AN INVESTIGATION 
OF THE BLOOD OF 
NECTURUS AND CRYPTOBRANCHUS 
(Necturus maculatus and Cryptobranchus alleghaniensis) 
by 
EDITH J. CLAYPOLE, Ph. B., M. S. 
This paper received the first prize in animal histology, awarded by 
The American Microscopical Society. 
[Extract from Proceedings. Voe- XV, 1893, pp. 39-71.] 
WASHINGTON, D. C. : 
JUDD & DETWEILER, PRINTERS. 
!893- TABLE OF CONTENTS. 
Introduction 39 
Division of the subject 39 
Coagulation 39 
Measurement and counting of corpuscles 47 
Measurement 47 
Counting 51 
* 
Ingestion of carbon by leucocytes 54 
Summary of paper 64 
Methods . 66 
Bibliography 67 
Plates and descriptions 71 , THE BLOOD OF NECTURUS AND CRYPTOBRANCHUS. 
(Necturus maculatus-Cryptobranchus a/leghaniensis.) 
Awarded First Prize in Animal Histology by the Society. 
Edith J. Claypole:, Ph. B. (Buchtel), M. S. (Cornell), Akron, Ohio. 
The investigation, the account of which is contained in these 
pages, is partly histological and partly physiological. Although 
there exists a large amount of literature on the subject of blood, it 
relates almost exclusively to that of mammals; the naturally pre- 
dominant interest of man in man has caused the nature, peculiari- 
ties, and influencing conditions of this important tissue of the body 
to receive careful and exhaustive study in higher forms of life. In 
the forms below mammals, in some relations at least, the blood has 
not been so critically studied, and consequently a great many points 
are as yet obscure and undetermined. It has been the subject of 
this investigation to attempt to take a step into this relatively unex- 
plored region and to make somewhat clearer the at present indefi- 
nite horizon. The conclusions have been drawn with as little bias 
as possible, yet the possibility exists that the facts as presented may 
bear other and better interpretations.* 
The subject, as here considered, divides itself into three principal 
heads: 
1. Coagulation. 
2. Measurement and counting of corpuscles. 
3. Ingestion of carbon by leucocytes and their subsequent dis- 
tribution through the tissues. 
1. Coagulation. 
In respect to the first part, all discussions on the subject of blood 
in general give no mention as to whether the conditions of coagu • 
* This paper contains the results of an investigation carried on in the 
Histological Laboratory of Cornell University, and was presented for the 
degree of M. S. in June, 1893. I wish to express my appreciation of 
the abundant material and facilities so generously put at my disposal. 
39 40 
PROCEEDINGS OF THE 
lation are similar in these low forms to those known to exist in the 
case of mammals; simply statements as to the rapidity with which 
the process takes place are made. The conditions are definite and 
very well known in the higher forms, and the various authors agree 
as to the results obtained under various physical conditions and 
when acted upon by various chemical reagents. 
Many difficulties exist in the investigation of amphibian blood, 
owing to its relatively small amount and its rapidity.of coagulation. 
The restraining or preventive agent must be mixed with the blood 
immediately on the issue of the latter from the heart or vessels, and 
only comparatively small quantities can be used to demonstrate its 
action. When, however, some skill is attained as accurate results 
can be got as when larger quantities are used and as definite action 
takes place in regard to the chemical substances used. 
Before entering on the discussion of coagulation macroscopically 
a few words as to the microscopical appearance of fibrin will here be 
appropriate. It is already known from the researches of Professor 
Gage, of Cornell University, that a great difference exists between 
the fibrin of mammalian and the fibrin of amphibian blood, in re- 
gard to the fineness of the constituent threads. The mammalian 
threads are much coarser than those of amphibian blood. As in the 
frog, the fibrin threads of Necturus and Cryptobranchus are so fine 
that they require careful observation with a -jL-inch oil-immersion 
objective to distinguish them. Even under the most favorable con- 
ditions a very fine network is apparent, rather than individual 
threads. Comparison with the fibrin of a mammal (see plate I) 
immediately shows the great difference between the two formations. 
Experiments were made with various per cent, solutions of neutral 
salts to determine, if possible, whether the delayed fibrin formed 
differently from that unmixed with chemicals. No difference in 
microscopical structure could be detected, either in fineness or any 
other respect. The fibrin thus formed had exactly the same appear- 
ance as that shown in pure blood preparations. The same methods 
of preparation were employed in preparing both mammalian and 
amphibian blood. Briefly, they were as follows: A small drop of 
blood, taken fresh from the animal, was put on a cover-slip; this 
was covered with another slip, leaving the two eccentric for con- 
venience of handling; the unused blood was pressed out; the prep- 
arations were put in a moist chamber for from io to 30 minutes. 
When a neutral salt solution was used, a small drop was mixed with 41 
AMERICAN MICROSCOPICAL SOCIETY. 
the blood on the cover immediately, the subsequent treatment of 
the two being exactly alike. For mounting, the covers containing 
fibrin were washed with normal salt solution (T per cent.) or water 
stained with eosin, dried thoroughly, and mounted dry in a cement 
cell. As both the mammalian and amphibian blood were subjected 
to precisely the same processes under similar conditions, the results 
obtained were quite comparable. 
During some experiments made with various per cent, solutions 
of the neutral salt, magnesium sulphate (chemically pure), to find 
the one that best prevented coagulation and preserved the form of 
the corpuscles, some interesting peculiarities were observed, i per 
cent., 2 per cent., 3 per cent., 4 per cent., 5 per cent., and 6 per 
cent, mixtures were used, and the first three proved to preserve the 
form of the corpuscles well, the 2 per cent, being on the whole 
preferable, as the weaker one decolorized and the stronger distorted 
the cells more or less. With increased strength came increased 
distortion, although the color was much better preserved. After 
12 to 14 hours the color was so far extracted from the corpuscles, 
even in the 2 per cent, solution, as to make the easy distinction of 
the cells impossible. With 5 per cent, the color was well preserved 
in the corpuscles even after 24 hours, and also in the 6 per cent, 
solution, but in the latter by that time a weak clot had formed, 
which increased slightly in 36 hours, remaining, however, distinctly 
marked even after the corpuscles were decolorized. On microscop- 
ical examination the characteristic appearance of fibrin was shown 
in the clot. Experiments were then made, keeping the same ratio 
of dilution, 1 part of blood to 50 parts of the salt solution, on the 
higher per cent, solutions of the salt, 7 per cent., 10 per cent., 15 
per cent., and 25 per cent. The clotting took place in these also 
and with greater rapidity than in the weaker solutions. A firm clot 
appears in 6 per cent, in hours; in 7 per cent, traces of a clot 
appear in 2 to 3 hours; in 10 per cent., 15 per cent., and 25 per 
cent solutions in 30 to 40 minutes, though in 6 hours a distinct 
clot forms in all the solutions. After 24 hours in the strongest so- 
lutions the whole mass becomes jelly-like, most strongly so in the 
25 per cent, solution, and decreasing to the 10 per cent., and only 
slightly appearing in the 7 per cent., the latter preserving most 
clearly the true appearance of the clot. Here it must be mentioned 
that in all the solutions the jelly-like formation increases slightly 
but definitely in time and the clot decreases in the same slow way. 
6 m 42 
PROCEEDINGS OF THE 
In order to find out whether other neutral salts behave in like 
manner a similar series of experiments was carried out, using solu_ 
tions of sodium sulphate (chemically pure) as the diluent. In this 
the only difficulty encountered lay in the much greater solubility of 
the magnesium salt; it takes 55 per cent, for saturation, while the 
sodium salt is saturate at 29 per cent. However, as the low per 
cents, were mainly used and the solutions made in an approximately 
uniform temperature, the difference between the two salts does not 
materially affect the results. 
As far as preservation of form in the corpuscles is concerned, 
Na2SO4 was not nearly so successful as the magnesium salt. Even 
with the low per cents, distortion takes place. This may be ac- 
counted for in part by the difference in the molecular weights of 
the two compounds, that of MgSO4 being 120, while Na2SO4 is 
142. The density of equal per cent, solutions is consequently 
different, greater in the sodium salt, producing a distortion which 
does not take place in the lighter magnesium salt. In clotting also 
a slight difference is present. Indications appear in 5 per cent, 
solutions, while in 7 per cent, there is a firm, solid clot; the firmness 
increases in 10 per cent., while in 20 per cent it is a jelly-like liquid 
rather than a clot. 
Experiments were made on the effect of these salts on frog's 
{Rana catesbianaj blood with approximately the same results. 
The clotting began in 6 per cent, and was very marked in 7 per 
cent. ; it formed slowly and rather weakly in 10, 15, and 20 per 
cent. Na2SO4 and MgSO4 in 2 to 3 hours. The jelly-like formation 
also took place less distinctly than in Necturus and Cryptobranchus, 
yet it was sufficiently clear to be unmistakable. A similar series of 
experiments was made on the blood of the ganoid fish, Amia calva; 
both salts were used with the characteristic results somewhat modi- 
fied and not so distinct as in the frog. In MgSO4 clotting began 
in 5 per cent, with a very slight trace, in 6 per cent, increased, in 
7 and 10 per cent, still weak, while not till 25 per cent, was a good 
clot observed, when the jelly formation also appeared. In 5, 6, 
and 7 per cent. MgSO4 and 7 per cent. Na.2SO4 the clot appeared 
fine and granular, while the strongest clot was in a saturate solution 
of Na2SO4. In addition, the same series of experiments was made 
on cat's blood ; the results were uniformly negative. No clot of any 
kind formed in any of these solutions, the mixture remained liquid, 
and even after the ninth day no tendency to clot was observed. That AMERICAN MICROSCOPICAL SOCIETY. 
43 
Necturus and Cryptobrauchus. 
Frog. 
Amia. 
Cat. 
MgSO4. 
i % ... 
No clot; form of red corpuscles well 
No clot 
No clot 
preserved. 
> 
2 " . . . 
3 " 
0 
4 " ••• 
No clot; form of red corpuscles not 
N 
well preserved. 
u 44 u 44 
fl> 
5 " ••• 
Slight trace of clot 
Slight trace of granular clot 
g 
6 " ... 
Weak clot in 6 hours ; jelly slight.. . . 
Weak clot • 
More traces of granular clot 
(T 
7 " 
Firm clot in 2 to 3 hours ; jelly slight; 
Weak clot and jelly 
Granular and weak clot; weak jelly. 
clot best preserved. 
IO " ... 
Firm clot in 30 to 40 minutes 
Weak clot and strong jelly 
Weak clot and weak jelly 
CZQ 
P 
in 2 to 3 hours. 
n> 
15 " ••• 
44 4 ( 4 ( 
I 4 4 4 4 4 
44 44 44 
0 
20 " ... 
Strongest jelly and clot in 
H. 
25 " 
Clot present; jelly strongest 
Strong jelly and clot 
o' 
2 to 3 hours. 
§ 
Na2SO4. 
i % ... 
No clot; form of red corpuscles not 
No clot 
No clot 
well preserved. 
0 
2 " ... 
1 4 
3 " • • • 
T 
4 " ... 
0 
5 " ••• 
Slight traces of clot 
n> 
7 " ••• 
Firm clot and weak jelly 
Firm clot; weak jelly 
Granular clot; jelly 
cn 
IO " ... 
Fess clot and stronger jelly 
Firm clot; stronger jelly. .. 
Clot; stronger jelly 
15 " ••• 
Strong jelly 
Weaker clot; strong jelly .. 
Stronger clot; stronger jelly 
25 " ■■■ 
Strong jelly and weak clot 
Weak clot; strongest jelly.. 
Strongest clot; strongest jelly 
Ta BEE I. 44 
PROCEEDINGS OF THE 
the peculiarities were not shared by mammalian blood was clearly 
demonstrated. 
The conditions under which this clotting takes place are exactly 
those which, according to all authorities on the coagulation of mam- 
malian blood, favor the prevention of the process : 
1. The addition of water to a greater amount than twice the bulk 
of blood. 
2. The addition of neutral salts, 2 to 3 per cent, and upward; a 
large proportion entirely prevents the formation of a coagulum. 
3. Agitation (shaking of vials to mix blood and liquid). 
The proportion of the salts usually given as best are 25 to 28 per 
cent. MgSO4, concentrated Na2SO4, saturate solutions of both. No 
statement as to the temperature under which the saturation takes 
place is given, however, and the amount of these salts soluble in water 
varies with the temperature. These solutions are commonly used 
in the proportion of per cent, of blood, but owing to the small 
quantities dealt with here and the very rapid coagulation their exact 
use is impossible, as only by immediate mixing of the two liquids 
direct from the vessels of the animal can coagulation be prevented. 
But a moment's consideration shows that the methods used-dilu- 
tion of 50 parts of water to 1 of blood, greater proportion than 2 
to 3 per cent, of neutral salts, and thorough shaking of the vials 
containing the mixtures-combined the various retarding agencies 
and should have exerted a retarding action or have totally prevented 
the formation of the fibrin. The facts, however, show that even 
under these conditions the formation of fibrin takes place in from 
thirty minutes to four hours, according to the strength, in all solu- 
tions over 5 per cent., those mixtures under 5 per cent, remaining 
clear and liquid. Together with the appearance of the clot in 
higher solutions comes the jelly-like change previously noted. As 
to the exact stage when the strongest fibrin formation takes place, 
if the jelly is debarred from that name, a considerable variation 
exists, according to the salt and blood used. In Necturus and 
Cryptobranchus, with MgSO4, 7 per cent, was approached as a maxi- 
mum, on each side being a decrease in the clot; but in the strong 
solutions the jelly increased with the lessening of the coagulum, and 
also with the passage of time the true clot became to some extent 
slightly obscured, but never disappeared-a fact which tends to show 
that the jelly was not a secondary formation resulting from the de- 
struction of the true fibrin, but rather an independent one, brought AMERICAN MICROSCOPICAL SOCIETY. 
45 
about by conditions additional to those favoring the production of 
the ordinary fibrin. This may be called additional fibrin, and, ac- 
cording to Semmer's researches, which are now to be considered, 
does not differ in its essential nature from fibrin obtained by ordi- 
nary methods. 
A paper by George Semmer, published as an octavo, in Dorpat, 
in 1874, entitled liUeber die Faserstoffbildung im Amphibien und 
Vogelblut," gives the results of the author's investigations in a sim- 
ilar direction, including, however, as indicated by the title, bird as 
well as amphibian blood. Very careful investigations were made 
on the blood of the frog and domestic fowl under conditions, how- 
ever, that vary somewhat from those given in the foregoing pages. 
In the first place, defibrinated blood alone was used for all the ex- 
periments; and, secondly, as a diluent a solution of soda. The 
jelly-like or additional clot formed under these circumstances also, 
and further investigations were made to determine, if possible, the 
exact origin of the substances from which it came. All the various 
known and possible sources of fibrin were eliminated-i. e., by 
whipping the blood to defibrinate it in the ordinary way, then care- 
fully decanting it to remove the serum and white corpuscles; and, 
further, by solution, the haemoglobin of the red cells. With these 
parts eliminated the author induced the additional fibrin by means 
of the salt solution. In consequence of these results he concludes 
that the fibrin owes its formation to substances contained in the 
protoplasm of the red cells in the blood and set free by the solvent 
action of the salt which had been been added. In all essential re- 
spects he believes there is no difference between the fibrin thus arti- 
ficially formed and that naturally produced, and that probably, if 
not exactly alike, they are very closely allied forms. These fibrin- 
forming materials belong to the red corpuscles of birds and 
amphibia, excluding those of mammals, which, under no similar 
conditions, show the least tendency to act as fibrin-bearing 
bodies. 
If the experiments with normal are compared with those on de- 
fibrinated blood some very interesting facts become apparent. The 
small clot that so uniformly appears in the stronger solutions, be- 
ginning usually with 6 per cent., is the true fibrin; with the in- 
creased amount of the salt the solution of the red elements begins 
to take place, and consequently the formation of an additional fibrin, 46 
PROCEEDINGS OF THE 
which attains its maximum quantity in the highest per cent, solutions. 
Whether the true fibrin in these solutions is slowly dissolved by the 
neutral salts or the elements forming it are converted to the addi- 
tional fibrin remains an open question. But probably both actions 
take place to some extent, since the clot in the higher solutions 
never appears as large as in the lower ones and also suffers a distinct 
though small decrease on the prolonged action of the salts. Another 
point which must also for the present remain unexplained is the 
much more rapid formation of the true fibrin in the stronger solu- 
tions. This is against all precedent in mammalian blood and is 
difficult to account for. That the neutral salts here play a signifi- 
cant part in hastening the fibrin-formation is beyond doubt, but 
their precise action on the different constituents remains for future 
investigation. 
As a result of the above experiments the following can be stated : 
1. In amphibian blood (Necturus, Cryptobranchus, Rana) and 
ganoid blood (Amia calva) true fibrin forms in the presence of 
strong solutions of neutral salts, (5 to 25 per cent. MgSO4, 5 per 
cent, to a saturate solution of Na.2SO4). 
a. The formation takes place in the presence of a large quantity 
of water, (50 parts to 1 of blood). 
b. It takes place with agitation in from 30 minutes to 6 hours, 
(shaking vials frequently). 
2. In all solutions above 5 per cent, there is an additional forma, 
tion of a fibrin-like body, the firmness of which increases in propor- 
tion to the strength of the salt solution. 
From these facts it is seen that a wide gap separates the forms of 
life here represented from those which are usually examined for co- 
agulation experiments. An interesting question arises as to the 
action of the same salts on reptilian and birds' blood. That the 
results would be comparable with those obtained from amphibian 
blood is highly probable, a probability that would make a general- 
ization as to the greater distinction between nucleated and non- 
nucleated corpuscle-bearing blood, in regard to fibrin formation, 
perhaps possible. However, there exists an immense field in the 
various forms of fishes, Marsipobranchs, Elasmobranchs, Ganoids, 
Teleosts, and Dipnoans, where practically nothing is known con- 
cerning the action of the blood toward these salts. No more than 
a suggestion is as yet warranted. AMERICAN MICROSCOPICAL SOCIETY. 
47 
2. Measurement and Counting of Corpuscles. 
A. -Measurement. 
A great difference exists between animals with non-nucleated and 
most of those with nucleated corpuscles as to the distribution of the 
blood corpuscles in larger or smaller elements. A difference also 
exists between reptiles and amphibia, and from this point of view, 
at least, the latter occupy a much lower position. 
In general appearance the red corpuscles of Necturus and Crypto- 
branchus do not differ much, excepting in size, from those of 
other amphibia. They are oval, nucleated and plate-like, slightly 
biconvex and of a pale yellowish color. The stroma is finely 
granular and darker than the nucleus, which shows more or less 
distinctly a dark network (Pl. I). After a fresh preparation (pre- 
pared by simply covering a small drop of blood with a cover-slip 
and sealing with a neutral oil) has stood for half an hour or more in 
a moist chamber, an overlapping tile like arrangement, which repre- 
sents the rouleaux in mammalian blood, appears (Pl. I). In addi- 
tion, a rosette form, commonly observed in the corpuscles of the 
frog, is also present, arranged about a larger or smaller mass of 
leucocytes. 
Fresh preparations were used exclusively, and when simply sealed 
with castor oil the elements preserved their form and color excel- 
lently for several hours. All measurements were made by the Zeiss 
micrometer ocular, with a movable scale and Reichert's no. 7 ob- 
jective The value of the divisions of the micrometer with this 
objective was determined by comparison with a Ewell stage micro- 
meter ruled to .01 mm. 
As in other questions of measurement, the results given in the 
following pages can be taken as of only relative correctness. The 
difference of instruments, circumstances, and of observers unite to 
form a considerable area for variations, even when the greatest care 
is taken to be accurate. The average size of fifty corpuscles in 
different individuals of Necturus and Cryptobranchus was obtained 
as shown in the following specimen table. L equals the long 
diameter, B equals the short diameter. In the ocular-micrometer 
scale one space equals three microns, with the combination of 
objective and eye-piece used. 48 
PROCEEDINGS OF THE 
Tabde 2. 
Measurement of 25 Red Corpuscles. 
Cryptobranchus. 
Necturus. 
B. 
L. 
B. 
L. 
B. 
L. 
B. 
L. 
10.9 
16.0 
9.8 
150 
9.0 
24-5 
9-5 
21-3 
10.0 
16.0 
12.0 
18.0 
11.0 
20.0 
9-2 
20.9 
10.7 
16.3 
10.0 
IS-1 
9-5 
21.3 
10.5 
20.0 
9-i 
16.1 
10.5 
17.0 
10.0 
21.2 
12-5 
17.0 
10.0 
16.0 
II.O 
18.0 
9.0 
22.0 
"•5 
17.0 
8.0 
i5-5 
II.0 
17.0 
8.0 
21.0 
10.0 
18.0 
10.0 
17.0 
I 1.0 
16.8 
8.0 
23-8 
11.1 
17.0 
10.0 
16.0 
11.0 
18.1 
9.0 
21.0 
9.0 
20.1 
10.2 
16.1 
9.0 
15.0 
H-5 
21.0 
8.6 
21.0 
10.0 
14.1 
I 1.0 
>7-3 
8.0 
25-5 
9.1 
25.0 
9.6 
14 4 
12.0 
18.4 
9.0 
250 
10.0 
24.0 
9-0 
16.0 
10.0 
15-3 
10.3 
20.5 
II.I 
22.0 
11.4 
156 
9.0 
16.5 
10.0 
22.0 
9-2 
23.1 
I 1.2 
17.8 
10.0 
17-5 
11.4 
21.3 
10.1 
24.0 
12.0 
16.0 
10.0 
16.6 
9-o 
21.0 
II.2 
25.6 
II.4 
18.0 
10 5 
'9-3 
10.0 
20.0 
10.6 
20.5 
10.2 
173 
10.1 
17.0 
10.0 
21.4 
8.0 
23-0 
9.8 
17.0 
10.3 
16.0 
II.O 
20.0 
9.0 
24.2 
9.4 
15.8 
9-o 
18.8 
8.0 
21.0 
10.1 
20.6 
10.4 
19.0 
10.0 
16.3 
I 1.0 
20.5 
11.4 
22.0 
I 1.0 
15-5 
II.5 
18.0 
7-o 
25.6 
8.1 
20.0 
I 1.2 
16.0 
10.0 
19-3 
II.O 
22.0 
9-3 
22.0 
I 1.0 
i7-4 
11.7 
15.0 
'i-3 
22.5 
11.0 
20.6 
IO. I 
17.2 
9.0 
15-2 
8-5 
22.0 
9-4 
19-7 
10.0 
17.0 
10.0 
14.6 
9-5 
21.0 
10.7 
23.6 
256.6 
409.1 
260.5 
401.1 
240.0 
547-1 
250.2 
532.0 
Av. of B = 10.35 X 3 - 3105 P- 
" L = 16.20 x 3 = 48.60p. 
Av. of B = 9 80 X 3 = 29.40 /z. 
" 17 = 21.58 X 3 = 64.74//. 
From fifty corpuscles in five different individuals of Necturus 
maculatus come the following results, (L = the long diameter and 
B = the short) : 
1. L =50.9/2 B = 30.6 p. 
2. L=55.5/z b = 3i.8/z 
3. L= 58.8/1 B = 31.5/2 
4. L=64.7/2 B = 29.4/2 
5. L=62. 1/2 B = 32.4/Z AMERICAN MICROSCOPICAL SOCIETY. 
49 
An average of these different figures gives L= 58.4/z and B = 
31.1 /z. While a considerable amount of uncertainty exists as to the 
exact truth represented by these figures, they probably express ap- 
proximately the true conditions. The cells exhibit so much varia- 
tion that a large number of individuals is required to obtain more 
approximately true results. Only two individuals of Cryptobranchus 
were examined, 100 corpuscles in each being measured with the 
following result: 
1. L=48.6/£ 13=31.0/2 
2. B =27.5/2 
The chief variation here, it is seen, appears in the short diam- 
eter, the long one being nearly constant. According to Dr. Leon 
Vaillant, in Siren lacertina a range of variation occurs between 
L= 54-58/2, B= 24-27/2. This author quotes Gulliver as giving 
L = 6 2/2, B = 33 ,<2, correcting the latter's figures by his own. Sup- 
posing the conditions of measurement to be approximately alike, the 
results obtained by both authors could be correct if the variations 
found to exist in the case of Necturus were assumed to be present in 
Siren also, an assumption by no means improbable. Brass gives the 
size of the corpuscle of Proteus anguineus as L = 58 /z, B = 33.7 /z, 
and those of Megalobatrachus maximits, the great Japanese salaman- 
der ,as L = 47 /2, B = 33 /2. Tabulating these results, the following 
is obtained by averaging the different results : 
Proteus 6=58.0/2 B = 33.7 fi 
Necturus 6=58.4/2 6 = 31.1/2 
Siren L = 58.0 /z B = 28.0 /z 
Cryptobranchus L = 48.7/2 6=29.2/2 
Megalobatrachus . ... 6=47.0/2 6 = 33.0/2 
If from these figures a generalization be made, it is seen that in 
the external gill-breathers the red cells average 10/2 longer than in 
the forms in which external gills are absent. In width, however, 
results are contradictory, and, moreover, Riddel gives the corpuscles 
of Amphiuma tridactylum as the largest known-one-third larger 
than in Proteus. This statement has been corroborated by Lamb, 
and corpuscles preserved in glycerine jelly have been examined and 
measured by the writer. Comparing them with the corpuscles of 
Necturus similarly prepared, the great difference in size could be 
seen, the relative shrinkage being probably the same in each case, 
7 m 50 
PROCEEDINGS OF THE 
If compared with the size of the red cells in the frog and largest 
mammals, the truly gigantic size of these corpuscles can more easily 
be realized. For the frog Ecker gives L = 24/2, B = 17/2; for the 
triton and salamander, L = 25 p, B = 16 p.; Brass, for the sturgeon, 
L = 13/1, B = 10 p ; Weicker, for the elephant, D = 9 p (the larg- 
est mammalian red blood corpuscles). In camels, L = 8 p, B = 4 p. 
Table 3. 
Size in Microns of the Red Corpuscles in Different Animals. 
Oval corpuscles. 
Circular corpuscles. 
Animal. 
L. 
B. 
Authority. 
Animal. 
D. 
Authority. 
Amphiuma ..... 
75 
Elenhant. 
9 
Brass. 
Proteus 
58 
35 
Brass. 
Goat.. .. 
4 
Brass. 
Siren | 
56 
27 
Vaillaut. 
Sheep . . 
5 
Brass. 
62 
33 
Gulliver. 
Beasts 1 
Necturus 
58.4 
48.7 
3i-i 
29.2 
E. J. Claypole. 
E. J. Claypole. 
of 
4-7 
to 
Cryptobranchus.. 
prey. 
I Brass. 
Megalobatrachus. 
47 
33 
Brass. 
Apes...' 
8.7 
Rana 
24 
17 
17 
Ecker. 
Man. . . 
R. esculenta 
22 
Stricker. 
Musk- 
R. teniporaria.... 
25 
16 
Brass. 
deer 
2 5 
Brass. 
R. viridis 
24.4 
25 
16.3 
16 
Hayem. 
Triton 
Brass. 
Lepidosiren 
41 
29 
Weicker. 
Carp 
15 
9 
Brass. 
Sturgeon 
13 
10 
Weicker. 
Lizard 
T5-9 
12 
9-9 
7 
Brass. 
Domestic fowl. . . 
Brass. 
Duck 1 
Pigeon J 
15 
6 
Brass. 
Camel 1 
Llama j 
8 
4 
Brass. 
To get comparative measurements of the leucocytes it is necessary 
for them to be spherical. By mixing the blood with a drop of i 
per cent, osmic acid or 4 per cent. MgSO4 the cells assume the de- 
sired form, care being taken only to measure those presenting true 
circular outlines. The number of cells measured was not equal in 
the two animals; in Cryptobranchus the average obtained from 100 
cells is D = 18.5 ; from only 20 of Necturus, D = 28.5. It can be 
seen that even by considering the latter but a rough approximation AMERICAN MICROSCOPICAL SOCIETY. 
51 
to the truth an appreciable difference exists between the two, com- 
parable with that already shown to exist between the red corpuscles 
in these animals. In the drawings also, made from dry preparations, 
it is apparent that such a difference exists, though no definite idea 
of the relative condition of expansion present can be obtained 
(Pl. II). In the study of living cells in amoeboid phases the differ- 
ence in size may also be noted. 
B.-Counting of Corpuscles. 
Before giving the results obtained from this part of the investiga- 
tion a few words as to the methods here used will be appropriate. 
For a diluent a 2 per cent, solution of MgSO4 proved to be the 
most successful, if used within two to six hours ; a stronger solution, 
although preserving the color better, produces such an amount of 
distortion and massing of the cells as to make accurate counting 
impossible. For preservation of form, 1 per cent., 2 per cent, and 
3 per cent, solutions of this salt were good, 2 per cent, being the 
best, as usually no distortion appeared in it, which occurred to a 
slight extent in 3 per cent., and also there was no great loss of color. 
(For further details see the discussion on Coagulation, page 42.) 
By means of a carefully graduated tube a definite amount of the 
liquid, 1,225 c- mm- was Put a small homoepathic vial. From this 
another smaller pipette, graduated to show 25 and 50 c. mm., was 
filled up to the 50 mark from the previously measured liquid, and 
then carefully emptied to 25 ; (this is to insure the subsequent filling 
up to 50 and the immediate mixing of the blood and salt solution.) 
The pipette ready, the heart of the specimen is exposed, a large ves- 
sel cut, the pipette inserted, and 25 c. mm. drawn in. Immediately 
this amount is mixed thoroughly with the MgSO4 in the small vial. 
By this process a mixture of 1 to 50 is obtained. After thorough 
mixing a drop is placed in the counting cell (25 mm. deep, ruled to 
1 mm. squares), covered, and allowed to settle on a plane surface. 
Then by taking an average from the different numbers contained 
in 100 of the squares and calculating the cubic contents of the 
cell, the number of corpuscles in a cubic millimeter of the blood 
and salt solution is found. As a dilution of 50 times exists, multi- 
plying the results obtained by the above process by 50, the actual 
number present in a cubic millimeter of pure blood can be found. 52 
PROCEEDINGS OF THE 
Table 4. 
Counting of Corpuscles. 
Necturus. 
Cryptobranchus. 
i 
4 
I 
1 
3 
2 
O 
3 
3 
2 
2 
3 
3 
4 
3 
3 
2 
4 
4 
6 
2 
4 
i 
6 
3 
2 
1 
3 
o 
3 
2 
4 
3 
o 
3 
4 
2 
2 
1 
3 
5 
2 
5 
2 
4 
6 
2 
4 
5 
4 
2 
4 
1 
2 
5 
i 
1 
3 
2 
2 
3 
3 
1 
3 
1 
6 
2 
3 
2 
3 
3 
3 
0 
4 
i 
3 
3 
2 
4 
3 
3 
2 
4 
3 
3 
3 
4 
5 
2 
2 
5 
5 
3 
3 
4 
5 
5 
3 
4 
5 
4 
10 
5 
7 
6 
8 
5 
6 
11 
7 
8 
7 
7 
8 
6 
6 
3 
6 
8 
6 
9 
8 
7 
6 
7 
9 
9 
6 
7 
8 
4 
9 
8 
10 
10 
6 
5 
7 
8 
6 
6 
6 
4 
4 
6 
9 
7 
6 
7 
6 
6 
10 
6 
10 
10 
8 
5 
5 
7 
7 
10 
8 
8 
6 
4 
7 
8 
8 
7 
7 
7 
9 
7 
4 
7 
7 
6 
6 
12 
9 
5 
7 
8 
7 
9 
8 
4 
9 
6 
6 
7 
6 
7 
9 
7 
7 
10 
t 
9 
7° 
72 
6i 
88 
171 
173 
181 
181 
291 in 100 squares. 
291 h- 100 = 2.91 in 1 square. 
Cubic contents of the cell - . 1 X • 1 
X .25 = .0025 cubic mm. 
2.91 = the average no. in 1 square. 
2.91 4- .0025 = 1,164, no. of cor- 
puscles in 1 cubic mm. of the 
diluted blood. 
1,164 X 5° (amount of dilution) = 
58,200, no. of corpuscles in 1 
cubic mm. of undiluted blood. 
706 in 100 squares. 
706 4- 100 - 7.06 in 1 square. 
Cubic contents of the cell =. 1 X • 1 
X .25 = .0025 cubic mm. 
7.06 - the average no. in 1 square- 
7.06 4- .0025 = 2,824, no. of cor- 
puscles in 1 cubic mm. of the 
diluted blood. 
X 50 (amount of dilution) = 
141,200, no. of corpuscles in 1 
cubic mm. of undiluted blood. 
The results obtained in Necturus from freshly caught, healthy 
animals are given below, the following precautions being observed: 
to make the dilution of the blood iminediately on opening the AMERICAN MICROSCOPICAL SOCIETY. 
53 
cardiac vessels, as the numbers resulting from dilutions made from 
the same individual before and after excessive bleeding are strik- 
ingly different, in all counts, to fill the cell twice to insure using 
a good sample of the mixture : 
1 58,200 
2 58,800 
3 56,800 
4 60,000 
5 53,600 
6 52,400 
339,800 ~ 6 = 56,633, average. 
In these results a large variation occurs, which arises from two 
causes at least: first, the greater or less uncertainty in methods; 
second, the condition of the animal at the time of examination. 
The first cause is unavoidable. As can be seen at a glance, a very 
small error in the first figures of the calculation will bring a large 
difference in the final result, and when the chances of misjudging the 
exact amounts measured are considered, there is seen to be reason 
enough for a variation of this amount; but when the second point is 
examined yet greater chances for variation are apparent. Even assum- 
uming the animals to be in the same condition, the following figures, 
obtained from the same individual in different stages of bleeding, are 
significant: The first count gave 53,600, the second 45,800, and the 
third 33,800 per c. mm.; a steady decrease is present, while in one 
specimen that had been bled for laboratory use and kept for a con- 
siderable time the small number of 21,000 was present. From these 
results it is apparent that a large amount of uncertainty exists in 
dealing with animals possessing so small an amount of blood and 
those in which coagulation occurs so rapidly. It is absolutely 
necessary to have some of the diluent in the pipette in which the 
blood is measured or else by the formation of a coagulum many cor- 
puscles become entangled, and consequently the results are vitiated. 
Moreover, after bleeding has occurred to some extent enough cor- 
puscles have been removed to leave the serum largely predominant, 
and small numbers again result from this cause. To obtain entirely 
satisfactory results a large number of experiments is needed on ani- 
mals in the same physical condition, and that as nearly normal as 
possible. 54 
PROCEEDINGS OF THE 
When these figures are compared with those given of other forms 
a great difference is seen to exist. In mammals the number varies 
between 3,000,000 and 18,000,000 per cubic millimeter, depending 
on the kind of animal used. In birds the average lies between 
£,000,000 and 4,000,000, while in fishes the number is still lower. 
From the researches of Professor Gage, of Cornell University, the 
numbers present in two forms of lampreys, Petromyzon marinus and 
Petromyzon branchialis, are, for the first, 362,889 as an average 
number, a variation existing between the two sexes, and 500,000 
for the latter form. From personal observation in the frog, Rana 
catesbiana, there.were found to be 229,000 per c. mm. Assuming 
that a maximum amount of haemoglobin or oxidizable material is 
essential, then the inference that the larger the corpuscles the smaller 
the number present is made safely, since there is a physical impossi- 
bility forbidding the occurrence of both large cells and large num- 
bers in the same animal, if the same amount of blood-forming ma- 
terial exists in both those possessing large and those having small 
corpuscles. On this principle, as would be inferred, a larger num- 
ber (138,600 per cubic mm.) is present in Cryptobranchus than in 
Necturus (see table on page 10); but as this is obtained from only 
one count, and so small a range of observation forbids the accept- 
ance of these figures otherwise than provisionally, they may, how- 
ever, be received as indicating the tendency which the above gen- 
eralization embodies. 
3. Ingestion of Carbon by Leucocytes and their Subsequent Distribution in 
the Tissues. 
The power of leucocytes by virtue of their amoeboid movement 
to take up foreign substances, giving to them as it were the office 
of scavengers of the animal body among their other duties, possesses 
great significance in the eyes of the physiologist, especially from 
pathological outlooks. The phagocyte theory of Metschnikoff, 
which to-day has so many adherents, is based on this power and a 
large amount of pathological investigation has been undertaken to 
prove, if possible, the true part played by these cells in diseases 
which owe their existence to the presence of foreign organisms and 
foreign matter in the body. 
In Necturus and Cryptobranchus the process of ingestion, both 
on account of the size of the'cells and their activity in the ordinary AMERICAN MICROSCOPICAL SOCIETY. 
55 
temperature of the room, can be most favorably observed. Mixing 
a little carbon with the blood, the leucocytes may be seen to take 
up the foreign matter while passing through their amoebdid phases. 
In Necturus especially these phases are most beautifully exhibited, 
and by quickly drying a preparation when the cells are well spread 
they can be preserved in their extended condition (Pl. II). The 
carbon is exclusively contained in the cell-body, although the nuclei 
also exhibit active amoeboid phases. From stained preparations 
these can be distinctly seen (Pl. II). Often two or sometimes 
three nuclei are present in one cell (Pl. II). In the one figured, 
two of the three nuclei are in amoeboid motion, while the one is in 
a resting state. From the active movement and continued exten- 
sion of the protoplasm into delicate pseudopods the carbon becomes 
massed in the central part of the cell in many cases, giving the ap- 
pearance of its being in the nucleus, but that such is not the case 
is shown by the gradual movement of the granules across the surface 
of the nucleus, and, if watched for a sufficient period, it will become 
evident that they are contained in the cell-body and not in the 
nucleus. Sometimes these cells containing two or even more nuclei 
divide while under observation ; such a one is figured in plate III. 
The division of the single nucleus had, of course, taken place pre- 
viously, but the gradual breaking away of the two parts of the pro- 
toplasmic body was observed and the times between the successive 
stages noted. Often in a double-nucleated cell division appears 
imminent, the two parts being united only by a thin thread of pro- 
toplasm; but this may gradually thicken and the cell remain undi- 
vided. Such observations give a vivid idea of the activity of life 
in these simple cells and make it better understood how they can 
play the part they are shown to take in the following experiments 
on ingestion of foreign substances. 
To ascertain the ultimate fate of foreign substances when they are 
introduced into the animal body and the means by which the ma- 
terial is disposed of has been the aim of an immense amount of 
investigation. Experiments have been made and existing patho- 
logical conditions studied, resulting in a great mass of literature 
and, as usually occurs, many conflicting statements. From the dif- 
ferent ways in which, pathologically, under natural conditions for- 
eign material comes into the system-that is, by inhalation and a 
consequent abnormal state of the lungs, by external contact as 
through wounds, and by the setting free from the blood itself mate- 56 
PROCEEDINGS OF THE 
rial which circulates freely-a condition resulting in melanosis-this 
question has been attacked from the following standpoints: 
1. By artificially introducing into the circulatory system, directly, 
different materials. 
2. By introducing foreign materials through subcutaneous injec- 
tion, indirectly, into the circulation. 
3. By compelling the inhalation under artificial circumstances of 
various dust-like materials and by forcing into the lungs substances, 
organic as well as inorganic. 
Especially in these researches is there a conflict of opinion as to 
the part played by the leucocytes or wandering cells, both as to their 
ultimate fate and their attitude toward the foreign bodies. Do they 
simply form a means of transport to the various tissues and there in 
some way discharge their load and pass again into the circulation, 
or do they suffer the same fate as the foreign substance, which is 
either deposition in the tissues or ultimate removal from the 
organism ? Is their presence essential or non-essential ? These are 
questions receiving various answers. Many questions also arise as 
to the relations of the cellular constituents of the various organs to 
the foreign matter, especially in those concerning whose individual 
structure controversy exists. 
To give here an adequate idea of the already published results is 
impracticable; merely a few of those whose works have been con- 
sulted will receive especial mention. In investigations on the effect 
of introduction through the lungs, the nature of the foreign material 
differs according to the different investigators. Some introduce 
into living or dead animals soluble substances; others use finely 
granular material suspended in a liquid, (Slavjansky, Schestopal). 
Another series of investigators adopted organic forms, (Sommerbrodt, 
Nothnagel), and, finally, the last class adopted the inhalation of 
dust-like materials suspended in the air, (Ruppert, Schottelius, 
Arnold). The latest researches have been made by Dr. Julius 
Arnold, whose results were published in 1885. A long bibliography 
of 264 writings on this subject is included in his publication, which 
makes it particularly valuable. 
In the method of vascular injection for the sake of easy recognition 
in the various organs, cinnabar suspended in normal salt solution 
received most general use. Ponfick, Hoffmann and Langerhans 
made investigations in this direction, the last two exclusively, the 
first chiefly, on mammals. The rabbit, guinea-pig, cat, dog, rat and AMERICAN MICROSCOPICAL SOCIETY. 
57 
other animals have been used by these investigators, to which list 
must be added the indispensable frog. 
The results of the different methods vary. In many respects they 
are contradictory, so that a general statement of the condition in 
which the work stands is impossible. Even the question as to the 
presence of a deposit of free coloring material remains in contro- 
versy. Ponfick emphatically states that no free deposit took place 
in his experiments, the foreign material being exclusively contained 
in cells of various kinds. On the other hand, Hoffmann and Lan- 
gerhans, in examination of the respiratory organs, which were un- 
touched by Ponfick, find the foreign coloring material in a free state. 
The distribution of the introduced substances also varies. Arnold 
does not think that transfer from the lungs to the general circulation 
takes place regularly, and from his extensive work the word of this 
author is of great weight. Slayjansky, on the other hand, considers 
this transfer a regular occurrence. 
One practice, however, is common to all the investigators whose 
works have been consulted ; that is the introduction into the system 
of a very large amount of foreign material-1.5 to 3 c. cm. into a 
frog and from 8 to 10 c. cm. into guinea-pigs and rabbits, in one 
case of a dog 210 c. cm. being introduced by successive injections. 
Under such circumstances, that free granules pass into the tissues is 
by no means surprising, nor, moreover, considering the weight of 
cinnabar, is it to be expected that the normal action of the various 
organs remains unimpaired. A pathological condition must result 
to a greater or less extent and the true action of the leucocytes 
toward the injected material be obscured. To ascertain with exact- 
ness the true normal relations existing between these elements and 
the foreign substances, no more than can be disposed of ought to 
be injected. If no free material enters the blood and afterwards 
free material or material in cells other than leucocytes is proved to 
exist, there is then a definite point on which to base a discussion. 
So far as possible these conditions have been preserved and the re- 
sults obtained are given in the following pages. 
The low forms represented by Necturus and Cryptobranchus have 
not (the frog excepted) been studied in regard to this point, in con- 
sequence, probably, of the strongly pathological aspect which the 
question presented to the investigators. From that standpoint the 
highest mammals available for experimentation showed conditions 
most similar to those encountered in human pathology; but on 
8 m 58 
PROCEEDINGS OF THE 
account of the relatively simple structure and large size of the 
constituent parts in these amphibian forms, they offer very favorable 
conditions for an investigation of this nature. Especially is this true 
in regard to Necturus. The presence of ectobranchise affords an 
excellent opportunity for examination of the circulation without 
any injury to the animal, and by careful watching the times of the 
appearance of the coloring substances in and the disappearance of 
the same from the blood can be determined. The following modi- 
fications were made in the methods usually adopted : 
1. The coloring material chosen was a mixture of gum arabic, 
lamp-black and normal salt solution (i gr. carbon, i gr. gum arabic, 
15 to 20 cc. H2O + .6 gr. NaCl). 
2. The injection was made in two places: 
a. Hypodermically into the abdominal cavity. 
b. Into the jugular vein. 
3. At no time was more than 1 cc. of the mixture injected; 
usually % to y2 cc. 
In cases of abdominal injection the time was necessarily prolonged 
beyond that required by venous injection, since some days must 
elapse before the foreign substance enters the blood through means 
of the lymph circulation. One fact, however, is ascertained : that 
the carbon particles must enter the blood this way in cells, as even 
four days after injection, when the injected material was not in the 
blood, no free carbon appeared in the abdominal cavity, all having 
been ingested by leucocytes. 
In a few words will be given the results of the experiments regard- 
ing the appearance of cells containing carbon in the circulation and 
the disappearance of free carbon from the blood. A variation to a 
considerable degree existed on the first point. The earliest time 
was six days after abdominal injection when ingested cells appeared 
in the circulation, and the longest nine days. After sixteen days a 
few cells still remained. In venous injection no free carbon was 
found after the second day, but ingested cells remained in a small 
number up to the eighteenth day, when the animal was killed. The 
tissues, however, in cases of venous injection were not so thoroughly 
investigated, so the results given apply chiefly to abdominal injec- 
tion. 
In the microscopic investigation of the different organs the first 
difficulty lay in the presence in nearly all the tissues of a large 
amount of natural pigment, which was confusing both on account AMERICAN MICROSCOPICAL SOCIETY. 
59 
of the similarity in color and the necessary obscuring by such large 
masses of the structural parts. Caustic potash destroys melanin, 
but boiling is necessary, a process which also destroys the tissues. 
From consultation with various authorities, ether, alcohol, acids, 
and strong alkalies were found to decolorize melanin, but the last 
two have a destructive action on tissues and the first two produce 
decolorization so slowly that they are practically useless. By means 
of hydrogen dioxide (H2O2) most successful results were obtained. 
Placing the sections, when cut and fastened to the slides, in a small 
vial of the liquid (io vol. or 2 per cent, sol.'), the melanin could be 
reduced to a pale yellow color in from 6 to 48 hours, depending on 
the amount of pigment present and the light in which the glass 
stands; strong sunlight materially hastens the process. If the treat- 
ment is continued longer the coloring material can be entirely 
removed, and by watching the process a little carefully the decolor- 
ization can be stopped at any point before absolute removal. Prac- 
tically it was found to be of great advantage to leave sufficient color 
to mark the position of the pigment cells. This liquid, strong as 
it is in action, does no appreciable injury to the structural elements ; 
when the tissues have passed through the processes necessary to 
prepare them for examination the details are as distinct as under 
ordinary circumstances and no chance remains to mistake the in- 
gested cells when they occur. That hydrogen dioxide likewise 
decolorizes the pigment of mammals, at least that in the choroid 
coat of the eye, was proved by experimentation on the eye of the 
sheep. A piece of the tissue was put in bulk into a vial of hydrogen 
dioxide and in a few days it presented a yellowish hue, the deep 
black color being entirely destroyed. 
This difficulty removed, the investigation to determine the loca- 
tion of the carbon in the different organs could be begun. The 
following parts were investigated: the spleen, kidney, ureter, liver, 
lungs, stomach, muscular and epidermal tissues. It was found to 
be necessary, in order to determine exactly the location of the car- 
bon, to make serial sections of the different organs; by this method 
the structure of the organs themselves also was much more easily 
studied and in many cases points otherwise obscure were made clear. 
In many cases what did not appear distinctly in one section became 
clear after a series had been examined, and facts were learned that 
without this method could not have been ascertained. In all cases 
investigated, if the carbon had made its way into the circulation, 60 
PROCEEDINGS OF THE 
the ingested cells were scattered through the various tissues. The 
largest amount appeared in the spleen, the next largest in the kid- 
neys, next in the liver, and in the remaining organs in about equal 
amounts. Considering each organ in turn on this basis of distinc- 
tion, the spleen receives first notice. 
In Necturus and Cryptobranchus, as in higher forms, this organ 
consists essentially of a strong, fibrous capsule, from which fibrous 
trabeculae pass in all directions, forming a supporting framework 
for the cellular structures. In this perplexing organ, owing to the 
obscurity of structural details, great difficulty exists in determining 
the exact relation between the carbon and the constituent elements. 
The cells containing carbon could be separated into two kinds: (i) 
leucocytes; (2) spleen-pulp cells. Those occurring in blood ves- 
sels were undoubtedly leucocytes, and from their peculiarity the 
spleen-pulp cells could be definitely distinguished. They were close 
to the fibrous tissue composing the malpighian bodies and often 
upon it; they were large, with very long protoplasmic processes, in 
which the carbon granules were contained (Pl. IV). The method 
of distribution of the carbon granules in the leucocytes was very 
different from that present in the splenic cells ; while in the latter 
the individual granules are scattered fairly evenly through the pro- 
toplasm, in the former they are massed into heaps and make the 
whole cell appear black. The peculiar, even distribution of the 
granules strongly suggested a free deposit in the fibrous tissue, the 
extreme transparency of the protoplasm in the splenic cells adding 
to the appearance, and only after long and careful observation was 
it ascertained that such was not the case. On the establishment of 
this fact the old vexed question arises as to how the carbon particles 
get into the true splenic cells. It has already been shown that all 
the carbon entering the circulation is inclosed in cells. By what 
means is a transfer made from these cells of the ingested material to 
the splenic-pulp cells? For the present this question must remain 
unanswered ; later a suggestion will be offered. 
1. Spleen. (PI. IK.) 
2. Kidney. (PI. K.) 
As in all Urodeles the kidney in Necturus and Cryptobranchus is 
an elongated club-shaped organ, with one renal vein between the AMERICAN MICROSCOPICAL SOCIETY. 
61 
two parts, receiving branches from both, and being continued into 
the post-cava. The malpighian bodies lie toward the .ventral sur- 
face, and usually are not completely filled by the glomeruli, leaving 
a lymph space between the two parts. The neck of the urinary 
tubule leading from the malpighian body is lined with small epithe- 
lial cells, bearing cilia (Pl. V) that exceed in length the diameter 
of the tube. In addition to these parts, which exist in the kidneys 
in higher forms, there are the nephrostomes opening into the ab- 
dominal cavity and communicating by tubes with the first part of 
the urinary tubule after it leaves the glomerulus. One funnel may 
communicate with two tubes or vice versa. The funnel-like aper- 
tures are clothed with epithelium that has long cilia, whose vibra- 
tions produce currents that carry the abdominal liquids and any 
substances contained in them down the funnels to the tubes and 
thence to the urinary tubules with the organ's peculiar secretion, 
and thence to the ureters (Pl. V).' Ingested cells were present in 
the blood vessels of the glomeruli, in the lymph space between the 
glomeruli and the malpighian corpuscle, in the tubules, in the 
nephrostomes before their union with the tubules, and in the ureters 
just before their opening into the cloaca (Pl. V). 
Three ways lie open for the passage of these cells, both ingested 
and free, into the urinary tubules: (i) through the nephrostomes 
direct from the abdominal cavity; (2) from the lymph space be- 
tween the malpighian capsules and the glomeruli; (3) from the fine 
capillaries of the glomeruli with the glandular secretion. Doubtless 
the majority find their way in through the first two ways, but their 
passage from the glomeruli also increases the number that leave the 
system through the kidneys. It is owing to the peculiar structure 
of the kidneys in Urodels that these organs rank so high in the pos- 
session of ingested cells. With the exception of the spleen, no 
other organs contained so many. In the ureters the cells were 
massed together and were invariably present. Neither ingested 
cells nor free carbon existed anywhere in the kidneys outside the 
blood vessels and tubules. 
3. The Liver. (PI. //.) 
The liver is composed of remarkably large cells, attaining some- 
times the length of 50 v, even after the tissue is hardened. Often 
the tubular structure is not evident, but in some surface sections the 62 
PROCEEDINGS OF THE 
details appear fairly distinct. Ingested leucocytes in this organ are 
confined to the blood vessels; no indication of extra-vascular 
presence could be detected (Pl. IV). 
Although in the remaining organs and tissues the ingested cells 
were not so numerous, their position affords valuable suggestions as 
to their ultimate fate. 
4. The Stomach. (PL VI.) 
In the stomach carbon-laden cells were present in the blood 
vessels, in the muscular tissue, in the submucosa, mucosa, and 
epithelium, and free on the mucous surface. No free carbon was 
present here (Pl. VI). 
5. Lungs. (PL IL.) 
Practically the same results were obtained from examination of 
the lungs-ingested cells appear in the blood vessels, in the inter- 
vascular tissue, among the epithelial cells, and free on the mucous 
surface of the lung cavity. The simple structure of the lung (it is 
a somewhat folded bag) makes the direct course of the cells through 
the tissues to the mucous surface easy to trace (Pl. V). 
6. Muscular Tissues and Skin. (Pi. VI.) 
In sections made of the abdominal walls and other parts of the 
muscular system ingested cells were present in the blood vessels and 
capillaries, between the muscular fibers; in the latter case they may 
either be in the endomysium or lymphatic system. From the mus- 
cular tissues they pass to the epidermal cells through the malpighian 
layer, where there are large mucous glands, and finally are free on 
the surface of the skin (Pl. VI). That those present in the last- 
named place did not come accidentally after death is proved by the 
presence of isolated cells in small folds of the skin, where one cell 
occupies all the space between the apposed surfaces. With the 
rapid coagulation of the blood that exists it would be impossible for 
one leucocyte to be alone. Red corpuscles must be present in greater 
or less numbers, and the combined mass would be incapable of 
penetrating to such fine crevices. 
In addition to the above locations with an abdominal injection, 
large masses of cells accumulated on all mesenteries, the outer sur- AMERICAN MICROSCOPICAL SOCIETY. 
63 
face of the stomach, the intestines, large and small, the lungs, and 
to some extent on all peritoneal surfaces; also at the point where 
the needle of the hypodermic syringe entered there was an accumula- 
tion of carbon-laden cells, giving evidence of an inflammatory 
process to some degree. 
The results of the investigation can be briefly summed up as fol- 
lows : 
1. No free carbon was present in any organ examined. 
2. All carbon was contained in leucocytes except in the spleen, 
where true splenic pulp cells also contained it. 
3. Ingested cells were both extravascular and intravascular except 
in the kidneys and liver. 
4. Ingested cells were free on mucous and epidermic surfaces- 
stomach, lungs, skin. 
5. Ingested cells were present in the excretory organs with waste 
products-kidneys. 
The one point in which the above results differ from those ob- 
tained by all the authors consulted is the absolute limitation, with 
the one exceptioh of the spleen cells, of the injected material to the 
leucocytes. This entire absence of free carbon in the tissues differs 
especially from the results of Hoffman and Langerhans, Arnold, and 
all those who, as the last-mentioned author, introduced coloring 
matter into the lungs. Ponfick, however, found no free cinnabar 
in his work. These authors show that the fixed connective tissue 
cells of most organs, the interstitial connective cells of all tissues, 
the epithelial cells of the lungs, some forms of pigment cells and 
bone-marrow cells, all can take up and retain the foreign material 
if the conditions of introduction remain practically the same. 
When, however, conditions are so radically changed in regard to 
the amount of material injected into the organism it is apparent that 
the results maybe greatly modified. In consequence it is believed, 
as the result of this investigation, that when no more material is 
present than can be disposed of by the leucocytes they exclusively 
ingest it and gradually dispose of it by wandering out on the mucous 
and cutaneous surfaces and by passing away with waste products. 
The presence of the carbon in the spleen is a secondary result, owing 
to the destruction probably of ingested cells in this organ, and either 
the subsequent taking up of the particles thus set free or else the 
ingestion of the corpuscles in toto by the splenic pulp-cells. It is 
believed, however, on the other hand, that when an overwhelming 64 
PROCEEDINGS OF THE 
amount of foreign material is introduced, these other forms of cells 
are pressed into service to help remove it from the circulation. 
Even free deposits of injected material may take place, owing to the 
inability of the cellular elements to cope with the large amount 
present. In this case the ultimate disposition of the injected mate- 
rial is not known, as usually conditions so profoundly pathological 
are produced that the experiments are prematurely ended by the 
death of the animals. 
Considering the leucocytes as scavengers of the animal body in 
addition to their other functions, if their individual fate is insep- 
arable from that of the waste material which they contain, they 
themselves become waste material when their active functions cease 
and must ultimately disappear from the organism. Already the two 
ways in which the leucocytes may ultimately pass away from active 
life have been suggested. In the first place, they wander from the 
circulation through the tissues to the epidermal and mucous surfaces 
and are carried away with the other waste products of the body. In 
the second place, they are destroyed in the spleen by the splenic 
cells. The large numbers found in various stages in the first con- 
dition and the relatively large numbers of ingested spleen-cells prove 
that the destruction of leucocytes is by no means insignificant. 
It is at present impossible to tell what becomes of the carbon 
contained in the splenic cells. In all problems connected with the 
blood and circulation this organ seems to play an important part. 
Only by prolonged experimentation could it be ascertained whether 
the carbon gradually disappears or remains permanently in these 
cells; but that is beyond the scope of the present series of experi- 
ments. The problems as to the time during which the ingested cells 
remain in the circulation and in the tissues are also not solved. 
These require more time. They are for the present left out of con- 
sideration. 
Summary of Whole Paper. 
From the action of different per cent, solutions of the neutral salts 
MgSO4 and Na2SO4 in relation to coagulation, an essentially different 
condition is found to exist in amphibian and fish blood from that 
known to be present in mammalian blood. True fibrin forms in 
the presence of neutral salts in solutions above 5 per cent. The 
rapidity of formation increases with the strength of the solutions, 
beginning in 6 per cent, in six hours and appearing in 15, 20, and AMERICAN MICROSCOPICAL SOCIETY. 
65 
25 per cent, in thirty to forty minutes. The quantity also varies; 
in amphibia it increases toward 7 per cent, solution as a maximum ; 
afterward a decrease in quantity is apparent. In the blood of Amia 
calva a slightly different condition is present (see pages 6 and 7). 
Simultaneously with the formation of this true fibrin an additional 
formation takes place. This is of a jelly-like nature and has been 
called the additional fibrin. This fibrin increases uniformly with 
the increased strength of the salt solutions. George Semmer has 
investigated a similar formation in the blood of the frog and do- 
mestic fowl under slightly different conditions. He considers that 
the additional fibrin is produced from the protoplasm of the red 
cells of the blood by the solvent action of the neutral salts. This 
additional fibrin is not formed in the blood of mammals, but occurs 
in that of birds, amphibia (Cryptobranchus, Necturus, Rana), and 
of a ganoid (Amia). This distinction perhaps makes it possible to 
draw a stronger line between the nucleated and non-nucleated cor- 
puscle-bearing animals. The experiments should be extended to 
the large field of work offered among the Teleosts and other forms 
of fishes, and also to reptiles, in order to make a sure generalization 
on this point. 
From the measurement and counting of the corpuscles in Necturus 
and Cryptobranchus the following facts were ascertained: The cells 
of Necturus are much larger than those of Cryptobranchus. Com- 
paring this result with the measurements given by different authors of 
the corpuscles in other forms of amphibia, a distinction can be made 
on the basis of size of cells between those animals possessing ex- 
ternal gills and those without; to which rule, however, a striking 
exception is found in Amphiuma. In enumeration, also, a differ- 
ence is shown to exist between the large and small corpuscle-bearing 
animals, the higher numbers of corpuscles per cubic millimeter 
belonging to the latter class of animals. 
From experiments made on Necturus and Cryptobranchus in the 
injection of small quantities of carbon into the abdominal cavity, 
hypodermically, the following results were obtained : In the first 
place, it was established that under the given conditions the 
leucocytes ingested all the foreign material and entered the blood 
circulation after varying periods. In the second place, it was found 
that from the blood the ingested cells are distributed to the tissues 
and ultimately pass away on mucous and epidermic surfaces with 
waste products of the body. Thirdly, that no deposit of free ma- 
9m 66 
PROCEEDINGS OF THE 
terial takes place, the foreign substance being contained exclusively 
in leucocytes, with the one exception of the splenic cells, where 
carbon is present. This latter position is a secondary result. From 
the above it is concluded that probably the fate of many leucocytes 
is to wander out on mucous and epidermic surfaces and to pass 
away with waste products. Many must also meet their end in 
splenic cells, as is shown by the considerable amount of carbon 
present there. The fate of this carbon is left undetermined from 
want of time to pursue the investigation further. 
Methods. 
Wherever it has been practicable, the methods of preparing the 
various objects to be studied have been inserted in the text. Those, 
however, have been omitted which concern the preparation of the 
tissue in toto, and also the details of the preparation of the sections 
when cut. The picric-alcohol method of hardening tissues was em- 
ployed throughout as used by Professor Gage, of Cornell University: 
95 per cent, alcohol 250 cc. 
Water 250 cc. 
Picric-acid crystals 1 gr. 
The whole animal, with the abdominal cavity opened, was putin 
a jar of the above mixture from 2 to 3 days, the liquid being changed 
at least once. From this it was transferred to 67 per cent, alcohol 
from 24 to 36 hours, and then into 82 per cent, alcohol. Here it 
can remain indefinitely. 
The following method of imbedding was used : Small pieces of 
the different tissues and organs were cut out; these were dehydrated 
in 95 per cent, alcohol 12 to 24 hours, soaked in chloroform 12 to 
24 hours, infiltrated 4 to 5 days in an incubator, and imbedded in 
small paper boxes by pouring a small quantity of pure, hot paraffin 
over the pieces of tissue. 
After the sections were cut they were fastened to the slides by a 
thin layer of albumen fixative. 
The paraffin is removed by soaking the preparations in a jar of 
xylol 15 to 20 minutes. The xylol is then removed by 95 per cent, 
alcohol; that is followed by 82 per cent, alcohol, and finally by 
water. 
For staining hydrochloric acid carmine was found to be most 
successful; haematoxylin proved to be too dark and was liable to be AMERICAN MICROSCOPICAL SOCIETY. 
67 
confused with the carbon. After staining the preparations, they 
were thoroughly washed, dehydrated with 95 per cent, alcohol, the 
alcohol removed by clearer (2 vols. of carbolic-acid crystals, 3 vols. 
of turpentine), and mounted in xylol balsam. 
The stained-blood preparations were made as follows : 
Most of the preparations were made from Cryptobranchus, because 
it proved to show the points most distinctly. The specimen was 
left ten days after injection before it was killed-longer than any of 
Necturus were left, the latter being examined to find the times of 
the appearance of the ingested leucocytes in the circulation. 
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71 
Plate !. 
Red Blood Corpuscles and Fibrin Filaments. 
A. Red blood corpuscles. 
I. Red corpuscles of Necturus. 
a. Overlapping tile-like formation. 
b. Cells seen flatwise. 
c. Cells seen edgewise. 
II. Red corpuscles of Cryptobranchus. 
a. Overlapping tile-like formation. 
b. Cells seen flatwise. 
c. Cells seen edgewise. 
Drawn from fresh preparations. 
B. Fibrin filaments. 
I. Mammalian fibrin. Three leucocytes lie between the meshes 
of the network. 
II. Fibrin of Necturus, with leucocytes closely connected with the 
threads. 
III. Fibrin of Cryptobranchus, showing two leucocytes closely 
united with the threads. 
"I" was drawn from a stained preparation. "II" and "III" were 
drawn free-hand. 72 
PROCEEDINGS OP THE 
Plate //. 
Leucocytes. 
A. Group of carbon-laden leucocytes, showing amoeboid phases. 
a, b. Leucocytes of Necturus. 
n. Nucleus. 
p. Cell-body. 
c, d. Leucocytes of Cryptobranchus. 
n. Nucleus. 
p. Cell-body. 
B. Group of Leucocytes, showing amoeboid cell-bodies and amoeboid 
nuclei. 
a, b, c. Leucocytes of Necturus. 
n. Nucleus. 
p. Cell-body. 
b. Shows three nuclei, two in amoeboid movement and one resting. 
d, e. Leucocytes of Cryptobranchus. 
n. Nucleus. 
p. Cell-body. 
e, Has three nuclei, two amoeboid and one resting. 
Drawn from stained preparations. PLATE II PLATE III AMERICAN MICROSCOPICAL SOCIETY. 
73 
Plate 111. 
Direct Division of the Cell-body of a Leucocyte.' 
Drawn from freehand sketches made when the division was in progress. 
The time taken to pass through the stages figured was thirteen min- 
utes. 
The unequal division of the parent cell is noticeable, the bud, as it 
were, being comparatively quite small. 
10 m 74 
PROCEEDINGS OF THE 
Plate IV 
A. Surface section of liver of Cryptobranchus. 
P. Capillaries of blood-vessels. 
T. Liver-cells. 
b. Red corpuscles in the capillaries. 
c. Ingested leucocytes in the capillaries. 
d. Small intercellular capillaries of the bile-duct. 
e. Epithelium of the larger bile-vessels. 
h. Hepatic cell-body. 
n. Nucleus of hepatic cells with nucleoli. 
Note the absence of extravascular ingested leucocytes. 
B. Vertical section of the spleen of Cryptobranchus. 
I. Part of the section near the surface. 
1. Peritoneum. 
2. Layer of fibrous tissue forming the capsule. 
3. Trabeculae passing from capsule among the splenic cells. 
c. Carbon-laden leucocytes. 
b. Red corpuscles. 
I. Leucocytes non-ingested. 
5. Splenic pulp-cells. 
II. Ental part of the section. 
Lettering as above. 
Note the ingested spleen pulp-cells and the different distribution of the 
carbon particles in them from that found in the leucocytes ; also the ab- 
sence of ingested splenic cells in the superficial part of the spleen. PLATE IV PLATE V AMERICAN MICROSCOPICAL SOCIETY. 
75 
Plate I/. 
A. Vertical section of the lung of Cryptobranclius. 
E. Ectal surface. 
R. Ental or respiratory surface. 
P. Blood capillary. 
c. Carbon-laden leucocytes. 
Note the presence of ingested leucocytes in extravascular tissue as 
well as on the ental surface of the lung. 
B. Section of kidney of Cryptobranclius. 
I. Transection of the ureters and cloaca, showing masses of in- 
gested cells. 
a. Ureters. 
c. Carbon-laden leucocytes. 
Z. Non-ingested leucocytes. 
II. Transection of urinary tubules. 
c. Carbon-laden leucocytes. 
III. Nephrostomic funnel, showing the ciliated mouth. 
c. Ingested leucocytes. 
Z. Non-ingested leucocytes. 
IV. Vertical section of the kidney near the ventral surface. 
G. Glomerulus. 
P. Capillaries of blood-vessels. 
T. Urinary tubules. 
S. Lymph space around the glomerulus. 
O. Origin of a urinary tubule, with small, ciliated epithe- 
lium. 
b. Red corpuscles. 
c. Carbon-laden leucocytes. 
Note the presence of extravascular ingested cells. 76 
PROCEEDINGS OE AMERICAN MICROSCOPICAL SOCIETY 
Plate VI. 
A. Vertical section of stomach of Cryptobranchus, near the pyloric part. 
I. Submucosa. 
II. Muscularis mucosse. 
III. Mucosa. 
c. Carbon-laden leucocytes. 
Note the presence of the extravascular ingested cells. 
The figure is diagrammatic in so far that the locations of the ingested 
cells are taken from different sections and put into one figure. 
B. Vertical section of the skin of Cryptobranchus. 
G. Targe mucous glands. 
c. Carbon-laden cells. 
The ingested cells are wandering to the external surface from the 
blood-vessels. PLATE VI