are collected at one spot in the cell (not near the border) and from
their resisting the action of acetic acid. It is possible, on the other hand,
that a plasmosoma may neither disintegrate into zymogen-like granules,
nor persist with the gradual loss of the eosinophilous character in the
cell protoplasm. I observed in the pancreas removed from an animal
one and a half hours after the injection of pilocarpin, the ductlets filled
with zymogen in a granular condition and containing here and there a
large plasmosoma-like mass. In this case no intra-cellular plasmoso-
mata were observed, although zymogen was still present in the cells. I
think this phenomenon indicates that the pancreatic cell can, under such a
strong stimulus as pilocarpin furnishes, throw out of itself all material
not part of its own mechanical structure, and that the extranuclear plas-
mosomata may, in some cases, be disposed of in this way.

That Ogata made the mistake he did in assuming that the extra-
nuclear plasmosomata become converted into nebenkerne and the latter
again into zymogen granules is very natural in view of what is described
above. The passage of plasmosomata from the nucleus to the cell, the
mingling of zymogen granules, either with the substance of the plaa-
modium-like mass or with the fibrillai of the degenerated parasite and
the occurrence of protoplasmic masses loaded with eosinophilous gran-
ules are demonstrable facts which Oj^.ita seems to have observed, and he
built up from these the theory outlined, a feat and a mistake which any
cytologist, who had paid as careful attention to the subject as Ogata did,
might have committed at that time. What was less excusable was the
construction of a theory of cell rejuvenescence, for although chromatolysis
was then unkric vn, or at least undescribed, and, therefore, the occurrence
in pancreatic cells of protoplasmic masses possessing chromatin unex-
plained, yet the knowledge concerning the indirect process of cell division
had then made a great advance and it was hardly necessary to postulate
the existence of another process. All things considered, however, Ogata's

[Vol. I.

1889-00.]

MOUl'HOLOGV AND PHYSIOLOGY OF TUK IKLL.

opiasm
e latter
ucts of
and by
bodies,
^or the
But
ightlx-
m the

work has been of great service in caUing s|)ccial attention to structures,
the further study of which may definitely establish a new function lor the
pancreas in cold-blooded animals, viz., a protective one against the
Ha^matozoic parasites.

In connection with these remarks on Ogata's views, I may mention
that I have frequently observed in some sections of the pancreas of
/^/V;;/;r/;'///.f examples of karyokinesis and that in the cells in this con-
dition there were neither nebenkerne, protoplasmic masses, nor plasmoso-
mata. Steinhaus gives an illustration of a pancreatic cell exhibiting
karyokinesis in which, apparently also, nebenkerne (parasitic) are present.
I have also frequently observed cell and nuclear division in the pancrea-
tic cells of the young Amblystomata and it was apparent that the nuclear
division might go on with the cell more or less filled with zymogen
granules.

4. Zymogenesis.

It has been known from the researches of Haidenhain and others that
ch uiges in the shape and staining power of the nucleus accompany the
change from the resting to the active phase of the secreting cell. What
the relations are which these changes bear to one another, were not divined,
but it was generally supposed that they were the results of increased or
decreased nutrition. The observations of Plainer and Steinhaus embrace
one aspect of these changes i.e., the staining power of the nucleus, and it
is to this that I propose to devote this section.

A summary of Platner's views as to the changes in the staining power
of the nucleus of the pancreatic cell has been given above in the histori-
cal sketch of the literature on the pancreatic nebenkerne. Steinhaus'*
observations, bearing more directly on the staining power, are of greater
interest to us and may be abstracted as follows :

The exhausted gland cells are small, indistinctly contoured, and defi-
cient in protoplasm and their arrangement in the form of alveoli is lost.
Their nuclei which are angular and crenated arc, when a double stain of
h.-ematoxylinandsafranin is employed, colored red, and their nucleoli are
.^afranophilous. When the active phase of the cells begins, the cyt iplasm
increases, the contour of the cell becomes distinct, the arrangement in
alveoli with central lumen is attained, while the form of the cell becomes
bluntly conical. At the same time the nucleus becomes oval and stains
readily with haimatoxylin. This dye stains one sort of nucleoli, the kary-
osomata, while the safranin colors the other and larger kind, theplasmo-

• op. cit. p. ni.

TRANSACTIONS OF THE CANADIAN INSTITUTE.

[Vol. I.

somata. At this point the formation of zymogen granules begins in the
part of the cell next the lumen and it proceeds till the cell is filled with
them. As to the origin and mode of production of the zymogen granules
nothing is known. When secretion begins these granules disappear and
the nucleus now tends to return to the condition found in the exhausted
cell.

My own observations coincide with those of Steinhaus. I may empha-
size here one or two points. The nucleus of the exhausted gland cell
stains readily and deeply with safranin, that of the cell in which the for-
mation of zymogen is going on vigorously, is colored deeply with h.-emato-
xylin, while its plasmosoma takes readily the safranin.

My explanation of this phenomenon is drawn from the results of my
observations on the formation of yolk in the ovarian ova of Necturus and
Rana, and a summary of these may therefore not be out of place here.

In the nuclei of the developing ova at a certain stage the chromatin
is principally collected in the form of nucleoli at the periphery immedi-
ately under the nuclear membrane. These nucleoli are usually spherical
and they may, though not usually, or very much, vary in size. All the
chromatin of the nucleus is not so situated, for there are long threads
which at certain points in the granular karyoplasma unite at angles with
one another. At this stage yolk spherules are absent from the cell. If
now sections of such an ovary are stained with the indigo-carmine
stain of Shakespeare and Norris, the significance of the peripheral
nucleoli is determined. Such sections show here and there an ovum in
which the peripheral nucleoli are stained deep blue, while the remainder of
the nucleus and cell is stained red. In other ova, again, the peripheral
nucleoli and the karyoplasma are stained blue, the cell red, while in others
again the peripheral nucleoli are smaller, the whole ovum, with its yolk
spherules which now begin to be formed, is stained blue, or blue green.

The origin of the subsfance which stains indigo-blue in this process is
certainly derived from the peripheral nucleoli, .'or it is possible to meet
with an ovum once in a while in which a portion of the karyoplasma in
the immediate neighborhood of and around each nucleolus is, like the
latter, stained indigo-blue, while the remainder of the karyoplasma is red.
The peripheral nucleoli generate a substance, therefore, which diffuses
gradually through the nucleus, then into the cell protoplasm, the point
in time of th° latter occurrence corresponding with the formation of the
yolk spherules. The mode of origin is through a process of deposition
from the nucleus of a substance allied to chromatin in the cytoplasm.

The diffusion of a substance produced from the nucleoli through the

[Vol. I.

1889-90.]

MORPHOLOGY AND PHYSIOLOCJY OF THE CEi^L.

nucleus and into the cell protoplasm, can also he determined by other
staining; reagents, (•..^.,'alum cochineal, but the different stajjes in this phe-
nomena cannot be thereby so readily detc-mined as with the other
method.

I regard the yolk spherules as formed by the union of a derivative of
the nuclear chromatin with a constituent of the cell protoplasm. This
derivative of the nuclear chromatin is, possibly, the same as the h.tma-
togcn which Hungc discovered in the fowl's egg united with an albumin.
The formation of yolk spherules in the cell protoplasm is analogous to the
the formation of zymot; a granules in the pancreatic cells and both arc
accompanied by changes in the nucleus and an increase in the cell
protoplasm. It is most natural to conclude that the processes underlying
the formation both of the yolk spherules and of the zymogen granules are
in a general way alike. We see many facts .supporting this view. In the
developing ovum there are phases in the elaboration of the chromatin and
the formation of nucleoli (plasmosomata) comparable to the production of
chromatin in the nucleus of the resting pancreatic cell, and to the appar-
ent conversion of this chromatin into safranophilous substance which
diffuses through the nucleus in the exhausted cell. We see a further
parallel to the formation of yoke spherules in that as the nucleus loses its
safranophilous substance the cell protoplasm acquires safranophilous
granules. If we accept the parallel so far as correct, we may then
assume that the chromatin of the nucleus of the pancreatic cell gives
rise to a substance which we may call " prozymogen," sometimes dis-
solved in the nuclear substance, sometimes collected in masses (plasmo-
somata), and finally diff*used into the cell protoplasm, uniting with a
constituent of the latter as zymogen. This is, I think, the true explana-
tion of the phenomena of secretion.

With the help of this theory we can explain why it is that in certain
pancreatic cells the protoplasmic masses contain, as described above, eosin-
ophilous granules of exactly the same size as those of zymogen, but
unlike the latter in that they are not dissolved out by solutions contain-
ing acetic acid. The protoplasmic masses swallowed by a pancreatic cell,
cannot be of the same composition as the cell protoplasm, and are not
amenable to the laws which govern the nutrition of the cell as a whole.
When the prozymogen diffuses from the nucleus to the ccU it invades
the protoplasmic masses enclosed, and it becomes united with a constitu-
ent of the latter, thereby forming a compound similar to zymogen in
some respects : the capacity for forming spherules, the eosinophilous and
safranophilous reaction, but differing from it, as already said, by being
insoluble in solutions of acetic acid.

uil

tC !i

TRANSACTIONS OP THE CANADIAN INSTITUTE.

[Vol. I.

That the nucleus is the seat of formative energy is shown by a number
of observations*, especially those which bear on the vegetable cell. In
the latter the first stages, at least of starch formation, are carried out in
the nucleus and this secretes a compound which is finally converted by the
the cytoplasm into starch. Korscheltf has also determined that in the
formation of the chitinous processes on the eggs of Ne/>i7 and Ranatra
the chitin necessary for each process is elaborated in a cavity between
and surrounded by two epithelial nuclei, and the only legitimate conclu-
sion from such a circumstance is that the chitin is derived from a nuclear
substance. I may also here refer to the fact that my own observations
have definitely shown that the h.emaglobin of the red corpuscles in
Xeciiirus SiXid Aiiiblystoiiia \s derived from the chromatin of the nucleus
both of the fully formed as well as of the developing red cell, and that the
ha;maglobin so formed diffuses through the nuclear membrane and be-
comes fixed in the cytoplasm. All these facts point definitely to the
prominent part played by the nucleus and if everything in connection
therewith is carefully studied, it will be admitted, I believe, that the inter-
pretation which I have given of the changes occurring in the nuclei of the
pancreatic cell during the various phases of glandular activity, is not a
strained or a far-fetched one.

APPENDIX.

After the foregoing was written, a paper containing the observations
of Eberthij: on the pancreatic nebenkerne in salamander came into my
hands. In this is advanced a new view of the relations of these bodies,
or pseudo-nuclei, as Eberth prefers to call them. He states that they
are developed out of the reticular fibrillcTe of the cytoplasm, the latter at
spots apparently becoming swollen, or thickened by fusion with their
neighbors, and at the same time altered in composition, whereby their

* See a resum^ of such researches in Strasburger : " Ueber Kern-und Zelltheilung im Pflan-
zenieiche nebst einem Anhang iiber Belruchtung," Jena, 1888, pp. 194-204.

t" Ueber einige interessante Vorgange bei ('er liildungder Insecteneier." Zeit. iiir wess, Zool.
Bd. 45.

JNeber Einschlussein Epithelzellen. Fortschritte der Medicin, Sept. i,, 1S90.

1 889-90. j

MORPHOLOGY AND I'lIYSlOLOCiY OF THK CKLl,.

capacity for absorbing staining reagents is inc/cased. Later several of sucii
bent fibrills approach one another and acquire the shape of a sickle, semi-
circle, or circle. The latter show all possible stages of transformation into
the laminated bodies and spherules, which possess a very irregular fibrilla-
tion appearing to consist of loose threads, while they may at times resemble
laminated colloid bodies. The pseudo-nuclei disappear during hunger,
while becoming gradually paler and less easily stainable. As to the
process and manner of disintegration Eberth could offer no cxplanat' p
He compares these bodies with structures described by Czcrmak as
occurring in the ethmoid cartilage of the calf, and with those found b\-
Solger in the cartilage cells of the shoulder-girdle of the pike. Ebcrtii
believes these structures to be normal, and in a sense, compar.ible to the
nodules of the nuclear network.

Eberth states that the employment of corrosive sublimate as a harden-
ing reagent and of paraffin for imbedding produces contraction and
shrinkage in these objects, and that then one obtains the peculiar shapc'A
which possess a certain resemblance to Cytozoa. He accordingly recom-
mends Rabl's Fluid or Flemming's Fluid for hardening and celloidin for
imbedding.

Now I have carefully gone over the whole of my preparations since
last October, and have during this winter made a number of new prepara-
tions from Dieviyctyli and young Ainblystoiiiata, using for this purpose
each of the three hardening reagents mentioned, frequently on pieces of
the pancreas from the small animal. I have found that Rabl's Fluid
often gives the appearance of coarse, parallel fibrillation in the pancreatic
cells, when neither Flemming's Fluid nor corrosive sublimate demon-
strated the presence of a single nebenkern in the parts of the pancreas
hardened with either of these reagents. Such a parallel arrangement of
coarse fibrillae is probably artificially produced. It appears also to cause
a swelling of the cytoplasmic fibrilkt, whereby these are rendered more
distinct, and I think that to this property is due the advantage obtained
by the employment of Rabl's Fluid in demonstrating the elements of the
achromatic spindles in dividing nuclei.

My later observations strongly confirm my view that the nebenkern c
are parasitic elements. In eight Amblystomata, killed during Januar)-
and February, there were nebenkerne in only one, and here very abun-
dant 'y. There could be no doubt about the sharply outlined form, as
Stc haus has figured it, often homogeneous but as often fibrillated. I
have seen quite distinctly the thickened portion ot the organism which
simulates a head. As the Ainblystoiiiata kept in the laboratory tank were

TKANSACTIONS OF THE CANADIAN INdTITUTE.

[Vol. I.

not rcffularly fed, I attribute the intact form possessrd by many of the
parasites to the lowered vitality of the host produced by want of food.

Kbcrth's views are directly oppo.sed to mine. He considers the fibril-
lation of the structures in question not as an evidence of their degenera-
tion, but as a stage in their formation. His observations, confined as
they were to one form, cannot, I think, be held as conclusive by any one
who has studied the changes in the pancreas of Amphibia as exhibited
throughout the year. I cannot share Eberth's views as to the action of
corrosive sublimate on the form of these bodies and that it does not
produce a contraction or shrinkage, as he maintains, is shown by
Figs. I, 2, 9, and lo^, n/>, which were drawn from preparations made
with this reagent. I would call attention to Fig. lo/;, )i/>, which
shows a form not at all uncommon in the specimen oi Aiiiblystoina re-
ferred to in the last paragraph and which is very like some of the
specimens of Drepanidiuin figured by Gaule.

I have, in this connection, made further observations on the elabo-
ration of the pancreatic ferment. The results of these are confirmatory
of the views already advanced by me and may be summarized as fo
lows : —

1. In the gland cell filling up with zymogen granulesj the latter are
largest at the border of lumen of the gland tubule, while the smallest
are found at that edge of the granular area nearest the nucleus. This
serves to show that the granules are increased in volume by the depo-
sition of a substance from the " protoplasmic " area of the gland cell.

2. While the eosinophilous substance disappears from the nucleus, the
"protoplasmic" zone becomes eosinophilous at a time nearly coin-
ciding with the commencement of the deposit of granules in the cell
In other words, the eosinophilous (or safranophilous) substance diffuses
out the nucleus to the protoplasmic zone of the cell, from which it is
ai)parently removed to be fixed in some way in the zymogen granules.

3. In the gland cell after exhaustion and when a restoration of its
active condition commences there is an absorption, apparently from
without, of chromatin, or ot a chromatin-like substance, by the protoplas-
mic zone, and it would seem that the nucleus increases its quantitj^ of
chromatin from this source.

[Vol. I.

any of the
t of food.

3 the fibril-
r dcgenera-
jonfined as

by any one
IS exhibited
le action of
it does not

shown by
itions made
_ „/;, which
iblystoma re-
some of the

n the elabo-
confirmatory
arizcd as fo

:he latter are
e the smallest
ucleus. This
by the depo-
- eland cell.

e nucleus, the
nearly coin-
2S in the cell
stance diffuses
)m which it is
logen granules.

iteration of its

pparently from

,' the protoplas-

its quantity of

'.^'^^'O

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l¥'

1889-90.]

MOKPnOI.OCiV AMI l'IIV>ililI,(i(JV OK TllK ( KLL.

•2\)

EXIM.ANATION 0|- ri.ATI I.

The illustrations arc dreiwn with the Ahbc camera lucida, ccmbiiied
with the 3111m. or the 2mm. apochroinatic objectives (Zei-is), and compen-
sation ocular 4 or K.

I'"igs. I- 1 1 are from the intestine of Dieinycty/i(s ^in'i/rsrri/s.

]'\<f^. 1. Three epithelial cells in each of which there are unusual struc-
tures — 1/> represents the cavity in uhicli, ap[)arently, a parasitic element
matured and whose spores are seen in the atljacent cytoplasm. In the
central cell are probably both spores and inva^inated. cjtolyseil material,
while in the cell to the left there are structures which from their sliape
appear to be parasitic. X720.

Fi{^. 2. Two epithelial cells in one of which the nucleus is degenerated.
In both cells arc seen structures exemplifying two stages in the develop-
ment of the same parasite. In the degenerated cell the parasite, /», is ma-
tured, but in th(; cytoplasm of the other cell, they are, apparently, all
comma-shaped. Xiooo.

Fig. 3. A single epithelial cell containing a fairly typical specimen of
the parasite, it, the cellular nucleus, fi/>, the nucleus of the parasitic organ-
ism. There is present a cavity and a rim of thickened protoplasm,/;'.

Fig. 3(?. An epithelial cell in which the parasite, p, is in the stage of
transition from the comma to the adult form. Xicoo.

Fig. 4. In this the parasite, /, possesses a central mass of protoplasm
in which is imbedded the nucleus and which sends processes toward the
periphery. The remains of the tail of the comma are still recognisable
in the denser portion of the periphery. Y 1000.

Figs. 5, 6, and 7. The sporulation stages of the parasite with the
tnibecular arrangement of the cell protoplasm pc, well marked The
horseshoe form of the spore is clearly shown in 6rt!. Xiooo. Oa. X2250.

Fig. 8. Represents five stages in the development of the sporulation
phase of the parasite. In a the thickened band of protoplasm at one
side represents the remains of the tail of the comma stage ; in l> the two
central rings probably represent a stage of mitosis which is further ad-
vanced in c; \n d the spores are formed each in a cavity of the ptoor
plasm and these are further developed in e. X 1000.

1*1

TRANHACTlD.NS (IK IIIK CANADIAN INSTITt'TK.

[Vol. I.

I''K- 9- Represents illustrations of comma forms met witli in tlic epi-
tliclial cells. In n coiled form is shown. Xiooo.

I'i^;. lo (i-(/. are illustrations showing,' tlie way in which the comma is
tr.uisAjrmcd into the adult parasite; <•-/ represent forins which show the
v.irious ways in wliich the nucleus, cavity and tail arc disposed in the
ailult or developing,' form. X lOOO.

I'ifj. I \(i. Represents a section of an epithelial cell in a cavity of which
arc eni;,miatical structures, the larjjer one probably bcid}^ parasitic, the
others may be cither parasitic or protoplasmic mas es with chromatin
spiicrulcs. Xiooo.

Fig. I \/>. In this cell arc a number of i»tructure^ all of which arc evi-
dently parasites. Xiooo.

Fig. 12. A cell found in the epithelial layer of the intestine of A\rtii-
yiis,ii, the nucleus,/, plasmosomata-like masses which may be parasitic.
n\ a nucleus in which the chromatin is principally massed at one side and
continued into the cavity in the form of doubly beaded rodlets ; a dumb-
bell shaped body, deeply eosinophilous, is shown in the act of migration
from the cavity. X66o.

Fig, 13. Epithelial cells of the intestine of Mr/wr/zj'; ;/, the epithelial
nuclei ; /, the nuclei of leucocytes ; /», two intracellular parasites lying in
cavities of the cell. XG60.

Fig. 14. Intestinal epithelial cells of .Vtr///r//j ; /, parasitic elements ;
/, the nucleus of a leucocyte, x 660.

Fig. 15. Intestinal cells of Nccturns ; p, parasites migrating from
from nucleus ; /', either in vaginal ed, cytolysed material or stages in the
development of the parasite. X600.

Fig. 16. A single epithelial cell of the intestine of AVf///r«j, showing
a large cavity in its proximal part occupied by a parasite /, and proto-
plasmic remains, /r. X600.

Explanation of Plate II.

The outlines of all the figures were made with Abbe's camera lucida in
combination with 2mm. apochromatic objective and compensation ocular,
4 or 8. In the case of Fig. 8 the drawing was made at the foot instead of
at the level of the stage of the microscope, hence the difference in the
magnification.

be, blood corpuscle.

ci)t, cytolysed masses.

[Vol. I.
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18«!)-90.]

MOKPHOLOGY AND IMIYSIOI.OOY OF THK CELL.

dim, chromatin masses or bodies derived from chromatolysed nuclei.

pmeg, protoplasmic bodies loaded with eosinophilous granules like
zymogen, but insoluble in acetic acid.

ul\ ncbenkcrn,

relic, remains of chromatolysed nuclei and cells.

"St zymogen granules.

Fig. I A resting pancreatic cell from Diciiiyctylus; iil, a large irregu-
lar plasmosoma ; the chromatin is very abundant. Corrosive sublimate.
l[;em., cosin. Xiooo.

Fig. 2. Two resting pancreatic cells from the same preparation as the
last. In the right hand cell the elasticity of the fibrils of the degenerated
nebenkern has sprung out the cell wall. X looo.

Fig. 3. From the active pancreas of Dieviyctylus. Illustrates the
invagination by normal cells of cytolysed material. The cavity in the
centre occupied by the round mass, rchc, was probably the site of the cylo-
l)'scd cell, and from this the cytolysed products have passed to the sur-
rounding cells. The part represented occupied the centre of the section
and the meshes of the cytoplasmic network were filled with zymogen
granules which were dissolved out by the acid hardening reagents. It is
to be noted that the nuclei here are large and rich in chromatin. Flem-
ming's Fluid. Hicm., eosin. Xiooo.

I'ig. 4. Taken from near the margin of a similarly prepared section
and therefore showing zymogen granules ; a, enlarged nuclei ; b, a
nucleus with a sickle-shaped clement, half within and half without the
cell. Xiooo.

I"'ig. 5. I'Vom the resting pancreas of Dieuiyctyhis. The part drawn
was from near the margin of the section. In the centre of the illustration
is shown a cavity or intercellular space partially occupied by cytolysed
material and the chromatin derived from it is found in the adjacent cell
(dim), whose nucleus is greatly enlarged. The other nuclei are somewhat
irregular and rich in chromatin. Flcmming's Fluid. Ha;m.,eosin, safra-
nin. Xiooo.

I'ig. 6. From the central part of a section from the pancreas of a
freshly captured specimen of Diemyctylus. Here also are shown free
intercellular masses, and in the adjacent cells spherules of chromatin and
cytoplasm ; a represents a single cell from the same preparation. Flem-
mings Fluid. Iliem., eosin. Xiooo.

Fig. 7. Three pancreatic cells from Diemyctylus. The formation of
zymogen has advanced somewhat, the chromatin is abundant and the

ki.\

TRANSACTIONS OF THE CANADIAN INSTITUTE.

[Vol. I.

karyosomata numerous and sometimes large (ks). The plasmosomata of
which there are two to each nucleus are usually large and irregular in
shape. Corrosive sublimate. Ha;m., eosin. x looo.

Fig. 8. From the pancreas of a specimen of Dieniyctylus deprived of
food for five weeks. Corrosive sublimate. Haem., eosin.

Hg- 9i From the pancreas of a specimen of Dicmyctylus removed
forty-five hours after an intra-abdominal injection of o.4mgrm of pilo-
carpin. Corrosive sublimate. Ha;m., eosin. XlCX)0.

Figs. lo and 1 1 . From the pancreas of specimens of Amblystoma pnnc-
tatum (developing into adult condition). Fig. lo, a-f, drawn from the
same pancreas. Corrosive sublimate. Haim., eosin. XIOOO'

Fig. 12. Cells lining the pancreatic ductlets of Dicmyctylus, showing
in their interior cytolysed and chromatolysed products a and b, from the
pancreas 24 hours and one hour respectively after the intra-abdominal
injection of pilocarpin. Corrosive sublimate. Ha^m., eosin. Xiooo

I !