REPORT OF INVESTIGATIONS 
INTO THE 
PATHOGENY OF DIPHTHERIA, 
CONDUCTED BY 
EDWARD CURTIS, M. D. AND THOMAS E, SATTERTHWAITE, M. D. 
New York, February n, 1877. 
To the Board of Health : 
GENTLEMEN—I have the honor to submit herewith a report of 
investigations made in accordance with a resolution of the Board 
of Health, passed November 24, 1874, as follows : 
“ Resolved, That Dr. Edward Curtis be requested, with the con- 
currence and under the supervision of the Sanitary Committee, to 
investigate the causes and nature of diphtheria by means of micro- 
pathological examinations and otherwise.” 
At my request Dr. Thomas E. Satterthwaite was associated with 
me in the investigation, and the experiments made were conducted 
by us jointly. 
Very respectfully, your obedient servant, 
EDWARD CURTIS, M. D., 
Honorary Microscopist to the Board. 2 
PART I.—GENERAL REPORT. 
By the broad instructions to investigate the “ causes and nature of diphtheria, ” we conceived 
that we were desired to work at some point in the pathogenesis of the disease, whose solution 
was important and at the same time seemingly within the means of laboratory experimenta- 
tion. Accordingly we took for our subject the question which has already attracted so much 
attention from its supposed solution in Germany, viz: what is the nature of the infectious 
frinciple of diphtheria, and what are the circumstances that determine the infection. 
Considering it already sufficiently proven that the false membrane of diphtheria is able to 
communicate the disease, we made our search for the infectious principle in this material. The 
first point was to know the morphological elements of the membrane. Concerning most of 
these there has never been any doubt; fibrin, epithelial cells, leucocytes and granular matter 
being easily recognizable. These elements we found in varying proportion in most of the 
sixteen1 membranes examined by us, and in three8 there was also the mycelium, and in one8 
of the same also the spores of a fungus, apparently an aspergillus or penicilium. But the 
important point was to determine the presence or absence in the membranes of bacteria, since 
these organisms have been alleged to be not only always present, but also to contain or be the 
noxious element. Accordingly we searched our membranes with special care for bacteria, with 
the following result: Small rod-bacteria, precisely similar in form and size to those found upon 
the mucous membrane of the healthy mouth, we found almost always present, though in greatly 
variable quantities, scattered throughout the tissue of the false membrane. Besides these rod- 
forms there also occurred in many, though not in all, of our membranes, distinct collections of 
closely packed bacteria, apparently either firmly glued together or else encased in a delicate 
invisible envelope. For these collections, or “ balls ” or “nests,” could be isolated and 
rolled about and subjected to heavy pressure by pressing upon the cover of the microscopical 
slide, without losing their form or coherence. Similar balls or masses have been repeatedly 
seen by us in non-diphtheritic matter infested with bacteria. On treating membrane contain- 
ing these balls with an alkali, the balls disappear as such, and the field becomes crowded with 
short rod-bacteria. From this fact it is likely that these balls are not, as their appearance 
first suggests, wholly collections of spherical bacteria, but that much of their contents at least 
is the ordinary rod-form of bacteria, which here looks spherical simply because the individuals 
are closely packed side by side with their ends presented to view. The same illusion of minute 
spheres is commonly met with wherever, as upon the papillae of the healthy tongue, rod-bac- 
teria are closely massed together. In addition, free spherical granules presenting the optical 
characteristics of bacateria were often met with, but rarely except in company with the free 
rods and the balls of massed bacteria already described. Upon these granules we tried the 
various chemical and coloring tests for bacteria proposed by Hiller, Letzerich and Eberth, 
but we found the tests so unreliable that we dared not base conclusions upon their showings. 
Inasinuch furthermore as the granules in question were almost always associated with unmis- 
takable bacteria, and were in no wise different in appearance from the similar granules found 
commonly in all bacteria-infested matter, whether diphtheritic or not, we did not attach much 
1 Several specimens were examined which do not appear in Part II., because no use was made of them for 
experimentation. 
2 Cases VI., XI., and XIII. See Part II., pp. 677, 689, 690. 
3 Case XI. importance to the determining whether or not the granules were themselves bacteria. When- 
ever, therefore, it may be remarked in passing, statements are made in this report about 
bacteria, either rods or balls of massed bacteria—both definitely recognizable by the .micro- 
scope—are always meant. 
So far then as regards the presence of bacteria in our diphtheritic membranes, we found 
abundant evidence of the existence of the form described by other observers, but these forms 
were in no wise different in optical or chemical behavior from the bacteria found in putrescent 
but non-diphtheritic animal matters. 
The morphological elements being thus determined, and being found to present nothing sui 
generis in form, we proceeded to our proposed experimentation, which was to inoculate living 
animals with diphtheritic membrane under varying conditions. The idea was that if, as 
alleged,1 inoculation will produce diphtheria in the inoculated animal, then, by varying the 
conditions, the nature of the poisonous principle and the determining circumstances of infection 
might be discovered. The animal chosen for experimentation was the rabbit, partly because 
we had not the facilities for dealing with larger or more troublesome animals, and partly 
because some German investigations, with which it was desirable that ours might be compared, 
were also conducted upon rabbits. 
The first point was to determine, as a basis for comparison, the effect upon the rabbit of 
simple inoculation of fresh diphtheritic membrane. Following Eberth,3 we first tried inocu- 
lating the cornea. This was done by pricking and scratching the cornea with a needle, then 
rubbing into the abrasions a bit of diphtheritic membrane, and finally holding the lids shut for 
a few minutes with the piece of membrane inside. According to Eberth8 this proceeding 
begets an extensive diphtheritic keratitis with constitutional symptoms and even termination 
in death. In our hands, however, nothing resulted but small local spots of inflammation at the 
site of each puncture, which appeared the day after infection, and speedily subsiding, left the 
eye well by the fourth or sixth day. No severe constitutional symptoms appeared, and none 
of the animals died except one whose thigh was also inoculated. These inoculations were tried 
with four4 different diphtheritic specimens. In three5 of these it is an open question if the 
material inoculated was active, but in the third8 case there is no such doubt, for the same 
membrane, which was thus almost wholly innocent upon six cornese, produced extensive 
lesions and proved fatal in forty-five hours when inoculated into the muscular tissue of the 
thigh.7 On account of these failures we abandoned trying the cornea, and in all subsequent 
inoculations resorted to the muscular or subcutaneous connective tissue. Where the material 
to be inoculated was solid, as with bits of diphtheritic membrane, it was inserted into the 
muscular tissue either of the fore or hind leg, by the following method: A snip is made 
through the skin and subcutaneous connective tissue an inch or more distant from the intended 
site of inoculation in the muscle; then the skin is drawn along till the hole exposes the muscle, 
when a cut is made into the latter tissue, and the inoculating material pushed into the wound. 
The skin being then let go, resumes its natural position, and the inoculated substance is 
retained in place without the need of a suture. Where the inoculating material was fluid it 
was simply injected subcutaneously by an ordinary hypodermic syringe. Thirty-eight rabbits 
1 Oertel, Eberth and others. 
2 Zur Kenntniss der Baeteritischen Mykosen. C. J. Eberth, Leipsig, 1872. 
3 Op. cit. 
t Cases I., II., III., IV. See Part II., pp. 671, 672, 673, 675. 
5 Cases I., II., and IV. 
8 Case III. 
1 Experiment 8. See Part II., p. 674. 4 
were thus inoculated with pure diphtheritic membrane from twelve different specimens.1 Of 
these animals twenty-seven were inoculated with bits of solid membrane, the size of the 
pieces varying with the amount of material on hand, but varying from one-sixteenth to one- 
fourth inch square. The other eleven animals were injected with aqueous infusion of the mem- 
brane, made by cutting the latter up fine, thoroughly triturating it with cold water, and then 
injecting the turbid infusion after the larger pieces of membrane had settled. Of the inocula- 
tions with solid matter all but three2 were double, that is, were made upon both the opposite 
legs of the animal, hind or fore, as the case might be. Of the infusion injections all but four3 
were similarily double. Of the thirty-eight animals inoculated, four4 showed no effects what- 
ever, local or constitutional. Of these, three5 had been inoculated with the membrane of 
Case IV.,6 which was a soft, glutinous material, made up of leucocytes and granular matter 
imbedded in mucus. It had formed in the nose of a three-year-old child on the second or third 
day of an acute nasal catarrh, and had become detached a day or two later, the child recover- 
ing without any alarming symptom. Both the appearance of the membrane and the history 
of the case thus made it doubtful if the disease in this instance were really diphtheria. All 
the other animals, inoculated, thirty-four in number, developed local lesions at one or both 
sites -generally both—of varying degrees of intensity. The great majority also showed con- 
stitutional symptoms, and twenty-two died. One death,7 however, is doubtful, as the rabbit 
was found half out of his cage, wedged between the bars, and may have injured himself. But 
this case is fairly offset by a doubtful recovery,8 where the local lesion was bodily removed on 
the fifth day, and the animal slowly recovered, whereas, four9 other rabbits inoculated with 
the same membrane and left untouched, all died. Of the twenty-one genuine deaths, five10 
took place between the first and second day after inoculation, thirty hours after being the 
earliest period—six11 between the second and third day, one each on the third,12 fourth,13 
sixth,14 fourteenth,15 nineteenth,16 and twenty-first,17 two on twenty-third,18 and one each 
on the thirty-first19 and thirty-eighth20 days. 
Our results thus confirmed those of other investigators, namely—that the matter of diph- 
theritic membranes inoculated into the muscular tissue of rabbits proves poisonous, and in the 
majority of cases fatal. But it still remained to be determined whether, as alleged, the disease 
produced, be actually diphtheria. To decide the question the first step obviously was, to com- 
pare the rabbit-disease produced by these inoculations with diphtheria as it occurs in the 
human subject. A study of our cases showed that the inoculation disease, though differing 
greatly in severity, course and results in different instances, yet was always the same process, 
as follows: At varying periods after the inoculation a lump appears under the skin at the 
inoculated spot. On cutting down upon it, a yellowish white mass is exposed protruding from 
the wound in the muscle into the connective tissue in the line of communication between the 
original skin and muscle-openings. When first formed, this mass is homogeneous, of pasty 
consistence, and when examined microscopically is seen to consist of the loose substance of the 
1 Cases III. to XIV. inclusive. 
2 Experiments 12, 13 and 24. 
3 Experiments 14, 31, 32, 33. 
4 Experiments 12, 13, 14, 60. 
5 Experiments 12, 13, 14. 
6 See Part II., p. 675. 
1 Experiment 62. See Part II., p. 691. 
8 Experiment 23. See Part II., p. 679. 
9 Experiments 21, 22, 24. 
10 Experiments 8, 17, 18, 19, 39. 
11 Experiments 20. 45, 52, 53, 54, 63. 
12 Experiment 40. 
is Experiment 36. 
11 Experiment 35. 
15 Experiment 58. 
16 Experiment 22. 
11 Experiment 21. 
18 Experiments 25, 68. 
19 Experiment 26. 
20 Experiment 24. 5 
connective tissue densely infiltrated with leucocytes, and also with greater or less numbers of 
bacteria according as the original inoculated material was rich or poor in these elements. 
Around this patch or lump of infiltrated connective tissue there will be found more or less 
evidence of an irritant action. In the severest cases there will be intense hypersemia of all 
the circumjacent parts, ecchymotic spots and extensive serous infiltration. In the animals 
that die early, these lesions are especially prominent, and in some cases death occurs before a 
lump has time to form at all.1 But in cases not speedily fatal, the symptoms of great irrita- 
tion are generally wanting, and the lump produced by formation of leucocytes develops slowly 
and apparently without any affection of the general health of the animal. In such case there 
will be but comparatively few bacteria to be found in the lump. The course of the disease 
in these cases follows two types. Either the lump, generally of small size, slowly disappears 
by absorption, leaving nothing but a cord of thickened connective tissue, or the material of 
the lump softens by fatty degeneration ; the skin gives way, and a running sore is established 
with more or less burrowing of pus into the adjoining tissues. When the latter course develops 
itself, the animal frequently becomes emaciated; its fur unclean and mangy ; it refuses food, 
and finally dies after a greater or less period, often extending over several weeks, with all the 
signs of general exhaustion. In cases where running sores and extensive burrowings of pus 
take place, the matter commonly becomes offensive and swarms with bacteria. In all types 
of the disease, there is a notable absence of lesions of any distant part. No exudations appear 
either upon any of the mucous membranes or upon any wounds which may be present, and 
there is no constant or even frequent lesion of any organ. In many cases we examined the 
blood, post-mortem, for bacteria, and while these bodies were found in about half the cases 
examined, their presence did not seem specially related to the presence of bacteria in the 
local lesion, nor did they occur more frequently than in the blood of non-inoculated animals, 
dead an equal length of time. 
In the above described disease we failed to see anything specifically resembling diphtheria 
as it occurs in the human subject. The whole story seemed to be one of local irritant poi- 
soning. which always tended towards the production of an abscess at the site of inoculation, 
with greater or less concomitant hypersemia, ecchymoses, and serous infiltration of neigh- 
boring tissues, according to the degree of virulence of the inoculated poison. According, 
also, to the severity of the primary lesions, the animals would die or survive the immediate 
effects; and according to the secondary history of the mass infiltrated with leucocytes— 
according, namely, as the mass was re-absorbed or softened and ulcerated—the animal sur- 
vived unaffected, or slowly wasted away and died of exhaustion. But it might, not without 
reason, be argued that a rabbit is a widely different animal from a man, and that a disease 
induced by subcutaneous inoculation might naturally differ in its manifestations from the same, 
occurring idiopathically; hence, that the described affection of rabbits might still be true 
diphtheria, although wanting in the specific characteristics of that disease as seen in the human 
subject. To determine this point we proceeded to try if effects similar to the foregoing would 
follow the inoculation of a material resembling diphtheritic membrane in its anatomical and 
chemical character, but yet not only not diphtheritic, but even incapable of producing any 
noxious effect under circumstances where diphtheritic membrane often proves highly infectious. 
Such a material presented itself in the scrapings from the upper surface of a somewhat 
“furred” tongue from a healthy person. These scrapings, while obviously not infectious 
when brought in contact with the pharyngeal mucous membrane, yet contain anatomical 
1 See Experiment 8. Part II., p. 674. 6 
elements of similar character, and in similar vital condition, to those of the diphtheritic mem- 
brane. They also swarm with countless bacteria. 
Such scrapings, inodorous when fresh, acquire a peculiarly offensive foetid smell even 
within a few hours. Inoculations with this pulpy material were then made in the usual way, 
with the following results : Three animals were inoculated with the matter freshly removed.1 
Of these, one8 died between the fourth and fifth day with a lump in each thigh, in all respects 
similar to that produced by the diphtheritic inoculation. One8 died on the fourteenth day, with 
similar lumps, which had softened and ulcerated; and the third4 on the twenty-fifth day, with 
widely extended burrowings of pus and secondary peritonitis. Four other rabbits5 were 
inoculated with tongue-scrapings twenty-four hours old and foetid. Of these, the first6 
developed a large lump of the usual character in twenty-four hours. This was then removed 
entire for study, and the animal survived with no further lesion. The other three animals 
were let alone; all developed the usual lesions, and all died on respectively the fifth,7 nine- 
teenth,8 and twenty-fifth9 days. A third series10 of two rabbits were inoculated with the 
mixed scrapings of five successive mornings, which was exceedingly offensive to the smell. 
One11 died on the sixth day with the usual lumps, but as this was a young animal from a litter 
some of which died without having been experimented on at all, it is an open question whether 
or not the death in this case was due to the inoculation. In the other rabbit18 lumps devel- 
oped, which ulcerated and discharged foul matter. The animal suffered also in its general 
health, but ultimately recovered. 
These series of experiments thus developed the important fact that certain non-diphtheritic 
and non-infectious matters will, when inoculated upon the rabbit, produce a disease closely 
similar to, if not identical with, the disease caused by the inoculation of diphtheritic membrane, 
and one, also, equally fatal in its effects. But inasmuch as tongue-scrapings are open to the 
suspicion of containing some peculiar poison derived from the animal body, we determined to 
experiment with a substance which should be free from such objection, while still resembling 
diphtheritic membrane and tongue-scrapings in the matter of being putrescent and swarming 
with bacteria. We accordingly chose Cohn's fluid, which had passed into a state of decom- 
position. This fluid is simply a solution in distilled water of ammonium tartrate, potassium 
phosphate, and magnesium sulphate, to which also a little calcium phosphate is added.18 
Upon exposure in a warm place, bacteria appear in this fluid in great numbers, and out of the 
salts present new compounds are formed of a strongly putrid smell. Inoculations were made 
with this putrescent material, as follows: Two animals 14 were inoculated in the usual way 
with the pulpy sediment forming at the bottom of the bottle, and obtained by straining the 
fluid. Both developed the usual purulent lumps, but with little attendant irritation or con- 
stitutional disturbance. In both, the lumps gradually disappeared by absorption and atrophy, 
and the animals survived. Four rabbits15 were inoculated by hypodermic injection with the 
lower stratum of liquid in another sample of the decomposed fluid. Lumps, as usual, 
formed at the site of each injection; one animal died on the tenth day, the other three sur- 
1 Experiments, 73, 74, 75. See Part II., pp. 694, 695. 
2 Experiment 73. 
3 Experiment 75. 
1 Experiment 74. 
5 Experiments 76-79. See Part II., pp. 695, 696. 
6 Experiment 76. 
1 Experiment 79. 
8 Experiment 77. 
9 Experiment 78. 
10 Experiments 83, 84. See Part II., p. 698. 
11 Experiment 84. 
12 Experiment 83. 
13 Ammonium tartrate, 1.0; potassium phosphate, 
magnesium sulphate, aa 0.5 ; calcium phos- 
phate, 0.05 ; distilled water, 100 c. c. 
11 Experiments 91, 92. Part II., p. 700. 
15 Experiments 93-96. Part II., p. 701. 7 
vived. F'our1 rabbits were injected hypodermically with the upper stratum of liquid in still 
another sample. Lumps again appeared at each inoculated spot, but they were small, and all 
the animals survived. The same procedure in four8 other rabbits with a fourth sample of 
fluid produced like effects in three of the animals, but in the fourth no lumps discoverable by 
feeling the spot, could be found. F'ourteen animals in all were thus inoculated with the putrid 
Cohn fluid. Thirteen of them developed lesions identical in appearance with the character- 
istic lesions from inoculations with diphtheritic membranes and tongue-scrapings, but the 
collateral effects were not so severe, and only one animal3 died from the effects of the inocu- 
lation. In short, then, putrid Cohn’s fluid, inoculated upon the rabbit, poisons after the 
same manner as diphtheritic membrane, though not to the same measure. 
It would seem therefore that the disease produced in the rabbit by inoculations of diph- 
theritic matter, is not only not specifically diphtheritic in character, but not even peculiar to 
the diphtheritic infection ; since a disease essentially similar, if it be not pathologically 
identical, is produceable, though in variable intensity, by inoculations of material at once 
non-diphtheritic and non-infectious to human mucous-membrane ; and even, it maybe, not of 
animal origin. This fact being apparently established, the important corollary follows, that 
pathological and pathogenetic conclusions drawn from the effects of diphtheritic inoculations 
of the rabbit, do not of necessity apply to the disease diphtheria as it appears in the human 
subject. The many current hypotheses in the premises therefore, which rest upon such animal 
experimentation, are built upon a quicksand. 
This conclusion being accepted, it was plain that to attempt to elaborate the pathology of 
the diphtheritic process, by study of the rabbit’s inoculation-disease, would be a waste of 
time, and all thoughts of the same were accordingly abandoned. But though the inoculation- 
disease is not necessarily, nor even probably, diphtheria, yet it is possible, and indeed 
probable, that the poisonous element in the diphtheritic membrane, which will produce diph- 
theritic infection in man, is the same kind of thing as that which produces the inoculation- 
disease in the rabbit. Hence, it seemed to us to be a useful research to try to discover the 
nature of the infecting principle, whether of diphtheritic membrane, tongue-scrapings, or 
Cohn’s fluid, which produces the inoculation-disease. 
It was, in the first place, conceivable, though not likely, that the disease in question 
might be due simply to mechanical irritation. To decide this point, we sought an inoculating 
material which should combine physical roughness with freedom from poisonous property. 
These qualities being obviously present in sand, we inoculated two rabbits4 with dry sand, 
after the usual method, and two others5 with a pulp made of sand mixed with Liebig’s 
meat-extract. Not the slightest symptom, local or constitutional, followed in any of the 
cases. At various subsequent dates, the tissues in the track of the original wound in the 
various animals were exposed and examined. The grains of sand were found scattered in 
the connective tissue; this tissue was slightly thickened, but there was no trace of suppura- 
tion or other sign of irritation. It was seemingly evident therefore, as expected, that the 
cause of the inoculation-disease is other than mere mechanical irritation. The next point to 
be determined was, whether the poisonous principle is in solution in the fluid of the inoculating 
materials, or whether it resides in the solid elements of the same. For this inquiry, aqueous 
infusions of the infecting matters were filtered, and comparisons made of the effects of inocu- 
lation of the filtered and unfiltered fluids. Filtrations were made through various thicknesses 
1 Experiments 97-100. Part II., pp. 701, 702. 
2 Experiments 106-109. Part II., pp. 703, 704. 
3 Experiment 94. 
1 Experiments 121, 122. Part II., p. 706. 
5 Experiments 123, 124. Part II., p. 706. 8 
of ordinary filtering paper, and also through porous clay. The experiments with paper- 
filtered fluids were as follows: An infusion of diphtheritic membrane1 was made and filtered 
through a double paper-filter.3 The filtrate, which was clear and odorless, was injected 
hypodermically into three rabbits.3 All survived, two without even any local lesion, but the 
third with a large lump of the usual character, containing living bacteria, upon one side. 
None of the animals seemed to suffer in general health. At the same time the unfiltered 
infusion was injected into three other rabbits,4 and all of them died before the end of the 
third day with severe lesions. With another sample of membrane,5 one rabbit6 was inocu- 
lated with the filtered infusion, and one7 with the original solid membrane. The former 
escaped with scarcely any effect, local or general, the latter died in forty-five hours with 
extensive and severe lesions. An infusion of a third membrane8 was allowed to putrefy for 
ten days, and was then filtered through a single paper filter. The filtrate contained living 
bacteria, and retained the putrid odor ; but it produced no effect at all upon injection into 
two rabbits.9 The unfiltered fluid was also injected into two rabbits,10 but produced only 
slight lesions. One of these animals died the day after the inoculation, but it had jumped 
from its cage and was found dead on the floor, and it is altogether probable that death was 
caused by the fall. A filtered infusion of still another membrane11 was injected into a single 
animal,13 but since in this case the original membrane, upon inoculation, proved to be itself 
innocuous, this experiment is to be disregarded. 
As may be at once seen, the general result of the above inoculations was, that paper- 
filtration tended to diminish the poisonous virulence of the infusions. But the experiments 
were not thorough, for the filtration was insufficient to entirely remove certain of the solid 
constituents (bacteria) from the infusions. The difficulty was that, from the small quantities 
of diphtheritic membrane at our disposal, we could never obtain a large enough bulk of strong 
infusion to permit of repeated paper-filtration, such as alone would separate, with reasonable 
thoroughness, all the solid elements of the infusion. We were forced therefore to turn to 
other infectious infusions, where the requisite bulk of fluid could be had to test the matter 
thoroughly. We accordingly took putrid Cohn-fluid and filtered it as follows, fresh filters 
being used at each successive filtration : Twice through single filters of ordinary gray filtering- 
paper ; twice through double filters of the same ; once through a triple, and once through a 
quadruple filter; and finally, thrice through quadruple filters of fine, white filtering-paper. 
The final filtrate, instead of the milky appearance of the original fluid, was but faintly opal- 
escent, and, what was interesting and suggestive, had entirely lost the strong putrid smell of 
the non-filtered fluid. Under the microscope (one-tenth inch immersion objective of J. Beck), 
the fluid appeared perfectly clear, except that about half a dozen small bacteria were to be 
found in each microscopic field. This filtrate was then injected into four rabbits,13 four other 
rabbits at the same time receiving injections of the unfiltered fluid.14 The latter animals all 
developed the usual lumps at each site of injection, but all those injected with the filtrate 
remained absolutely unaffected. No lumps whatever could be felt upon the thighs at any 
period; and, to make sure, the skin over one thigh was snipped on the fourteenth day, and 
through the “ window” thus made, the whole subcutaneous territory was thoroughly inspected. 
1 Case X., Part II., p. 687. 
2 Part II., p. 689. 
3 Experiments 55-57. 
■t Experiments 52, 53, 54. Part II., p. 688. 
6 Case III., Part II., p. 673. 
6 Experiment 11. 
1 Experiment 8. 
8 Case IX., Part II., p. 685. 
9 Experiments 48, 49. 
10 Experiments 46, 47. 
11 Case IV., Part II., p. 675. 
12 Experiment 16. 
13 Experiments 102-105., Part II., p. 703. 
11 Experiments 97-100., Part II., p. 701, 702. 9 
Not the slightest lesion was to be found ; it could not have been told that the region had 
been inoculated at all. Here, then, we seemed to have positive proof, so far as the experi- 
ments went, that thorough paper filtration would remove entirely the infectious element of 
the putrid Cohn-fluid. But here the following possible source of error was acutely pointed 
out by a friend.1 During the very numerous filtrations above described, the fluid, falling 
slowly from the funnel drop by drop, had been submitted to a very thorough aeration as well 
as filti-ation; and, since it was conceivable that the infectious element might be a volatile 
chemical compound, it might be that in the experiment the innocuousness of the filtrate 
resulted from dissipation of a volatile principle by the aeration, instead of separation of a 
solid element by the filtration. To determine this point, a funnel was plugged with a rubber 
stopper pierced with a small hole, through which fluid could pass only slowly and drop by 
drop. Some putrid Cohn-fluid was then passed through this funnel as many times as the 
number of filtrations of the sample used in the last-described experiment. This aerated but 
unfiltered fluid was then injected into four rabbits,8 four other rabbits® being at the same time 
injected with the original fluid fresh from the bottle. Lumps were produced at each site of 
injection in the rabbits treated with the aerated fluid, exactly as in those injected with the non- 
aerated. This test experiment thus removed the possible flaw in the evidence obtained by the 
filtration injections, and left no doubt that by the separation of the solid elements of infectious 
infusions by thorough filtrations, the infectious principle tends to be removed. Further 
corroboration was afforded by experiments with a putrid infusion of calfs liver. Injections 
of the unfiltered infusion4 produced lumps as usual, but the fluid, when filtered,5 even through 
a single filter, failed to produce any effect. 
Simultaneously with the above described experiments with paper-filtrates, injections were 
practiced with some infusions filtered through porous clay. To obtain such filtrates, a porous 
clay battery-cylinder was made use of. A piece of wide rubber tubing, connected with an 
exhausting syringe, was slipped over the closed end of the cylinder, and by exhausting the air 
in the tubing, a little of any fluid in the clay cylinder would be drawn through the bottom of 
the same, of course suffering very perfect filtration in the passage. Infusions of three separate 
diphtheritic membranes,6 a sample of putrid Cohn fluid and the infusion of calf’s liver, already 
mentioned, were severally thus filtered. In all cases the filtrate was perfectly clear, entirely 
free from odor, and, examined microscopically with a 1-10-inch object-glass, apparently 
totally devoid of all solid elements of any kind. The three filtered diphtheritic infusions were 
severally injected into three,7 two,8 and again two9 rabbits, the filtered Cohn fluid10 and liver 
infusion11 each into a single rabbit. Not the slightest sign of a local lesion or of any consti- 
tutional disturbance followed any of these injections, while in all cases the unfiltered infusions 
were found upon injection to be more or less virulent. 
These various experiments thus seemed to demonstrate beyond reasonable doubt that the 
infectious element in diphtheritic membrane, putrid Cohn fluid, and the like, is separable by 
filtration from an aqueous infusion. And when the filtration is thorough, as by means of the 
clay filter, the removal of all noxious principles from the infusion is apparently absolute. 
From this fact the inference may be fairly drawn that the infectious principle or principles is 
1 Dr. John S. Billings, U. S. Army. 
2 Experiments 110-113. Part II., p. 704. 
3 Experiments 106-109. Part II., pp. 703, 704. 
4 Experiment 114. Part II., p. 705. 
5 Experiments 116, 117. 
s Cases VII., VIII., and IX. 
1 Experiments 28-30. 
8 Experiments 37, 38. 
9 Experiments 42, 43. 
10 Experiment 101. 
u Experiment 115. resident in some solid element or elements of the gross material, and cannot be separated 
therefrom to any recognizable extent by the action of cold water. 
This being assumed to be true, the next important point for differentiation seemed to be 
to determine if the noxious principle be specially connected with the bacteria found in the 
infectious materials, and if it be co-extensive with the number and with the degree of vital 
activity of these organisms. The great practical importance of the question thus raised is 
obvious. For if, as has already been claimed,1 the poison resides wholly in these organisms, 
and infection of the human system with diphtheria occurs only by bodily entrance of living 
bacteria into the blood, the whole current of preventive treatment will be turned to devising 
means for either destroying the vitality of such poison-bearing bacteria, or to prevent their 
passage through the tissue of the mucous membranes into the blood-vessels. In attacking 
this question by experimentation it was plain that a roundabout method must be adopted, since 
the direct plan of bodily separation of the bacteria from the other solid elements of the infec- 
tious material was obviously impossible. Hence the procedure that seemed to us most prom- 
ising, was to treat a piece of diphtheritic membrane in such manner as to destroy as far as 
possible the vital activity of the contained bacteria, without otherwise affecting the elements 
of the membrane, and then observe the comparative effects of inoculation of the materials so 
treated on the one hand, and of the original pure membrane on the other. For carrying out 
the projected treatment, the obvious procedure of boiling the material was deemed improper, 
for, though by this means the bacteria would doubtless be killed, yet at the same time all albu- 
minous matter would be permanently altered by coagulation. Failure to infect, on the part 
of boiled matter, might therefore with equal likelihood be assigned to the coagulation of an 
albuminous chemical poison, as to the killing of an assumed infecting living organism. For a 
similar reason, in resorting to the only other possible procedure, namely, impregnating the 
material with some agent fatal to bacterial life, all substances chemically disorganizing to animal 
matters were debarred from use. For an obvious reason furthermore it was necessary that the 
agent used should, itself, when inoculated, be practically free from all poisonous and even irri- 
tant effect. From all these considerations it seemed to us likely that the necessary conditions 
would be best fulfilled by impregnating the infectious matter with Salicylic acid, for we would 
seem to have in this substance one at once non-poisonous, slightly irritant only, with as little 
chemical action on animal products as could be expected, and yet with very great power to 
arrest more or less permanently the vital activity of bacteria and allied organisms. We 
accordingly determined to use this substance for the purpose described. But inasmuch as at 
the time of instituting these experiments {now nearly two years ago), much less was known 
than now of the properties of salicylic acid in its relation to living forms and substances, we 
deemed an investigation into these properties a necessary preliminary to the proposed inocu- 
lation experiments. We accordingly instituted two sets of experiments, one to determine the 
effect of salicylic acid upon the life and health of the rabbit, and the other to test exactly the 
degree of its power over the vital activity of bacteria. For the former purpose we inoculated 
a paste of salicylic acid and water into the muscular tissue of the thighs of three rabbits,2 in 
the usual manner. No effect whatever followed, either constitutional or local, except a lim- 
ited disintegration of the tissue impregnated with the acid.3 But even here there was no 
reaction in the surrounding tissues, nor the slightest appearance of hyperaemia or infiltration 
of leucocytes. Next, to test more thoroughly the possible poisonous effect of the acid after 
absorption, we injected hypodermically salicylic acid dissolved in an aqueous solution of sodic 
1 Oertel. 
2 Experiments 125-127. Part II., p. 707. 
3 See Experiment 125. Part II., p. 707. phosphate.1 A litter of young rabbits weighing 1 pounds apiece was used in the experi- 
mentation, and setting aside a death where the hypodermic needle accidentally pierced the 
liver, the result was that the animals which received five, ten, and fifteen grains, respectively, 
of salicylic acid, survived, but one which received twenty-four grains died in about twenty 
minutes. But in order to dissolve the twenty-four grain dose of the acid, seventy-two grains of 
sodic phosphate and nearly three fluid drachms of water were required, and to administer this 
bulk of fluid ten separate injections, of a syringeful each, had to be practiced one after the 
other at different points upon the back and belly of the animal. Conceiving therefore that 
the death after administration of this dose might be due, not to the salicylic acid, but either to 
the sodic salt or the physical violence of the repeated injections, the operation was repeated 
on another animal with a simple sodic solution, containing no salicylic acid ;s death followed 
as in the former instance. From all these facts therefore there seemed no further question 
that in the comparatively small quantities in which it was intended to be used, salicylic acid 
was practically harmless to the rabbit. To test next the power of the acid over the vital 
activity of bacteria, test-tubes containing various putrefiable solutions were charged with grad- 
uated percentages of salicylic acid, set aside in a warm place and watched for the first begin- 
ning of turbidity, which, as is well known, is always a sign of beginning bacterial develop- 
ment. The results were as follows: In the first series of experiments Cohn’s fluid was 
used,3 and it was found that when the charge of salicylic acid was less than 1 part to 2,500 or 
3,000, the solution became turbid almost as fast as plain Cohn’s fluid, to which none of the 
acid had been added, namely, in three or four days. Where the charge was from 1 to 2,coo 
or 3,000 (the results varying slightly with different samples of the salicylic acid), clouding was 
postponed for several days later, and charges of 1 to 1,500 and upward kept the fluid perfectly 
clear during the twelve days that the observation of this series lasted. In another series human 
urine which had been boiled and filtered was used,4 with the result that a charge of 1 to 2,500 
was necessary to effect any postponement of the normal period of beginning turbidity, and 
that a charge of 1 to 1,000 was the lowest that kept the urine clear during the eight days of 
the observation. In another series where the observation was more prolonged a weak meat 
decoction already inoculated with living bacteria was employed.5 This was made by first 
preparing the graduated solutions of salicylic acid in distilled water (the solution being effected 
by addition of a little sodic phosphate), and then inoculating a fluid drachm of each solution 
with a single large drop of a strong decoction of meat in which bacteria were already swarm- 
ing. The results were as follows: Test-tubes charged respectively with 1 part of salicylic 
acid to 100 and 250 remained clear during the whole seventy-eight days of observation. Those 
containing 1 to 500 and 750 were clear on the twenty-eighth day, but were found cloudy when 
next examined on the seventy-fourth day. A charge of 1 to 1,000 postponed the clouding 
twenty-eight days, but charges of 1 to 2,500 and less had scarcely any influence. The gen- 
eral results of these experiments then was that even in practically minute proportion the 
addition of salicylic acid to putrefiable solutions prevents and suspends bacterial development. 
These various experiments thus seem to show that in salicylic acid we had an available 
material for our present purpose, namely, a substance practically harmless to the rabbit, of 
little chemical action upon animal matter, yet of great power to suspend or prevent the vital 
activity of bacteria. We accordingly proceeded to institute comparative observations 
between inoculations of pure diphtheritic membrane and tongue-scrapings on the one hand, 
1 Experiments 128-133. Part II., pp. 707, 708. 
2 Experiment 137. Part II., p. 709. 
3 Experiments 139-175. Part II., p. 710. 
4 Experiments 176-186. Part II., p. 712. 
5 Experiments 187-206. Part II., p. 712. and on the other, of the same substances thoroughly triturated before inoculation with 
salicylic acid. The experiments were as follows: In one case,1 fourteen grains weight of 
diphtheritic membrane was first triturated with fourteen minims of water containing two 
per cent, of salicylic acid, and six per cent, of sodic phosphate in solution. Next, five grains 
of solid salicylic acid was added, and the whole thoroughly triturated in a mortar for half an 
hour. The pulpy mass thus formed was then inoculated in the usual way upon three rabbits.2 
All developed the usual lumps at the point of inoculation, but the lesions were not extensive, 
and the general health of the animal did not seem to suffer. One animal was accidentally 
killed a month after the inoculation, but the other two survived. The same membrane 
without the addition of salicylic acid was also inoculated upon two3 rabbits, as a control 
experiment. One of these animals was found dead on the second day, and the other on the 
third, but in the latter case, the animal was found partly out of the cage, between the bars, 
and the death may have been caused by injury. In both there was diffuse inflammation with 
the usual pasty infiltration at the sites of the inoculation, but the lesions were not severe. In 
another case,4 a piece of a different specimen of diphtheritic membrane was treated with 
salicylic acid in the same way as the foregoing, and inoculated upon three rabbits.6 One 
animal was found dead on the fourth day, and examination showed the usual pasty infiltra- 
tions, although there was not much circumjacent inflammation. The second died on the 
fifteenth day, much emaciated, with large lumps of the usual character, and about the average 
degree of inflammation of surrounding parts. The third animal survived, but also developed 
large lumps which ulcerated and discharged a white creamy matter, almost devoid of smell. 
The same membrane, pure, was inoculated upon three rabbits6 for comparison. All three 
developed lumps which ulcerated and discharged offensive matter. Two survived, one 
becoming somewhat thin and mangy, but subsequently recovering; and the third died on the 
twenty-third day, much emaciated, and with abscesses at the sites of inoculation. Analogous 
experiments with tongue-scrapings were as follows: Six grains weight of pulpy tongue- 
scrapings from a furred tongue, three hours after removal, was rubbed thoroughly with one 
grain of salicylic acid, and inoculated upon three rabbits.7 The first animal presented on 
the sixth day an extremely emaciated and dull appearance, and had thick lumps in each thigh. 
These were pricked and the matter examined for bacteria, but only a few motionless rod- 
forms could be found. The animal died the next day. Rabbit No. 2 died on the sixteenth 
day, and post-mortem examination showed an extensive patch of yellow material at each 
inoculation site, with, however, no signs of circumjacent irritation. No living bacteria, and 
but very few motionless ones were to be found in the matter composing the patches. The 
third rabbit survived, and never lost his fine general condition, but large lumps, attaining on 
one side the size of a pigeon’s egg, formed at the inoculated spots. The matter composing 
these was examined on the seventh and again on the twenty-fifth day, but no bacteria could 
be found on either occasion. These experiments were repeated with another specimen of 
tongue-scrapings, as follows : Some of the mixed scrapings of five successive mornings from a 
furred tongue was treated with salicylic acid solution, and then with the solid acid exactly as 
were the diphtheritic membranes in the experiments with the same last described. And it 
was an interesting and suggestive observation that, whereas the mixed scrapings before this 
treatment had, from their staleness, a horribly offensive odor, all smell disappeared after the 
1 Case XIII. Part II., p. 690. 
2 Experiments 64-66. 
" Experiments 62, 63. Part II., p. 691. 
* Case XIV. Part II., p. 692. 
5 Experiments 70-72. Part II., p. 693. 
6 Experiments 67-69. Part II., pp. 692, 693. 
1 Experiments 80-82. Part II., p. 697. salicylic acid was thoroughly incorporated with the mass. Six rabbits1 were now inoculated 
with the acid-treated mixture. All developed lumps, but not very large ; two died, one on 
the twelfth, and one on the nineteenth day, the other four survived. Some of the same scrap- 
ings, not treated with the salicylic acid, was inoculated into two other rabbits.8 Both 
animals were affected about as those last described, and one died on the sixth day ; but as this 
was a young rabbit, and others of the same litter died about the same time without having 
been experimented on at all, it is doubtful if the present death were due to the effects of the 
inoculation. 
Reviewing, now, these experiments with material treated with salicylic acid, it will be 
seen that the original matters, both the specimens of diphtheritic membrane and of tongue- 
scrapings—varied a good deal in virulence, but that such poisonous property as they had, was 
scarcely affected by the thorough trituration with the acid. The only obvious effect of the 
salicylic acid treatment seemed to be to prevent to a certain extent the matter composing the 
lumps from becoming offensive, and to prevent much multiplication of bacteria in the same. 
Although not bearing directly upon the matter under investigation, yet it seemed 
to us important to know if other and specific animal infectious matter would be equally 
unaffected by admixture with salicylic acid. Accordingly it occurred to us to mix vaccine 
lymph with a solution of salicylic acid, and then see if after such treatment the lymph would 
preserve its activity. In pursuance of this idea we requested Dr. Frank P. Foster to allow 
some bovine vaccinations to be made at his establishment after the manner indicated. He 
kindly consented, and vaccinations were accordingly made by Dr. W. F. Cushman and Mr. 
H. Lawrence, under direction of one of us (T. E. S.), as follows: Salicylic acid was dissolved 
in water by the help of sodic phosphate, two solutions being made, one containing the acid 
in the proportion of 1 to 250, and one in the proportion of 1 to 500. All being ready for the 
vaccination, some of the lymph to be used was mixed with each of these solutions, measure 
for measure, and the mixture allowed to stand for one minute before inoculation. Then 
both the pure lymph and the mixtures were inoculated upon the same animal.3 The result 
was that the admixture of the salicylic acid had not the slightest effect upon the virulence of 
the lymph, just as perfect pocks forming where the mixtures had been inoculated as where the 
pure lymph had been used. These experiments then, so far as they went, were in harmony 
with those preceding. 
So far, now, as regards the inoculations on the rabbit of the salicylic-treated diphtheritic 
membrane and tongue-scrapings, we have the fact that an admixture of the acid in over- 
whelming quantity (overwhelming from the point of view of its power to suspend the vital 
activity of bacteria), was yet almost powerless to affect the poisonous property of the matter 
subjected to treatment. Hence the important conclusion suggests itself that this same quality 
of infectiousness in the diphtheritic membrane and tongue-scrapings must be independent of 
the vital condition of the bacteria present in the matter. 
In this connection it becomes an interesting question to review the histories of the cases 
from which the membranes used for inoculation were drawn, and see what relation appears 
between facts connected with the disease in the human subject and the disease produced in the 
inoculated animals. In this analysis, cases I. and II. must be disregarded, because no inocu- 
lations were made with the material derived from them upon the muscle of the rabbit, and there 
is, therefore, no means of rating the comparative virulence of such material with the mem- 
branes from the other cases which were all used for muscle-inoculations. Taking, then, the 
1 Experiments 85-90. Part II., pp. 698, 699. 2 Experiments 83, 84. Part II., p. 698. 
3 Experiment 138. Part II., p. 710. other twelve cases, and studying the results of inoculation of the solid membrane or unfiltered 
aqueous infusion of the same in the thighs of rabbits, the membranes may be rated as follows 
in order of inoculable virulence, beginning with the most active: V., X., VIII., III., XIII., 
VI., VII., XI., IX., XIV.. XII., IV. 
Studying, now, the cases in this order the following points become manifest: I. There is 
no relation between inoculable virulence of a membrane and the time after detachtnent that 
has elapsed before the material is used for inoculation. For, of the two membranes heading 
the list for virulence, one (case V.) was used on the very day of detachment, and the other 
(case X.) not until three days old and already beginning to putrefy. On the other hand the 
membrane that proved the least virulent of all (case IV.) was used the day after detachment, 
and among the feebly virulent (case XIV.) the membrane was three days old. II. There is 
no relation between inoculable virulence of a membane and the quantity of bacteria present 
in the same. For, bacteria were abundant among both the most and least virulent of the 
membranes, and on the other hand the membrane third on the list for virulence (case VIII.) 
contained very few bacteria. III. There is a rough relation between inoculable virulence on 
the part of the membrane and the severity of the original disease in the human subject, so 
far as this can be estimated by the issue of the case in death or recovery. For, taking the 
cases in the order of inoculable virulence of the membranes as given above, the terminations 
of the cases in death or recovery read thus: Death, recovery, death, death, death, recovery, 
death, death, unknown, recovery, recovery, recovery. 
Besides the foregoing work, we had proposed for ourselves many other experiments and 
observations, notably the making of careful post-mortem examinations on human subjects dead 
of diphtheria, and the sanitary inspection of houses where many cases of the disease had occurred. 
Whenever opportunity offered we followed out our intentions in these regards, but during the 
period covered by the investigations these opportunities, in spite of our best efforts, were so 
few, that the desultory observations made are not numerous enough to generalize from. 
Other laboratory experiments were also planned and begun. These looked to the treatment 
of infectious matters by various chemical and physical agents, with a view of determining the 
nature of the poisonous principle by observing how the infectious quality might be affected by 
such treatment. But the investigations had to be brought to a close before enough of these 
later experiments had been made to draw conclusions from. 
The results of our investigations may therefore be summed up as follows : 
I. Inoculation of diphtheritic membrane into the muscular tissue of the rabbit produces 
severe local lesions, and even constitutional disturbance and death. But these effects differ so 
in their pathology and clinical history from diphtheria in the human subject, that there is no 
warrant for defining them as diphtheria, or for applying conclusions drawn from observation 
of this inoculation-disease in the rabbit to the case of diphtheria in man. 
II. Effects exactly similar to the foregoing and of equal severity can moreover be produced 
by inoculation of a material not only non-diphtheritic, but non-infectious to the human sub- 
ject under conditions where diphtheritic membrane is infectious, i. e., when brought into 
contact with the mucous membrane of the mouth and throat. The material referred to is the 
pulpy scraping of the upper surface of the healthy human tongue. 
III. Effects generally similar to the foregoing, though not of equal intensity, can further- 
more be produced by inoculation of a putrescent matter which is not even of immediate 
animal origin, namely Cohn’s fluid, allowed to spontaneously decompose. (Cohn’s fluid is 15 
simply an aqueous solution of ammonic tartrate, potassic and calcic phosphates and magnesic 
sulphate.) 
IV. The foregoing inoculation effects are not due to simple mechanical irritation, for 
inoculations of sand produce no effect whatever. 
V. Thorough filtration of a proven virulent aqueous infusion of diphtheritic membrane 
or of putrid Cohn’s fluid removes the infectious property of the same. Hence in such diph- 
theritic infusion the poisonous quality probably inheres in some particulate thing, from which 
it is not separable by the action of cold water. 
VI. Thorough trituration of proven virulent diphtheritic membrane and tongue-scrapings 
with a high percentage of salicylic acid fails not only to remove, but even markedly to modify, 
the intensity of the infectious quality of those substances. Hence, since salicylic acid in even 
a minute percentage is capable of permanently suspending the vital activity of bacteria, the 
inference is that the infectious quality of diphtheritic membrane upon the system of the rabbit 
is not correlated to the vital activity of the bacteria present in such membrane. 
VII. If. as is not improbable, the noxious principle in diphtheritic membrane which pro- 
duces in rabbits the effects described, be the same with or even analogous to the principle 
which produces diphtheria in man by direct infection, then the conclusion of VI. will apply 
to the infectious quality of such membrane in its relation to the reproduction of diphtheria in 
the human subject. If this be the case, it follows as an important practical corollary that 
there is no theoretical ground for assuming that preventing the bacteria of a diphtheritic 
patch from making their way through the underlying mucous membrane will, per se, 
prevent general diphtheritic infection of the system. 
VIII. There is no relation between inoculable virulence of a diphtheritic membrane and 
the period, within three days, that has elapsed between the detachment of the membrane and 
the inoculation with the same, nor between inoculable virulence and gross amount of bacteria 
present in the membrane. 
IX. There is a rough relation between inoculable virulence of a diphtheritic membrane 
and the severity of the original case of diphtheria, so far as this can be estimated by the termi- 
nation of the case in death or recovery. 
But it must be distinctly understood that these nine propositions are not put forth as 
proven, but merely as the results of our experiments and observations so far as the latter go, 
stated in abstract form. Before the propositions can be considered proved as truths, a large 
number of corroborative experiments will have to be made. 
PART II.— RECORD OF EXPERIMENTS. 
I.—INOCULATIONS WITH DIPHTHERITIC MATTER. 
Case I. 
March 29, 1875, received from Dr. Harwood Wakeman a piece of diphtheritic membrane, 
wrapped in damp cotton, and this encased in rubber tissue. Accompanying was the following 
memorandum : “ Fresh at 4 P. M. ; has been pretty well soaked with an application to the throat of the child of diluted muriatic acid ; child four years old ; sixth day since first appear- 
ance of membrane.” Not stated whether removed from living throat or post-mortem. The 
specimen, on soaking a bit in glycerine, and examining microscopically, showed a general 
infiltration with minute punctate bodies over the surface, and, imbedded in the substance, 
spheroid collections entirely composed of such bodies closely packed (nests or balls of bacteria). 
Twenty-four hours after removal the following inoculations were made, the animals being 
young rabbits of the same litter. 
a. Inoculation of cornea with pure membrane. 
Experiment i : 
March 29, 1875. Right cornea of a rabbit pricked and scratched In many places with a 
clean needle, and a piece of diphtheritic membrane thoroughly rubbed into the 
wounds. Piece finally left in cul de sac of upper lid. Left eye similarly scratched, 
but no inoculation made. 
March 30. Left eye, punctures healing, without irritation. Right eye, lids glued with 
catarrhal secretion, forming thick crusts on the outside. On opening lids, points of 
puncture cloudy and elevated. General catarrhal conjunctivitis, but no thickening 
of the conjunctiva nor appearance of a diphtheritic patch. 
March 31. Left eye, punctures healing. Right eye, catarrhal secretion less, but gray 
points of infiltration at punctures are perhaps slightly more distinct, with some 
general corneal haziness in that neighborhood. Animal feeds well, and is lively. 
April 1. All the morbid symptoms subsiding. 
b. Inoculation of cornea with aqueous infusion of membrane. 
Experiment 2 : 
March 29, 1875. A small piece of membrane was thoroughly triturated withf~_ ij of cold 
water, and steeped in the same for half an hour. Both cornese of a rabbit were then 
inoculated with the infusion in the manner described above. 
March 30. Right eye, punctures apparently healing, without irritation. Left eye, 
points of puncture cloudy ; general conjunctivitis, but less than in Experiment r. 
March 31. Right eye healing. Left eye, general improvement. 
c. Inoculation of cornea with aqueous infusion of membrane, boiled. 
Experiment 3 : 
March 29, 1875. A little of the foregoing infusion was boiled in a test tube for five 
minutes, and then allowed to cool. Both cornese of a rabbit were inoculated with 
the same, when cold. 
March 30. Both eyes healing, without irritation. 
March 31. Both eyes nearly well. 
Case II. 
April 4, 1875, received from Dr. J. Lewis Smith the trachea, bronchi, spleen and kidneys 
of a child supposed to have died of diphtheria twenty-four hours previous. The tissues seemed well preserved and had no bad smell. The mucous membrane of the trachea and bronchi 
was reddened, but there was no appearance of a membrane. The child from whom the speci- 
mens were taken had a moderate inflammation of the glands of the neck. On the third day 
there was a catarrhal discharge from the nostrils, and an epistaxis. A trace of albumen 
appeared in the urine, and the child died on the sixth day. 
a. Inoculation of cornea and conjunctiva with reddened mucous membrane of trachea. 
Experiment 4 : 
April 4, 1875. Inoculated right cornea of a rabbit, with the scrapings of the 
reddened trachea ; in left eye, a piece of the conjunctiva was snipped out, and a. 
piece of the mucous membrane inserted in the wound. 
April 5. Right eye, hardly any reaction. Left eye, considerable conjunctival inflamma- 
tion. 
April 6. Right eye almost well; left eye, conjunctivitis diminishing, but the piece of 
mucous membrane is still in situ, and appears to be firmly imbedded in the con- 
junctiva. No constitutional disturbance. 
April 7. Right eye well; left eye, conjunctivitis subsiding; piece still in situ; rabbit 
doing well. 
Experiment 5 : 
April 4, 1875. Right cornea of a rabbit inoculated with a piece of the kidney. 
April 5. A good deal of keratitis. 
April 6. Keratitis increased. Rabbit does not feed as well as usual. The keratitis is 
limited to a small area, by % inch, and the cornea is quite opaque. 
April 7. Rabbit eating better. Corneal redness diminishing, opacity as before. Removed 
with forceps as much matter as could be gotten off the cornea, some of the 
epithelium coming off with it. There was a slight layer of fibrine on the surface, 
and the epithelial cells contained numbers of brownish granules. 
April 8. No increase in inflammation. Conjunctivitis and keratitis subsiding. Opacity 
as before. Rabbit’s condition good. 
April 10. Rabbit well. 
b. Inoculation of cornea with kidney-tissue. 
Case III. 
April 13, 1875, received from Dr. Horace T. Hanks a piece of diphtheritic membrane of 
soft consistence and grayish yellow color, taken from the tonsils of a girl four years old. About 
April r, patient was taken with loss of appetite and high fever. April 4, she was first seen by 
Dr. H. and a membrane was then observed on the tonsils—later it appeared in the nares. It 
became detached on the sixth day of the disease, but symptoms of blood poisoning set in and 
the child died on the seventeenth day. There had been no obstruction of the larynx or 
paralysis. 18 
a. Inoculation of cornea with pure membrane. 
Experiment 6: 
April 13, 1875. Both corneae of a rabbit inoculated with the pure diphtheritic membrane. 
April 14. Slight keratitis and conjunctivitis only. 
April 15. Eyes improving. 
April 17. Eyes nearly well. 
Experiment 7 : 
April 13, 1875. Same procedure as foregoing. 
April 14, 15, 17. Results same as in foregoing. 
b. Inoculation of muscular tissue with pure membrane. 
Experiment 8 : 
April 13, 1875. Muscular tissue of both thighs of a rabbit inoculated with pieces of pure 
membrane. (For method of these and all subsequent inoculations into the muscular 
tissue, see ante, Part I.) 
April 14. No reaction of any moment. 
April 15. Died about forty-five hours after the inoculation. Autopsy twenty-seven 
hours later: Serous infiltration of the subcutaneous connective tissue over the 
thighs as far back as the lumbar vertebrae; isolated patches of the same over the 
abdomen as high as the foreshoulder. Right thigh: Both the aponeurosis and 
muscular substance of the entire thigh closely stippled with small ecchymoses. On 
vertical section through inoculation site, find a grayish-white patch about equal in 
size to the original inoculated material; this, under the microscope, is found to be 
composed of cellular bodies undergoing fatty degeneration, much free granular 
matter and a few bacteria. Around the muscle wound the tissues were reddened 
and ecchymosed, and there was much serum in the intermuscular connective tissue. 
The muscle-substance adjoining the wound when compared with that of a non- 
inoculated rabbit that died at about the same time was found in no wise different, 
both were undergoing post-mortem changes ; the strige were still well marked, and 
a few bacteria were found in both cases, but most numerous in the tissue from the 
non-inoculated animal. Considerable infiltration of the muscular fibres with fine 
oil-drops became apparent after three hours soaking in glycerine. The appearances 
in the left thigh were in all respects similar to those in the right. The blood drawn 
from the right ventricle of the heart contained a few small rod-bacteria, but far 
fewer than the blood of the non-inoculated rabbit already mentioned. The liver 
contained small echinococcus cysts. The kidneys were carefully examined for 
bacteria, fresh, and after soaking pieces in caustic potassa. No bacteria were found 
nor any appearances different from what obtained in the kidney of the non-inocu- 
lated rabbit. 
c. Inoculation of cornea with aqueous infusion of diphtheritic membrane. 
Experiment 9 : 
April 13, 1875. Both corneseof a rabbit were inoculated with an aqueous infusion of the 
membrane. 19 
April 14. No result of any consequence. 
April 15. Same condition. 
April 17. Nearly well. 
d. Inoculation of muscular tissue with boiled aqueous inf usion of diphtheritic membt ane. 
Experiment 10: 
April 13, 1875. Similar procedure to foregoing with the infusion after boiling and 
cooling. 
April 14, 15, 16. Results as in foregoing. 
e. Inoculation of muscular tissue with filtered aqueous infusion of diphtheritic 
membrane. 
Experiment 11 : 
April 13, 1875. Thigh of rabbit inoculated with the unboiled infusion after filtering 
through one thickness of filtering paper. 
April 14, 15, and 17. Results as in foregoing. 
Case IV. 
April 14, 1875, received from Dr. Horace T. Hanks another piece of membrane taken 
the night before from the nares of a girl three years old. The child was taken with an acute 
nasal catarrh, and on the second or third day this membrane formed in the nares. It became 
detached a day or two later and the child recovered in a week without having had any alarm- 
ing symptoms. The specimen was not firm and tough like genuine diphtheritic membrane, but 
soft and glutinous. Under the microscope it was seen to be composed of leucocytes and gran- 
ular matter held together by tough mucus. 
a. Inoculation of cornea and muscular tissue with pure membrane. 
Experiment 12 : 
April 14, 1875. Both, cornese and one thigh of a rabbit were inoculated with the pure 
membrane. No obvious effect was produced. 
Experiment 13 : 
April 14, 1875. Same procedure and same result as foregoing. 
b. Inoculation of cornea and muscular tissue with aqueous infusion of pure membrane. 
Experiment 14 : 
April 14, 1875. Both corneae and one thigh of a rabbit were inoculated with an aqueous 
infusion made by triturating in one fluid ounce of water a piece of the membrane 
about square. No recognizable effect followed. 20 
c. Inoculation of cornea and muscular tissue with aqueous infusion of membrane, boiled. 
Experiment 15 : 
April 14, 1875. Both cornese and one thigh were inoculated with the foregoing infusion 
after it had been boiled for three minutes, No recognizable effect followed. 
d. Inoculation of cornea and muscular tissue with aqueous infusion of membrane, filtered. 
Experiment 16: 
April 14, 1875. Both cornese and one thigh were inoculated with the infusion of “ b ” 
after filtration through a single thickness of filtering paper. Death forty-five hours 
later. Autopsy: Small whitish spot in the muscular substance at the site of the 
thigh inoculation ; strise obliterated in the circumjacent muscular fibres, and numerous 
rows of fat globules developed. After soaking the fibres in 10 per cent, caustic 
potassa for twenty-four hours, the sarcous matter and fat globules both wholly dis- 
appeared. Nothing abnormal to be found in any other part of the body. Cornese 
entirely healed. (N B.—This animal was one of a litter of young rabbits, others of 
which, not subjected to experimentation, died at about the same time.) 
Case V. 
April 23, 1875, received from Dr. George W. Rachel a piece of diphtheritic membrane 
removed the same day, and wrapped in moist rag and enveloped in rubber tissue. The case 
was that of a boy three and a half years old, whom Dr. Rachel first saw on the third day of 
his illness. The fauces and nasal cavity were then almost wholly'covered by a membrane, and 
the urine was albuminous. Difficulty of breathing began on the seventh day and continued 
till death, which took place four days later. The patient became finally comatose, and the 
temperature reached as high a figure as 106.25.° Others in the same family were attacked 
by the disease, and there were other cases still in the neighborhood. The specimen of mem- 
brane as received was apparently in excellent preservation ; it contained bacteria. 
a. Inoculation of muscular tissue with pure membrane. 
Experiment 17: 
April 23, 1875. Both thighs of a rabbit inoculated with the pure membrane. 
April 25. Dead forty-five hours after inoculation. Autopsy.—Left thigh : Skin wound 
closed, but attached to the inner surface of the skin at this point was a small yellow- 
ish mass about the size of a No. 8 bird-shot. At the point of inoculation in the 
muscle was a yellowish mass spread out beneath the muscle sheath. The muscular 
tissue itself was mottled red and white, but there was no extensive inflammatory 
change in the adjacent parts. Right thigh: Skin wound closed. Over the whole 
hinder half of the back and belly the subcutaneous tissue was infiltrated with serum, 
and unnaturately adherent to the skin and sheaths of the muscles. The muscles and 
their sheaths were stippled with numerous eccliymoses. Track between the skin and 
muscle-wounds was filled with a yellowish pasty material. This, under the micro- 
scope, was found to consist of the elements of connective tissue stuffed with leucocytes 
with a little granular matter. No recognizable bacteria. The same matter from 
the right thigh was essentially similar in composition. Blood from the right ventricle 
contained numbers of rod shaped bacteria. Trachea showed post-mortem conges- 
tion, but no signs of a membrane. Kidneys normal ; no signs of bacteria. Experiment 18: 
April 23, 1875. Both thighs inoculated with pure membrane, as in the foregoing 
experiment. 
April 25. Death between thirty and forty hours after inoculation. Autopsy.—Left thigh: 
Skin wound closed. At point of inoculation was a yellowish white mass, partly upon 
the surface of the muscle, and partly infiltrating the substance of the same, the 
overlying tissues adherent to one another and to the skin. The muscle itself, at the 
point of inoculation, was not much affected, but further away it was spotted with 
ecchymoses, and there was a large ecchymosis, one-half inch in diameter, on the 
back. Right thigh : Appearances generally similar, but less inflammation in the 
connective tissue, though more in the muscular. Blood from the right heart full 
of rod-bacteria, and the corpuscles much changed. Many of them had become 
adherent, forming irregular masses. Kidneys not congested. Trachea : Unnatur- 
ally red, but no membrane had formed. Microscopically, the yellow material 
from the sites of inoculation consisted of connective tissue stuffed with leucocytes and 
granular matter. 
Experiment 19: 
April 23, 1875. Both thighs inoculated with pure membrane, as in the two foregoing 
experiments. 
April 25. Dead between thirty and forty hours after inoculation. Autopsy: Skin-wounds 
closed on both sides. Subcutaneous connective tissue of thighs much thickened, 
and of a mottled yellow, red and white color. Small collections of yellow material 
at points of inoculation, and the adjacent muscular tissue much reddened. Appear- 
ances generally similar to those in Experiment 17. 
Case VI. 
May 2, 1875, received again from Dr. George W. Rachel a piece of diphtheritic mem- 
brane. The patient was a girl, two years old, sister to the patient of Case V. She was 
taken with vomiting, fever, and drowsiness, and on the following day, membrane formed upon 
the left tonsil and pharyngeal wall. It was detached the same day by forceps. No albumen 
in the urine, larynx not obstructed, recovery after four days’ illness. The specimen was sev- 
eral days old when received. It was found to be made up of a meshwork of the mycelium of 
a fungus stuffed with masses or “balls” of closely-packed bacteria, comparatively few leuco- 
cytes, a little fibrin, and a considerable quantity of large fat globules. After treatment with 
an alkali, the balls of seemingly spherical bacteria disappeared, and the field became crowded 
with rod-forms, free. 
a. Inoculation of muscular tissue with pure membrane. 
Experiment 20: * 
May 2, 1875. Both thighs of rabbit inoculated with pure membrane. 
May 3. Animal apparently well in the morning, but towards evening became languid 
and refused food. 22 
May 4. Found dead. Autopsy, at least fifteen hours after death : No extensive local 
lesions on either side. The piece of inoculated membrane had disintegrated, its 
leucocytes were undergoing fatty degeneration, and the compressed bacteria-balls 
had disappeared, giving place to a general infiltration of free rod-bacteria. Very 
little disturbance in the muscular tissue at the point of inoculation. Blood contained 
an abundance of rod-bacteria and granular matter, the latter dissolving on addition 
of caustic potassa solution. Kidneys contained bacteria. (N. B.—It will be 
remembered that this examination was at least fifteen hours after the death of the 
animal.) 
Experiment 21 : 
May 2, 1875. Both thighs inoculated with pure membrane, as in the foregoing. 
May 4. A vein in the ear was pricked, and the blood examined microscopically for 
bacteria, but none were found. 
May 19. Animal’s general condition pretty good. A lump could be felt under the skin 
at the point of inoculation on the right side. The skin was snipped, and the parts 
beneath inspected. The lump was a collection of yellowish-white material about the 
size of a pea; it had no connection with the skin-wound, which was healed. The 
material of this lump was found to consist of connective tissue stuffed with leuco- 
cytes, with a few large corpuscles containing good-sized oil-drops, a very moderate 
number of free rod-bacteria, and some granular matter. The substance had no 
offensive odor. 
May 23. Found dead. 
May 24. Autopsy.—Right thigh: The lump described above had diminished in size. 
Its substance was of the same composition, except that the bacteria had almost 
wholly disappeared. No new appearances since previous examination. Left thigh : 
Long, narrow collection of yellow material in the subcutaneous connective tissue, 
one-half inch long by one-tenth inch wide ; consists of enormous numbers of closely- 
packed fat-globules of various sizes, with a few corpuscles, themselves stuffed with 
oil-drops. No bacteria. Site of inoculation in the muscle could not be found. 
Skin-wound healed, and no hyperaemia or infiltration of tissues adjoining the adven- 
titious mass. Blood from right and left hearts free from bacteria. Internal organs 
all apparently healthy. 
: 
May 2, 1875. Both thighs inoculated with pure membrane, as in foregoing. 
May 21. Dead. Autopsy: Body greatly emaciated. Left thigh: Circular, thin layer 
of yellow material, inch in diameter, closely adherent to the outer surface of the 
aponeurosis of the muscle; material pulpy, and found to consist of leucocytes in an 
advanced stage of fatty degeneration, with a good deal of free oil-drops of various 
sizes. Beneath the aponeurosis, immediately under this layer, was a linear yellow 
streak about long, found to be almost entirely made up of closely packed 
minute oil-drops of various sizes. Right thigh : Yellow layer similar to above, but 
smaller, in the deep connective-tissue. In neither of these patches were there 
any bacteria, nor was there on either thigh any serous infiltration or hyperaemia of 
any of the tissues. Blood contained no bacteria. Internal organs apparently 
healthy. 23 
Experiment 23 : 
May 2, 1875. Both thighs inoculated with pure membrane, as in foregoing. 
May 7. Lump felt at point of inoculation on left side ; movable with the shin, and seemed 
to be about the size of a pea. On exposing the parts, a small, round, white, semi- 
solid mass, the size of a pea, was found lying in the loose subcutaneous tissue, over 
the thigh. The connective tissue immediately surrounding the mass was slightly 
thickened and reddened, forming a sort of capsule, but was not adherent to the 
aponeurosis of the muscle beneath. The mass was carefully dissected out, and bodily 
removed. No lesion of the aponeurosis of the muscle or of the muscle itself beneath 
the site of the tumor could be found. The material composing the tumor consisted 
of closely packed leucocytes undergoing fatty degeneration, free granular matter, 
free fat-globules, and sc moderate number of rod-bacteria in active motion. No 
“ball” or “nests” of bacteria. 
May 24. Animal somewhat poorly, but no lumps. 
July xo. Animal still living, but in poor condition. 
Experiment 24: 
May 2, 1875. Right thigh only inoculated with pure membrane, as in foregoing. 
June 10. Found dead. Autopsy: Body greatly emaciated. In inoculated side a small 
yellow mass inch in diameter; circumjacent skin and connective tissue thickened 
and reddened. Tumor composed of connective-tissue elements swollen and infiltrated 
with oil-globules. Wound of inoculation in the muscle wholly healed. Blood con- 
tained a very few rod-bacteria. Lungs normal. Liver moderately fatty. Kidneys 
showed a moderate number of small bodies apparently possessed of independent 
motion, and possibly bacteria. 
Case VII. 
May 21, 1875, received from Dr. Lewis W. Oakley, of Elizabeth, N. J., a piece of 
diphtheritic membrane detached from the throat of a child eight years old on the sixth day of 
the disease. When first seen, both tonsils, the uvula and most of the soft palate were already 
covered by membrane. There was no albumen in the urine, no marked symptoms of laryngeal 
obstruction, but decided and constant signs of constitutional infection. The case terminated 
in death fifteen days after it was first seen by the physician. The specimen of membrane was 
two days old when the following inoculations were made with it: 
a. Inoculation of the muscular tissue with pure membrane. 
Experiment 25 : 
May 21, 1875. Both thighs of a rabbit inoculated with the pure membrane. 
May 22. Animal well. 
June 8. Animal thin, but lively. The skin wound on the right side presented a small 
ulcer with puffy red sides. At site of inoculation a lump the size of a large pea 
could be felt. The skin was snipped, the yellow tumor pricked, and the exuding 
pasty matter examined. It consisted of degenerating leucocytes, granular debris, and 24 
a moderate number of dumb-bell-shaped bodies in dancing motion, apparently bac- 
teria. Left side : No lump felt. The skin was snipped and the parts beneath 
inspected. A shred of slightly thickened and yellowish connective tissue was found 
attached to the inner surface of the skin wound, and reaching toward the muscle. 
It was about a quarter of an inch long, and was found to consist of connective 
tissue elements stuffed with large oval sacs crowded with air-globules; no leuco- 
cytes ; no bacteria. 
June 13. Dead. Autopsy.—Right thigh: At site of inoculation, a cylindrical yellow 
mass connecting the skin and muscle-wounds, and ending in an enlargement the 
size of a hazel-nut; it consisted of a connective tissue wall, with soft, yellowish- 
white contents. The latter material was composed as above described, except that 
the bacteria had disappeared. Left thigh : Same appearance as at previous examin- 
ation. Lungs, liver, spleen, and larynx normal. Kidneys rather congested. 
Body greatly emaciated. 
Experiment 26 : 
May 21, 1875. Both thighs inoculated with pure membrane, as in foregoing. 
May 22. Animal well. 
Tune 8. Animal well. Both thighs presented lumps the size of a bean. That on the 
left thigh was pricked and the matter examined; it consisted of degenerating leuco- 
cytes and granular debris. 
June 21. Found dead in the morning. 
June 22. Autopsy: Body in good condition; fur sleek and clean. Right thigh : 
Yellow mass the size of a bean joining the skin and muscle-wounds, composed of 
fatty and granular leucocytes. Left thigh : Yellow mass extending from skin-wound 
into and through a thin thigh muscle, and communicating with a large lobulated 
yellow mass f-inch long by f-inch wide and deep, situated in the lax intra-muscular 
tissue. The material of this mass consisted of connective-.tissue elements, densely 
infiltrated with fat granules, and the usual granular leucocytes. Only here and there, 
in the matter from either thigh, could a motionless rod-bacterium be seen. 
Experiment 27 : 
May 21, 1875. Both thighs inoculated with pure membrane, as in foregoing. 
May 22. Animal well. 
May 28. Skin of right thigh was snipped and parts inspected. Yellowish-white mass 
connecting skin and muscle wounds. On opening, contents fluid, consisting of 
leucocytes, some free bacteria, and others collected into large masses. 
June 8. Lump still to be felt on right side as big as a bean. Pricked, yielded yellow, 
pasty matter composed of closely packed leucocytes in good preservation, granular 
debris; few bacteria. 
June 24. Animal in good condition ; cord-like thickening only to be felt ; not opened. 
July 7. Animal in good condition. 25 
b. Hypodermic injection of aqueous infusion of pure membrane after filtration through 
a porous clay cylinder. 
A piece of the membrane weighing six grains was cut up fine, thoroughly triturated with 
two fluid drachms of distilled water, and allowed to steep for twenty-four hours. The infusion 
was then poured into a clay cylinder, such as come for u«e in galvanic batteries. The closed 
end of this cylinder was covered with a rubber cap, by which communication was made with 
an exhausting syringe. By exhausting the air by means of the syringe, the fluid was drawn 
through the cylinder to the extent of eleven minims. This filtrate was perfectly clear ; and, 
examined microscopically, proved wholly free from bacteria or any kind of solid particles. 
Experiment 28: 
May 22, 1875. Three minims of above clear filtrate injected hypodermically into left 
thigh of a rabbit. 
May 28. Animal in excellent condition ; not the slightest sign of a lump to be felt. 
June 26. Excellent condition ; there has been no development of a lump. 
July 10. Excellent condition ; never any sign of a lump. 
Experiment 29 : 
May 22, 1875. Four minims of above clear filtrate injected hypodermically into right 
thigh of a rabbit. 
May 28. Excellent condition ; no sign of a lump. 
July 10. Excellent condition ; there has been no lump. 
Experiment 30: 
May 22, 1875. Four minims of above clear filtrate injected hypodermically into left 
thigh of a rabbit. 
May 28. Excellent condition; no sign of a lump. 
July 4. Dying. 
July 5. Dead. Autopsy: Not the slightest indication of there having ever been any 
effect produced at the site of the injection. Death probably from some other cause. 
Many animals died during the warm weather without having been experimented upon. 
c. Hypodermic injection of unfiltered aqueous infusion of pure membrane. 
The residue of the infusion used to obtain the filtrate above mentioned, was allowed to 
stand till the larger particles had settled. The turbid supernatant fluid, examined microscopi- 
cally, was found to contain small broken fragments of diphtheritic membrane, and to be swarm- 
ing wuth actively moving bacteria. 
Experiment 31 : 
May 22, 1875. Four minims of the above turbid infusion injected hypodermically into 
left thigh of a rabbit. 
May 28. Animal has been sickly. Lump the size of a pea felt at site of injection. Skin 
opened, and yellow tumor found adhering to the superficial fascia. It was pricked, 
and the pasty contents examined microscopically: found to consist of leucocytes and 
granular debris, with very few bacteria. 26 
June 26. Animal in excellent condition. The lump seems to have diminished in size. 
July 10. Animal in excellent condition. 
Experiment 32 : 
May 22, 1875. Four minims of turbid infusion injected in right thigh of rabbit. 
May 28. Animal in good condition ; lump felt under skin. 
June 8. Lump, size of a large pea, felt freely movable under skin at site of injection. 
Skin snipped; tumor pricked, and yellow contents examined. Consisted of leucocytes 
in good preservation, and granular debris; only an occasional bacterium. Lump 
left in place. Animal in good condition, though badly bitten by mate. 
July 1. Lump increased to size of an almond-kernel. Pricked, and contents examined, 
consisted of corpuscles undergoing fatty and granular degeneration. No bacteria. 
July 10. Animal in good condition. Lump shrunk to size of a split pea. 
Experiment 33 : 
May 22, 1875. Five minims of turbid infusion injected into left thigh of a rabbit. 
May 28. Animal in good condition; lump felt under skin. Skin snipped, and parts 
explored. A yellow patch, half an inch long, and a quarter wide, found in the 
superficial fascia. When cut into, it proved tenacious and elastic ; no matter could 
be squeezed from the cut surface. Material found to consist of connective tissue, 
stuffed with granular matter, a moderate number of leucocytes and cells full of oil- 
globules. A very limited number only of bacteria could be found. 
July 1. Animal in fair condition; has been shedding his hair. Lump felt to have 
shrunk considerably in size. 
July 10. Animal in good condition. 
Case VIII. 
May 29, 1875, received from Dr. A. S. Church, a piece of diphtheritic membrane, 
detached from the throat of a patient, on the sixth day of the disease. The patient, a boy 
nine years old, was taken with a chill, followed by fever. The next day, membrane was found 
on the tonsils; on the following day, on the uvula also, and the day after that, also, in the 
nares. The larynx did not become obstructed, but there was constitutional infection, and the 
patient died on the ninth day of the disease. The specimen was examined and used two days 
after it was received, being in the mean time kept wrapped in a damp cloth, protected by 
rubber tissue. It was found to consist of a fibrinous exudation containing a moderate number 
of leucocytes, multitudes of oil-globules, with very few bacteria. 
a. Inoculation of muscular tissue with pure membrane. 
Experiment 34: 
May 31, 1875. Both thighs of a rabbit inoculated with the pure membrane. 
June 26. Animal in good condition. Right thigh : a small lump. Left thigh : small 
open abscess, discharging freely. Matter found to consist of normal pus, with only 
a small amount of granular matter, and an occasional bacterium. 
July 8. In good condition. 27 
Experiment 35 : 
May 31, 1875. Both thighs inoculated with the pure membrane. 
June 7. Animal died during forenoon. Autopsy at 2 P. M.—Right side : At site of 
skin-wound a scab % inch in diameter closely adherent to underlying tissues. On 
exposing the subcutaneous tissue a flat layer of yellow material was found reaching half 
down the thigh, backwards over the rump, and upwards to the median line of the back 
where it met a similar exudation from the opposite side. This plaque was quite 
closely adherent to the skin. The muscle at the site of inoculation was gangrenous 
over an area ]/2 inch by Ja- Adjacent to this area the muscle was mottled red and 
yellow, and at one point a sharply defined yellow line of demarcation separated the 
diseased from the healthy muscular tissue. Microscopic examination showed the 
material of the yellow plaque to consist of a fibrinous meshwork, containing numer- 
ous deformed corpuscles about the size of leucocytes and a large quantity of granular 
matter. The yellow spots in the muscular tissue contained oil-globules and smaller 
granules strung together, so as to give the appearance of dumb-bells or chains. Liver 
large and containing echinococcus cysts. Kidneys swollen, soft; tube-cells inclined 
to be fatty. In the debris were similar chains and figures-of-eight to those seen in 
the muscular tissue. Blood contained a few of the same elements. Lungs normal. 
Experiment 36: 
May 31, 1875. Both thighs inoculated with pure membrane. 
June 4. Dead 96 hours after inoculation. Autopsy.—Left thigh: Yellow patch f inch 
in diameter in subcutaneous connective tissue. Surrounding this, a white streak 
extending to the median line of the back. Beyond this again, a general reddening 
of the tissues for the space of half an inch, and considerable serous infiltration. 
Thigh itself, mottled with red spots. Material of yellow patch found to consist of 
fibrous network holding leucocytes, abundance of granulaf matter and a few motion- 
less bacteria. What looked like balls of bacteria found in the blood-vessels in the 
muscular tissue at the site of inoculation. Right thigh : Small yellow patch over- 
lying muscle-wound, with surrounding serous infiltration of the subcutaneous 
connective tissue. The muscle-wound was lined with a dirty, friable, yellowish- 
white material, and the muscular tissue below was transformed into a soft, cheesy 
t matter, found to consist of muscular fibres in advanced fatty degeneration, with here 
and there a motionless bacterium. For an area an inch in diameter around this 
affected spot the muscular tissue was reddened. Liver and kidneys rather red and 
swollen. Blood contained no bacteria. 
b. Hypodermic injection of aqueous infusion of pure membrane after filtration through 
porous clay. 
A piece of the membrane was triturated with water, infused for twenty-four hours and then 
filtered through a porous clay cylinder in the manner described in “ b ” case VII. (see ante, 
p. 681). The filtrate was perfectly clear and free from solid elements. 
Experiment 37 : 
June 1, 1875. Five minims of the clear filtrate injected into each thigh of a rabbit. 28 
June 5. Animal in excellent health and fat. Skin snipped at site of each injection and 
parts beneath thoroughly explored. Not the slightest sign of any lesion, nor even 
hypereemia. 
June 8. Died during the night. Autopsy : No lesion whatever discoverable. Was a 
young animal, and death was probably unconnected with the experimentation. 
Experiment 38 : 
June 1, 1875. Six minims of the clear filtrate injected into each thigh of a rabbit. 
June 5. Animal in excellent condition. Examination made as in foregoing, but nothing 
whatever found. 
June 11. Animal got out of his cage and fell a considerable height, breaking his leg. 
June 12. Died about noon. Autopsy two hours later : Body in excellent condition. 
No lesions whatever to be found at sites of the injections. Larynx, lungs, liver, 
normal. Blood from right heart contains no bacteria. Death was probably due to 
injury from the fall. 
c. Hypodermic injection of unfiltered aqueous infusion of pure ?nembrane. 
The residue of the infusion used to obtain the filtrate of “b” was allowed to stand till 
the larger particles settled ; the turbid supernatant fluid was then taken for the injection. 
Experiment 39 : 
June 1, 1875. Six minims of the turbid infusion injected into the right thigh, and eight 
into the left, of a rabbit. 
June 3. Died in convulsions at 3.30 P. M., about forty-eight hours after the injection. 
Autopsy half an hour later.—Right thigh: General serous infiltration of connective 
tissue, area of thickened connective tissue two inches in diameter, inclosing a yellow 
patch by of an inch in area. Interior of this patch was semi-fluid yellow mat- 
ter made up of granular matter, and great numbers of dumb-bell and rod-shaped 
bodies looking like degenerate bacteria, but not in motion. Underneath this patch 
the aponeurosis of the muscle was cloudy and thickened, but the muscle itself was 
not affected. Left thigh : No patch, but intense congestion of the tissues over the 
thigh and of the muscle itself to the depth of Jg inch with great serous infiltration 
of the subcutaneous connective tissue. Liver large, smooth, and congested, 
Kidjieys congested. Lungs, larynx, and heart, normal. Blood from right ventri- 
cle contains no bacteria. 
Experiment 40: 
June 1, 1875. Six minims of the turbid infusion injected into each thigh of a rabbit. 
June 4. Dead. Autopsy: Subcutaneous connective tissue over whole aspect of both 
thighs intensely congested, and skin unnaturally adherent. Where the injection had 
been delivered the connective tissue was infiltrated with specks of yellow material, 
found to be composed of debris of corpuscles and innumerable ordinary rod-bacteria. 
The appearance suggested that these specks were the solid portions of the original 
infusion as injected. Extensive serous infiltration over buttocks and between the 
muscles of the thighs. Aponeuroses, and muscles themselves, for the depth of two 
lines, and over an area of an inch and a half in diameter, intensely congested. Blood 
of both sides of the heart contained moderate number of minute rod-bacteria. Case IX. 
June 2, 1875, received from Dr. Harvey S. Gaya fine, fresh specimen of diphtheritic mem- 
brane, one-eighth of an inch thick. It was composed of fibrinous material containing few, if 
any, leucocytes, multitudes of minute oil-globules, but no actively moving bacteria. 
No history of the case was furnished. 
a. Inoculation of muscular tissue with pure me7tibrane. 
Experiment 41 : 
June 2, 1875. Both thighs of a rabbit inoculated with the pure membrane. 
July 1. Lump on each side as big as a filbert. That on the left side has ulcerated and 
is discharging yellow matter. The material of both composed of leucocytes, granu- 
lar matter and a few rod-shaped and ball-headed bacteria. 
July 10. Animal in excellent condition. 
b. Hypodermic injection of aqueous infusion of pure membrane after filtration through 
porous clay. 
An aqueous infusion of the membrane was made and filtered the same day through the 
porous clay cylinder. (See ante, page 68r.) The filtrate was perfectly clear. 
Experiment 42 : 
June 2, 1875. Five minims of the clear filtrate injected into the right thigh, and four 
minims into the left, of a rabbit. 
June 22. Animal somewhat thin but healthy; fur clean ; feeding well ; no lumps to be 
felt. 
June 29. Found dead. 
June 30. Autopsy: Body in good condition. No sign of a lesion discoverable. 
(N. B.—The weather has been intensely hot.) 
Experiment 43: 
June 2, 1875. Six minims of the clear filtrate injected into the right thigh, and five into 
the left, of a rabbit. 
June 22. Animal somewhat thin but healthy ; no lumps to be felt in either thigh. 
July 10. Animal in good condition. 
c. Hypodermic injection of unfiltered aqueous infusion of pure membrane. 
The turbid infusion of “ b,” after the heavier particles had settled, was used for the injec- 
tions. 
Experiment 44 : 
June 2, 1875. Six minims of the turbid infusion injected into each thigh of a rabbit. 30 
June 26. Animal in good condition. On each side, a lobulated hard lump, as big as a 
filbert, apparently not connected with the muscular tissue. Pricked, a yellowish 
white curdy matter exuded, found to be composed of coarsely granular leucocytes, 
granular matter and rod-bacteria. 
July 10. Animal in good condition. 
Experiment 45 : 
June 2, 1875. Six minims of the turbid infusion injected into each thigh of a rabbit. 
June 5. Animal died during night. Autopsy.—Right thigh: General congestion of 
connective tissue and aponeurosis of thigh-muscles, with presence of extensive clots 
of blood, apparently from injury to a vessel by the hypodermic needle. Atone spot, 
a circle of yellow infiltration of the connective tissue half an inch in diameter. 
Through the aponeurosis underlying this patch, a small hole could be traced, and 
on exposing the muscle beneath, the latter presented a ring of yellow infiltration 
corresponding in position with that of the patch in the connective tissue above, in the 
centre of which was a grayish, sloughing cavity. Apparently, the needle had forced the 
muscular tissue. The yellow infiltration of the tissues contained abundant leucocytes 
and living rod-bacteria, and the gray pultaceous muscular slough was composed of 
muscular tissue undergoing fatty degeneration, and swarming with living rod-bacteria. 
Left thigh : Appearance the same except that there was no lesion of the muscle. 
Blood, from both sides of the heart, contained no bacteria. 
d. Hypodermic injection of aqtieous infusion of pure membrane ten days old and putrid. 
The unfiltered infusion of uc” was allowed to stand for ten days. Evaporation was 
compensated for by adding two fluid drachms of distilled water. The infusion was now 
thoroughly putrid, being a turbid brown fluid, having an extremely offensive smell, and 
swarming with actively moving bacteria. 
Experiment 46: 
June 12, 1875. Six minims of the putrid infusion injected into each thigh of a rabbit. 
June 13. Found dead on the floor, having escaped from its cage and fallen a considerable 
height. Autopsy: Body limp; hind legs have an offensive smell, but not the fore- 
legs. Left thigh: No lesion except faint pinkish discoloration in connective tissue 
at site of injection. A bit of the lax connective tissue examined microscopically, 
appeared normal, except that there were numerous rod-bacteria to be seen. Right 
thigh : Same condition, bacteria far more numerous. Liver : Large clot on under 
surface; organ passively congested. Kidneys : Congested. Blood of right heart fluid 
and containing an exceedingly small number of minute rod-bacteria. 
Experiment 47 : 
June 12, 1875. Six minims of the putrid infusion injected into each thigh of a rabbit. 
July 10. Animal in good condition. Skin snipped over both thighs and parts explored. 
Right thigh: Small flattened sac, about j£~inch in diameter, covered with newly 
formed vessels, found in the subcutaneous connective tissue. Sac when opened, 
found to contain about a drop of soft yellowish material, composed of leucocytes, 
flattened corpuscles stuffed with oil-globules, and an immense amount of free oil- 
globules aggregated together here and there into chains. No bacteria. Left thigh : 
Appearances same, but more free oil and a few motionless rod-bacteria. 31 
e. Hypodermic injection of putrid infusion of pure membrane, after filtration through 
paper (.single filter). 
The putrid infusion of “ d” was filtered through a single ordinary paper filter. The 
filtrate was clear, but of a yellow color, foul smelling, and contained a few actively-moving 
bacteria in each microscopic field. 
Experiment 48: 
June 12, 1875. Six minims of the filtrate injected into each fore leg of a rabbit. 
June 24. Animal in excellent condition; no lump to be felt in either leg. 
July 10. Animal in excellent condition. 
Experiment 49: 
June 12, 1875. Six minims injected into each thigh of a rabbit. 
July 10. Animal in excellent condition. No lump to be felt in either thigh. 
f. Hypoder7tiic injection of putrid inftision after boiling. 
Some of the putrid infusion of “dn was boiled in a test-tube for three minutes and 
allowed to cool. All the contained bacteria were then motionless. 
Experiment 50: 
June 12, 1875. Six minims of the boiled infusion injected into each thigh of a rabbit. 
June 18. Found dead. Autopsy: Body emaciated. Right thigh: Small, rather 
thick, patch, one-third by one-eighth inch in area, in the connective tissue at site of 
injection. Material composed of large flattened corpuscles filled with oil-globules 
and granules, but no bacteria; the muscle beneath in a state of fatty degeneration. 
Left thigh : Soft, yellow patch in connective tissue at sight of injection, the size of 
a three-cent piece. Composition same as above. 
Experiment 51 : 
June 12, 1875. Six minims of the boiled infusion injected into each thigh of a rabbit. 
June 29. Found dead. No autopsy. 
Case X. 
June 5, 1875, received from Dr. W. H. Vermilye a piece of diphtheritic membrane 
detached by coughing on the seventh day of the disease. The patient, a girl six years old, 
was attacked with what at first seemed to be a simple follicular tonsillitis. On the sixth day 
the tonsils, palate, and pharynx became covered with a thick exudation, which peeled from 
the palate quite easily, leaving a bleeding surface behind. The next day the same surfaces 
became again covered, but thinly and in patches. Some of these patches were pulled upon 
and loosened, and immediately afterward the sample furnished was detached and discharged 
by expectoration. The patient had already begun to improve, and without having had any 
symptoms of laryngeal obstruction or of constitutional infection, made a good recovery after 
two weeks illness. Albumen was detected in the urine on the twelfth day, but the urine was 
not examined regularly, the patient being a dispensary case. Two sisters of the patient were 
also attacked, but with them the disease was of even a milder type. The specimen was 32 
received wrapped in moist rag, encased in oiled silk. It was not opened till the second day 
after receipt. It was then yellowish-red, soft, and pultaceous, and had a putrid smell. 
Microscopically, it was found to consist of corpuscles of various forms matted together and 
infiltrated with dense masses'of bacteria, which swarmed in the field as free rods of various 
sizes. 
a. Hypodermic injection of aqueous infusion of pure membrane. 
Eighteen grains weight of the membrane was ground up with three fluid drachms of water 
and infused one hour. The turbid, supernatant fluid, after settlement of the larger pieces of 
membrane, was used for the injections. It had no smell. 
Experiment 52 : 
June 7, 1875. Six minims of the turbid infusion injected into each thigh of a rabbit. 
June 10. Died in the previous night. Autopsy in the afternoon : Subcutaneous con- 
nective tissue over whole region of each thigh hyperaemic, cedematous, and diffusely 
yellow. Yellow spots, composed of connective-tissue densely stuffed with bacteria 
in closely-packed masses. Swarms of bacteria, both dumb-bell and rod forms, 
actively moving about in the field of the microscope. Blood, of right heart, con- 
tained no bacteria. Kidneys: Tubes contained a great deal of fat in small granules. 
In the free fluid were numerous bodies, in pretty active motion, resembling bac- 
teria. Spleen: Normal. Liver: Quite fatty. 
Experiment 53: 
June 7, 1875. Six minims of the turbid infusion injected into each thigh of a rabbit. 
June 10. Died in the afternoon. Autopsy immediately : Both thighs in same condi- 
tion in all respects as foregoing, only the yellow matter in this case contained, in 
addition to the elements above described, many leucocytes in a state of degeneration. 
Blood from right heart contained no bacteria. Liver: Cells slightly fatty. Kidneys : 
Tubes normal; no bacteria. 
Experiment 54 : 
June 7, 1875. Six minims of the turbid infusion injected into each thigh of a rabbit. 
June 10. Died during previous night. Autopsy in the afternoon.—Right thigh : Skin 
at site of injection gangrenous for an area as large as a three-cent piece ; great infil- 
tration of subcutaneous connective tissue. Muscular tissue for an area two inches in 
diameter reddened by small punctate spots; the tissue at these spots found to consist 
of broken down muscular fibres, but with no bacteria present. Left thigh : Appear- 
ances similar, except in addition a thin, yellow patch in the subcutaneous connective 
tissue, found to consist of connective-tissue elements, with leucocytes, large cells con- 
taining oil-globules and vast numbers of bacteria in active motion. Bacteria were 
also found among the muscular fibres in the affected spots in the muscle. Blood 
from right heart contained a moderate number of bacteria. Kidneys : Normal, but 
fluid scrapings from a cut surface contained a large number of free bacteria. Liver : 
Large and fatty. Spleen : Normal. 33 
b. Hypodermic injection of aqueous infusion of pure membrane after filtration through 
paper (double filter). 
The infusion of “ a ” was filtered through a double filter of ordinary filtering-paper, 
made by inserting one filter within another, so that four thicknesses of paper were everywhere 
presented to the fluid. The filtrate was clear and odorless, but of a yellow color. 
Experiment 55 : 
June 7, 1875. Six minims of the clear filtrate injected into each thigh of a rabbit. 
July 1. No sign of a lump to be felt on either thigh ; no adhesion of skin to parts, 
beneath ; animal fat and healthy. 
July 10. Animal in good condition. 
Experiment 56 : 
June 7, 1875. Six minims of the clear filtrate injected into each thigh of a rabbit. 
July x. No sign of a lump to be felt on either thigh. Animal in excellent condition. 
July 10. Animal in good condition. 
Experiment 57 : 
June 7, 1875. Six minims of the clear filtrate injected into each thigh of a rabbit. 
July 1. Animal thin, but apparently healthy. Right thigh : No lump to be felt. Left 
thigh: Large lump, the size of a pullet’s egg. Pricked, was found to contain the 
usual yellow creamy matter, composed of malformed leucocytes, granular matter, 
with some moving bacteria. 
July 10. Animal in good condition. 
Case XI. 
June 19, 1875, received from Dr. Alexander Buchanan a small scrap of membraniform 
material taken from the throat of a scarlet fever patient, on the ninth day of the disease. 
The patient, a boy six years old, was first seen by Dr. B. on the eighth day of 
the disease. The pulse was then 188, temperature 104° Fahr. ; there was enormous 
swelling of the parotids, and from the tonsils upwards to the posterior nares the 
mucous membrane was covered with exudation. The small piece furnished was removed 
the next day. .Patient died on the fourteenth day, having from the first refused all 
nourishment. The specimen of membrane was about x-x6 inch thick, greenish yellow in 
color, and so friable as to be easily broken into pieces. Microscopically, the material was 
composed, in greatest part, of the thalli and spores of a fungus, apparently penicilium glaucum. 
Rod-bacteria and deformed, flattened corpuscles, probably lymphoid cells, were also present 
in considerable number. There was only enough of the material for the inoculation of two 
animals. 34 
a. Inoculation of muscular tissue with membrane from scarlet fever patient. 
Experiment 58: 
June 20, 1875. Both thighs of a rabbit inoculated with the solid membrane. 
July 4. Dying. Right thigh showed a small lump, containing cheesy material, as in 
cases where pure diphtheritic membrane was inoculated. 
Experiment 59 : 
June 20, 1875. Both thighs of a rabbit inoculated with the solid membrane. 
July 10. Animal in good condition, but somewhat thin. Left thigh : Small lobulated 
yellow tumor in the connective tissue, at the site of injection. Mass composed of 
deformed leucocytes, large, flat corpuscles, containing fat and granular debris. No 
rod-bacteria. Right thigh: No lump to be felt. 
Case XII. 
June 19, 1875, received from Dr. A. S. Church a second piece of diphtheritic membrane. 
The patient, a boy sixteen months old, was taken with fever and convulsions. Dr. C. saw 
him first on the third day, and membrane was then already present upon the tonsils and 
uvula. It subsequently appeared in the nose. Small pieces were detached on the sixth day, 
and larger ones on the ninth. There was no obstruction of the larynx, but there were con- 
stitutional symptoms, with albumen, and granular and blood-casts in the urine. Recovered, 
after three weeks’ illness. The specimen furnished was a broad, leathery strip, over two 
inches long, nearly an inch wide, and from 1-16 to 1-8 inch thick. It was made up of a dis- 
tinctly fibrillated network, with flattened corpuscles, containing oil-drops. Numerous bacteria 
were also present. 
a. Inoculation of muscular tissue with pure membrane. 
Experiment 60 : 
June 20, 1875. Both thighs of a rabbit inoculated with pure membrane. 
June 26. Animal thin, but healthy. No lumps to be felt at sites of inoculation. 
July 10. Animal in good condition. 
Experiment 61 : 
June 20, 1875. Both thighs of a rabbit inoculated with pure membrane. 
June 26. Animal thin, but healthy. No lumps to be felt. 
July 8. Killed by a fall from a considerable height. 
July 9. Autopsy.—Left thigh: Small lump. Right thigh : Nothing to be found. 
Case XIII. 
January 21, 1876, received from Dr. Francis H. Weismann a piece of diphtheritic mem- 
brane, coughed up by a patient on the sixth day of the disease. The patient, a boy eleven 35 
years old, was taken with slight chills and cough, followed by swelling of the glands and high 
fever. On the fourth day membrane formed in the throat, involving the cavity of the mouth 
and extending into the larynx, producing obstruction to breathing. Pieces were first coughed 
up on the sixth day, and from time to time many large pieces were thus detached. The prog- 
nosis was bad from the first, the urine being slightly albuminous and constitutional symptoms 
present. A small cut on the finger became covered with membrane. Paralysis was already 
beginning in the right fore-arm when the patient died, on the fourteenth day of the disease. 
Others of the same family were beginning to show symptoms of diphtheria when this boy 
died. The specimen of membrane was examined two days after detachment. It was then 
firm, tenacious, of a yellowish-white color, and was made up principally of the interlacing 
filaments of the mycelium of a fungus. There were also numerous minute spherical bodies, but 
no rod-bacteria. 
a. Inoculation of muscular tissue with pure membrane. 
Experiment 62: 
January 22, 1876. Both thighs of a rabbit inoculated with the pure membrane. 
January 25. Found dead in the morning. Animal had partly succeeded in getting out 
of his cage. 
January 26. Autopsy in the afternoon.—Right thigh : Spreading from site of inocula- 
tion, a diffuse inflammation, the subcutaneous connective-tissue being infiltrated with 
yellow pasty material, and the veins of the region congested. The appearances, in 
short, were the same as have been so often noted, only the lesions were not severe. 
The yellow material was composed of swollen, distorted corpuscles, with granular 
debris. Left thigh : Diffuse inflammation of connective-tissue, and fatty degenera- 
tion of the muscle at site of inoculation. The strings of fat granules between the 
muscular fibres looked much like round bacteria, but many were too large, and others 
were dissolved by even a weak solution of caustic potash. Viscera normal. 
Experiment 63: 
January 22, 1876. Both thighs of a rabbit inoculated with pure membrane. 
January 24. Found dead in the morning. Autopsy: Microscopic appearances same as 
in foregoing case. No microscopic examination made 
b. Inoculation of muscular tissue with mixture of pure membrane and salicylic acid. 
Fourteen grains weight of the membrane was triturated with fourteen drops of water, 
containing in solution two per cent, of salicylic acid and six per cent, of sodic phosphate. 
Next, five grains weight of salicylic acid itself was added, and the whole mixture thoroughly 
triturated in a mortar for half an hour. 
Experiment 64 : 
January 22, 1876. Both thighs of a rabbit inoculated with the mixture of membrane 
and salicylic acid. 
January 26. Animal in good condition. 
January 29. Animal in good condition. 
February 22. Killed in a fight with another rabbit. There were lumps found at the site 
of inoculation. 36 
Experiment 65 : 
January 22, 1876. Both thighs of a rabbit inoculated with the mixture of membrane 
and salicylic acid. 
January 29. Animal in good condition. 
February 22. Animal in good condition. 
February 25. Animal in good condition. 
March 11. Animal in very good condition. Very small lumps to be felt on both sides 
at sites of inoculation. 
Experiment 66: 
January 22, 1876. Both thighs of a rabbit inoculated with the mixture of membrane 
and salicylic acid. 
January 26. Animal in good condition. 
February 23. Animal in good condition. Small lumps to be felt on both sides at sites 
of inoculation. 
March 11. Animal in fair condition, though rather thin. Lumps still to be felt. 
Case XIV. 
February 25, 1876, received from Dr. William E. Bullard two pieces of diphtheritic 
membrane. The patient, a boy six years old, had had enlarged tonsils for some weeks. Next, 
the whole pharyngeal surface became very red, and the following day membrane formed upon 
both tonsils, uvula, and anterior nares. Four days later much of the membrane came awayf 
leaving the left nostril and one of the tonsils free. At this date the temperature was high, 
the pulse irregular, and the prostration great. But, in six days more, all signs of membrane 
had disappeared, and the patient recovered after an illness, in all, of thirteen days. A sister 
of this boy was in the fourth day of an attack of diphtheria when he himself first fell ill. 
The larger specimen of membrane was about one-eighth inch thick, soft, friable, and of a 
grayish-yellow color. It consisted of a fibrillated matrix containing large numbers of corpus- 
cles of various sizes, and also numerous bacteria. The smaller specimen was still more 
distinctly fibrillated; contained large numbers of granules, but few bacteria. 
a. Inoculation of muscular tissue with pure membrane. 
Experiment 67 : 
February 25, 1876. Both thighs of a rabbit inoculated with pure membrane. 
February 28. Animal in good condition. 
March 11. Ulcers at sites of inoculation, discharging freely. Animal fat, and seemingly 
in good condition. 
Experiment 68 : 
February 25, 1876. Both thighs of a rabbit inoculated with the pure membrane. 
February 28. Animal in good condition. 37 
March n. Animal very weak but has good appetite. .Open ulcers at sites of inocula- 
tion, discharging freely an offensive matter. 
March 19. Dead. Autopsy: Body greatly emaciated. Abscesses over both thighs, 
extending into the muscular tissue; contents consisting of pus-corpuscles, large gran- 
ular corpuscles and granular matter, but no bacteria. 
Experiment 69 : 
February 25, 1876. Both thighs inoculated with pure membrane. 
February 28. Animal in good condition. 
March 11. Animal thin, but pretty active. Coat rather dirty and rough. Ulcers at 
sites of inoculation discharging freely an offensive matter. 
June 13. Animal in good condition. Ulcers healed ; no lumps or thickening to be felt. 
b. Inoculation of muscular tissue with mixture of pure membrane and salicylic acid. 
Seven grains weight of membrane was treated first with salicylic acid solution, and then 
with the solid acid in exactly the same manner and proportion described in the last case. (See 
ante, page 691.) 
Experiment 70: , 
February 25, 1876. Both thighs of a rabbit inoculated with the mixture of membrane 
and salicylic acid. 
February 28. Animal in good condition. 
February 29. Found dead at 3 p. M. 
March 2. Autopsy: Moderate sized collections of yellow material at sites of inocula- 
tion in the muscles, but not much inflammation of surrounding tissues. 
Experiment 71 : 
February 25, 1876. Both thighs of a rabbit inoculated with the mixture of membrane 
and salicylic acid. 
February 28. In good condition. 
March 11. Found dead. Autopsy.—Left thigh : Large patch at site of inoculation ; 
matter pasty in consistence and of a rather reddish tinge. Considerable inflamma- 
tion of surrounding tissues. Internal organs apparently healthy. Body much 
emaciated. 
Experiment 72: 
February 25, 1875. Both thighs of a rabbit inoculated with the mixture of membrane 
and salicylic acid. 
February 28. Animal in good condition. 
March 11. Large lumps on both sides at sites of inoculation. Had begun to ulcerate 
and discharge externally. Contents white, creamy and almost odorless. II.—INOCULATION OF SCRAPINGS FROM THE HEALTHY HUMAN 
TONGUE. 
a. Inoculation of muscular tissue with pure tongue-scrapings. 
The upper surface of a healthy human tongue with an habitual tendency to be “ furred,” 
was thoroughly scraped with a knife. The pulpy yellowish-white scrapings consisted of the 
ends of the filiform papillae covered by epithelium, with the latter in turn densely covered and 
infiltrated with packed layers of bacteria. The scrapings were used at once for inoculation. 
Experiment 73 : 
April 29, 1875. One thigh of a rabbit inoculated with fresh tongue-scrapings. 
May 3. Dead. Autopsy: Large lump of dirty yellow color at site of inoculation; 
material containing free bacteria and also round balls or nests of cloudy packed bac- 
teria exactly similar to those seen in diphtheritic membrane. Blood contained 
only a few bacteria. 
Experiment 74 : 
April 29, 1875. Both thighs of a rabbit inoculated with fresh tongue-scrapings. 
May 4. No effect observed. 
May 7. Lump felt at site of inoculation on one side. Skin snipped and parts explored. 
A flattened yellow tumor, the size and shape of a kidney-bean, found in the connec- 
tive-tissue, immediately over the muscle-wound. By pricking its thickened wall of 
connective-tissue, a white pasty substance was obtained, found to consist of broken 
down leucocytes, some rod-bacteria and an immense amount of granular matter. No 
balls of bacteria were to be seen. Tumor left in situ. 
May 17. Very considerable general swelling of thigh and leg on the left side, and fluc- 
tuation detected over this whole area as far, even, as the toes. Animal evidently 
sickly. 
May 24. Dead. Autopsy.—Right thigh: An extensive cavity, whose contents had 
been discharged by ulceration, found under the skin, extending over the whole area 
of the thigh. The skin itself formed the outer wall of the sac, and internally the 
muscles and periosteum had disintegrated so as to expose nearly the whole length of 
the femur, and part of the ilium, denuded, at the bottom of the sac. Left thigh ; 
A large livid, flattened, fluctuating tumor, covering the whole area of the thigh and 
leg, and reaching to the toes. Tumor consisted of a wall of thickened connective- 
tissue enclosing a dirty, yellowish, semi-fluid material, consisting of deformed leuco- 
cytes, granular matter, and large numbers of actively moving bacteria. Examina- 
tion of abdomen showed general peritonitis, loops of intestine being adherent to the 
abdominal wall and to each other by fresh, moderately firm, adhesions. Liver: 
Deeply congested, anda containing echinococcus ova. Lungs: Deeply congested. 
Blood: From right heart, vena cava, and from lungs, liver, and kidney contained a 
moderate number of rod-bacteria of larger size than common, but none of these ele- 
ments were found in blood taken from the left heart. 39 
Experiment 75 : 
April 29, 1875. Both thighs of a rabbit inoculated with fresh tongue-scrapings. 
May 4. Animal ing ood condition; small lumps to be felt at each site of inoculation. 
Pricked, and contents found to be a yellow pasty material, composed of broken 
down leucocytes, granular matter, actively moving rod-bacteria, and balls of closely- 
packed bacteria. 
May 7. Appearances same in all respects, but in addition there are open ulcers at the 
sites of the skin-wounds. 
May 12. Dead. Body considerably emaciated. The lumps have increased a good deal 
in size. 
b. Inoculation of muscular tissue with pure tongue-scrapings. 
Tongue-scrapings obtained in the same way as in “a,” but twenty-four hours after removal, 
were used for the following inoculations. The material had already acquired an offensive 
smell. 
Experiment 76: 
May 6, 1875. Both thighs of a rabbit inoculated with pure tongue-scrapings. 
May 7. Skin over right thigh snipped and parts explored. Oblong yellow tumor, one 
inch long by three-eighths of an inch wide, found connecting the skin and muscle- 
wounds. On opening the tumor, the contents were found to be a yellow pasty 
material, made up of leucocytes and enormous multitudes of bacteria. Debris of 
the inoculated material could also be found in the mass. The entire tumor, walls 
and all, was carefully dissected out and bodily removed. 
June 24. Animal in good condition. No lumps to be felt in either thigh. 
July 10. Animal in good condition. 
Experiment 77 : 
May 6, 1875. Both thighs of a rabbit inoculated with pure tongue-scrapings. 
May 9. Skin of right thigh snipped and parts explored. Skin-wound closed; yellow 
oblong tumor connecting skin and muscle-wounds; contents white and semi-fluid 
containing myriads of bacteria. A drop of blood, obtained by pricking a vein in 
the ear, showed no bacteria. 
May 25. Dead. Autopsy.—Right thigh: The above-mentioned tumor found to 
measure one inch by one-eighth inch in area; material consisted of leucocytes 
undergoing fatty degeneration, and a great deal of free fat in globules, but no 
bacteria were now to be seen. Left thigh: The whole muscular tissue was infil- 
trated with distinct collections of yellow, semi-fluid material, consisting of leucocytes 
undergoing fatty degeneration, and a moderate number of rod-bacteria, the latter 
being most numerous in the foci nearest the skin-wound. Blood contained no 
bacteria. 40 
Experiment 78: 
May 6, 1875. Both thighs of a rabbit inoculated with pure tongue scrapings. 
May 10. Skin of left side snipped and parts explored. Yellowish-white tumor an 
inch long connecting skin and muscle-wounds; contents, leucocytes undergoing fatty 
degeneration and a moderate number of free bacteria. No “ balls ” of bacteria. 
May 19 Collections of yellowish matter in both thighs which have ulcerated externally 
and have been discharging. Site of ulcers now covered by scabs. Discharged 
matter consisted of moderate number of leucocytes and abundant and active bacteria. 
May 31. Dead. 
June 1. Autopsy.—Left thigh: Multilocular tumor in the subcutaneous connective- 
tissue, as large as a large filbert; not attached to the aponeurosis of the muscle, 
. nor could the muscle wound be found. Contents of tumor white and semi-fluid, 
consisting of leucocytes undergoing degeneration, granular debris, and shreds of dis- 
organized connective-tissue containing large numbers of minute bacteria. Right 
thigh: Presented nothing abnormal except a slight thickening of the connective-t:ssue 
immediately underlying the skin at the site of the original wound. The visceral 
cavities and organs were healthy. Blood: From both sides of the heart showed 
crenation of the red corpuscles, and a massing of the white into groups of from six to 
thirty. No more bacteria observable than is usual in the blood of animals thirty 
hours dead. 
Experiment 79: 
May 6, 1875. Both thighs of a rabbit inoculated with pure tongue-scrapings. 
May 10. Animal sick. 
May 11. Dead. Autopsy between 12 and 17 hours after death.—Left thigh : Large 
flat collection of white pasty material in the subcutaneous connective-tissue, an inch 
in diameter ; material composed of granular leucocytes, free granules and rod- 
bacteria in considerable number. At site of muscle-wound the muscular tissue 
showed close lines of white glistening aspect running parallel to the direction of the 
muscular fibres. These consisted of the sarcolemma of muscular fibres filled with 
closely packed granular debris in place of the usual sarcous substance. Brightly 
shining spherical bodies (too glistening for bacteria) of varying size, occasionally 
arranged in rows, also appeared scattered in the sarcous substance of the muscular 
fibres near these streaks of white. Right thigh : Large diffuse collection of yel- 
lowish-white material of irregular shape, measuring three by one-and-a-half inches 
in area, in the subcutaneous connective-tissue. This patch was attached to the skin 
and the muscle at the sites of the original wounds in the same. Material composed 
of connective-tissue elements infiltrated with degenerating leucocytes and enormous 
numbers of bacteria. The cavity of the muscle-wound contained a dirty pasty sub- 
stance in small quantity, consisting of broken down muscular debris and closely 
packed masses of bacteria. Around this spot were white streaks similar to those 
above described as found in the other thigh. Blood: From a vein in the neighborhood 
of the muscle-wound showed no bacteria, but that from the right heart contained 
bacteria. There were general pericarditis, peritonitis, and inflammation of the 
tunica vaginalis of the testicles with fibrinous friable soft exudation, composed of a 
fibrinous network in whose meshes were myriads of bacteria. 41 
c. Inoculation of muscular tissue with mixture of tongue-scrapings and salicylic acid. 
Six grains’ weight of pulpy tongue-scrapings, three hours after removal, was thoroughly 
triturated with one grain of Squibb’s unbleached salicylic acid. 
Experiment So: 
May 28, 1875- Both thighs of a rabbit inoculated with the mixture of tongue-scrapings 
and salicylic acid. 
June 4. Animal extremely emaciated and dull. Thick lump on each side at sites of 
inoculation. Pricked, exuded yellow semi-fluid matter consisting of granular leuco- 
cytes, granular debris, and a few motionless rod-bacteria. 
June 5. Dead. By a mistake the body was thrown away before an autopsy was made. 
Experiment 81: 
May 28, 1875. Both thighs of a rabbit inoculated with the mixture of tongue-scrapings 
and salicylic acid. 
June 4. Animal in fine condition. Skin of right thigh snipped and parts explored. 
Large thick lump an inch long and half an inch wide. Contents pulpy, consisting 
of very granular leucocytes, but no bacteria. On left side a smaller thickening 
could be felt under the scab of the skin-wound. Pricked, exuded pulpy yellow 
matter, consisting of granular leucocytes, and a very small number of motionless 
bacteria. 
June 13. Dead. Autopsy four hours after death: Animal had been in good condition 
though somewhat thin; fur in good order. Left thigh : Yellow tumor over two 
inches long and half an inch thick and wide, connecting the skin and muscle-wounds; 
thickest over muscle-wound ; composition same as found June 4 ; no bacteria. No 
signs of circumjacent serous infiltration or congestion. .Right thigh : Exactly the 
same condition of things ; the tumor was nearly two inches long, and half an inch 
wide and thick. Blood of right ventricle contained a very few club-shaped bacteria, 
motionless. The red globules in many instances were beset with processes project- 
ing radially and having exactly the appearance of short rod-bacteria. But on 
adding water these processes became pale like the rest of the substance of the cor- 
puscle, and as the latter swelled under the action of the water these processes were 
rapidly retracted, till they disappeared wholly, leaving the usual smooth, round 
contour to the swollen corpuscle. 
Experiment 82 : 
May 28, 1875. Both thighs of a rabbit inoculated with the mixture of tongue-scrapings 
and salicylic acid. 
June 4. Animal in fine condition. Thick lump felt on each side. Pricked, exuded 
yellow semi-fluid matter containing leucocytes and granular debris, but no bacteria. 
June 22. Animal in fine general condition. On the right side the lump is as large as a 
pigeon’s egg. On squeezing, a yellowish material exuded from under the scab cover- 
ing the skin-wound. Contained granular leucocytes and debris, but no bacteria. 
July 10. Animal in excellent condition. 42 
d. Inoculation of muscular tissue with putrefying tongue-scrapings. 
The scrapings of five successive mornings were mixed as obtained, and used for inoculation 
on the fifth day. The mixed material had acquired a horribly offensive smell. 
Experiment 83: 
January 4, 1876. Both thighs of a rabbit inoculated with the foetid tongue-scrapings. 
January 10. Lumps felt on both sides. 
January 13. The lumps have ulcerated externally; discharging freely an extremely foul, 
offensive matter. Animal not as lively as others, but fat, and fur in good condition. 
January 26. Animal in good condition. 
February 22. Killed in a fight with another rabbit. Autopsy: Large collection of the 
usual matter at sites of inoculation, but no lesion of any internal organs. 
Experiment 84: 
January 4, 1876. Both thighs of a small rabbit inoculated with the foetid tongue- 
scrapings. 
January 10. Dead. Autopsy: On both sides the usual lumps of yellow material. 
(N. B.—This rabbit was one of a litter of six small animals under three months of 
age, two of which, not experimented upon at all, died about the same time 
from unknown causes.) 
e. Inoculation of muscular tissue with mixtures of putrefying tongue-scrapings and 
salicylic acid. 
Fourteen grains’ weight of the foetid tongue-scrapings of “ d" was treated with salicylic 
acid solution, and then with the solid acid in exactly the same manner as the diphtheritic 
matter in series Case XIII. (see ante, page 691). After mixture with the salicylic acid 
the excessively offensive odor of the stale tongue-scrapings almost wholly disappeared. 
Experiment 85 : 
January 4, 1876. Both fore-shoulders of a rabbit inoculated with the mixture of tongue- 
scrapings and salicylic acid. 
January 10. Animal in good condition. Lumps the size of a large marble on each 
side. 
January 13. Lumps as large as a filbert on each side. Skin-wound scabbed over. On 
detaching the scab the contents of the lump were exposed and were found to consist 
of the usual yellowish pasty material. Animal in good health. 
January 16. Animal in good condition. 
March 11. Animal in good condition and fat. Ulcers have healed. 
April 18. Animal perfectly well. Experiment 86 : 
January 4, 1876. Both fore-shoulders of a rabbit inoculated with the mixture of tongue- 
scrapings and salicylic acid. 
January 10. Lumps on both sides the size of a large marble. Animal in good condition. 
January 13. Animal in good condition. Lumps the size of a filbert, and discharging 
matter externally. 
January 26. Animal in good health, but paralyzed. 
March 11. Animal perfectly well. Ulcers healing. 
Experiment 87 : 
January 4, 1876. Both fore-shoulders of a rabbit inoculated with the mixture of tongue- 
scrapings and salicylic acid. 
January 10. Condition and lumps same as the two foregoing. 
January 13. Lumps the size of a small marble; yellowish pasty matter exuding from 
under scab over skin-wound. 
January 26. Animal in good condition. 
February 23. Animal in good condition. 
March 11. No marks of ulceration ; animal fat and in good health. 
Experiment 88 : 
January 4, 1876. Both thighs inoculated with the mixture of tongue-scrapings and 
salicylic acid. 
January 10. Animal lively and in good condition ; small lumps on both sides. 
January 13. Same condition as the others. 
January 26. Animal in good’condition. » 
February 20. Killed in a fight with another rabbit. 
Experiment 89: 
January 4, 1876. Both thighs inoculated with the mixture of tongue-scrapings and 
salicylic acid. 
January 10. Small lumps on each thigh. 
January 13. Small lumps on both sides. Skin-wounds scabbed; yellowish material 
easily squeezed out from under scabs. 
January 23. Dead. No autopsy. 
Experiment 90 : 
January 4, 1876. Both thighs of a rabbit inoculated with the mixture of tongue- 
scrapings and salicylic acid. 
January 10. Small lumps, but with boundaries not very well defined, on both sides. 
January 13. Large scabs at site of skin-wounds ; yellow creamy matter exuding from 
under the scabs ot nearly as offensive odor as in Experiment 83. 
January 16. Dead. No autopsy. 44 
III.—INOCULATIOxNS OF DECOMPOSED COHN’S SOLUTION. 
a. Inoculation of muscular tissue with sediment from bottle of decoinposed Cohn's solution. 
A large bottle of Cohn’s solution1 was prepared and allowed to spontaneously decompose. 
It became turbid, acquired a strong putrid smell, and deposited a thick pulpy sediment. Fluid 
and sediment swarmed with actively moving bacteria. The sediment was separated by strain- 
ing and used for the inoculations. 
Experiment 91 : 
May 15, 1875. Both thighs of a rabbit inoculated with the pulpy sediment from the 
decomposed Cohn’s solution. 
May 19. Skin of right thigh snipped and parts explored. Thick yellowish tumor, one- 
fourth inch wide, connecting skin and muscle-wounds ; material composed of granular 
leucocytes and a moderate number of free rod-bacteria. 
May 27. Right leg again explored. The yellow tumor has considerably increased in 
size; leucocytes undergoing fatty degeneration ; bacteria very few. Left leg ex- 
plored : long yellow mass of the usual character, over 1 inch long by inch wide; 
contents contain granular leucocytes and a very moderate number of bacteria. 
June 8. The patches still to be felt from without. Skin not opened. 
June 22. Left leg explored : the tumor has shrunk to a thin yellowish streak running 
from the site of the muscle-wounds forwards into the connective-tissue elements in- 
filtrated with minute oil-globules. Right leg explored : Thin yellowish layer attached 
to inner surface of skin-wound. Site of muscle-wound not discoverable. Animal 
thin but otherwise seemingly healthy. 
July 8. Died suddenly. Autopsy. — Right thigh: Small, very thin yellow deposit just under 
the skin. Left thigh: No action to be found. (N. B.—The weather at this time 
was intensely hot, and animals that were not experimented upon died.) 
Experiment 92: 
May 15, 1875. Both thighs of a rabbit inoculated with the pulpy sediment from the decom- 
posed Cohn’s solution. 
June 8. Animal in excellent condition; no lumps felt. Right leg: Explored in the usual 
way; nothing near the skin-wound, but at the muscle-wound was a lobulated yellow 
tumor, the size of a medium-sized pea, lying in the subcutaneous connective-tissue, 
and connected through the original cut in the aponeurosis with the muscular sub- 
stance below. Material of the tumor contained leucocytes, minute oil globules, and 
large sacs stuffed with the same. Only here and there an occasional motionless rod- 
bacterium. No inflammation or congestion about the tumor. Left leg explored : 
Flat yellow patch in the connective-tissue near skin-wound; material elastic and 
coherent; contains connective-tissue elements undergoing fatty degeneration, leucocy- 
tes and sacs filled with oil-globules. Only rarely a bacterium to be seen motionless. 
The site of the muscle-wound could not be found. 
July 10. Animal in excellent condition. 
l Formula for Cohn’s solution. Amnionic tartrate i gramme, potassic phosphate, magnesic sulphate, aa. 5 
grammes ; calcic phosphate .05 gramme, distilled water 100 c. c. 45 
b. Hypodermic injection of lower stratum of decomposed Cohn's solution. 
The supernatant fluid of a quart bottle of decomposed Cohn’s solution that had stood for 
two months was carefully drawn off and the thick semi-fluid lowest stratum used for the 
injections. From four to ten minims was the quantity injected in each limb. 
Experiment 93 : 
March 6, 1876. Both thighs injected with decomposed Cohn’s solution. 
April 13. Lumps on each side as big as pullet’s eggs. 
Experiment 94: 
March 6, 1876. Both thighs injected with decomposed Cohn’s solution. 
March 26. Dead. 
March 28. Autopsy 48 hours after death : Lumps on both sides; material contained 
leucocytes and bacteria in tolerable abundance. 
I ’ 
Experiment 95 : 
March 6, 1876. Both thighs injected with decomposed Cohn’s solution. 
April 13. Lumps on both sides; material contained leucocytes, bacteria and granular 
matter. 
Experiment 96: 
March 6, 1876. Both thighs injected with'decomposed Cohn’s solution. 
March 26. Lumps on both sides. 
April 13. Lumps have disappeared. Animal well. 
c. Hypodermic injection of decomposed Cohn's solution. 
A sample of Cohn’s solution was inoculated, upon making, with a few drops of old, 
decomposed Cohn’s solution, and allowed to stand one month. It was then itself decomposed, 
had a putrid smell, was turbid, and swarmed with bacteria. The supernatant fluid was used 
for the injections, sixteen minims being injected into each limb. 
Experiment 97 : 
April 18, 1876. Both thighs of a rabbit injected with the decomposed Cohn’s solution. 
April 25. Animal in fair condition ; somewhat thin, and coat not so sleek as others. 
Perhaps small lumps, but not very distinct. 
May 2. Animal in good condition. Right side lump size of pea. Left side lump size of 
bean. Latter pricked, exuded yellow material, containing granular and fatty 
leucocytes, and debris; no plainly marked bacteria. 
May 9. No change. 
May 22. Both lumps seemingly disappeared. Left leg explored, and yellowish thickened 
patch of connective-tissue found at site of wound. 
June 13. Nothing to be felt. 46 
Experiment 98: 
April 18, 1876. Both thighs of a rabbit injected with the decomposed Cohn’s solution. 
April 25. Animal in excellent health. Apparently small lumps on both sides. 
May 2. Lump on each side size of bean. Left leg explored : Material yellowish, pasty 
composition, as in foregoing, but more shreds of connective-tissue. Animal in good 
condition. 
May 9. Right lump disappeared. Left, now only a small diffuse thickening. 
May 22. All thickening gone. 
June 13. Nothing to be felt. 
Experiment 99 : 
April 18, 1876. Both thighs of a rabbit injected with the decomposed Cohn’s solution. 
April 25. Animal hearty, apparently. Small lumps in both thighs. 
May 2. Animal in good condition. Right side, no lump to be felt; left side, diffuse 
lumpy thickening of the size and shape of a Lima bean; composition as in Experi- 
ment 97. 
May 9. Lump on left side same size. 
May 22. Lump on left side slightly smaller. 
June 13. Lump on left side diminished to size of split pea. 
Experiment 100 : 
April 18, 1876. Both thighs of a rabbit injected with the decomposed Cohn’s solution. 
April 26. Animal thin, but otherwise well. Lumps on both sides. 
May 2. Left side, lump twice the size of a bean, with ulcerated and scabbed skin over 
area X inch long by X inch wide. On lifting top of scab pasty material exuded, 
containing granular and fatty leucocytes and debris and swollen minute oblong 
bodies, possibly bacteria. Right side, lump the size of a marble, not opened. 
Animal thin and mangy. 
May 9. Right side, lump size of bean. Left side, slight thickening only. 
May 22. Right side, lump size of small pea. Left side, thickening disappeared. 
June 13. Right lump also disappeared. 
d. Hypodermic injection of decomposed Cohn's solution after filtration through porous 
clay. 
Some of the same sample of Cohn’s solution as was used in “ c " was filtered through 
the porous clay cylinder (see ante, page 701). Two minims of fluid was obtained. This 
filtrate was clear, odorless, and wholly free from bacteria or other solid elements. 
Experiment 101 : 
April 18, 1876. The two minims of clear filtrate was injected into the right side of a 
rabbit. 
April 26. Animal in excellent condition. No sign of a lump. 
May 2. Animal in fine condition. Nothing to be felt. 
June 13. Same as on May 2. 47 
e. Hypodermic injection of decomposed Cohn's solution, after filtration through many 
thicknesses of paper. 
The same sample of Cohn’s solution used in “c” and u d " was filtered through suc- 
cessive paper filters as follows, a fresh filter or filters being taken each time : Single, twice ; 
double, twice ; triple, once; quadruple, four times. The filtrate was faintly opalescent only, 
and had entirely lost the original putrid smell. It contained but a few active bacteria. 
Experiment 102: 
April 18, 1876. Eight minims of the filtrate injected into each thigh of a rabbit. 
April 25. Animal in excellent condition. No lumps to be felt. 
May 2. Same as on April 25. Left leg explored; nothing to be found. 
June 13. Every thing perfectly normal. 
Experiment 103 : 
April 18, 1876. Same procedure ; same history; same results as in Experiment 102. 
Experiment 104: 
April 18, 1876. Same procedure ; same history ; same results as in Experiment 102. 
Experiment 105 : 
April 18, 1876. Same procedure; same history ; same results as in Experiment 102. 
f. Hypodermic injection of decomposed Cohn's solution. 
A sample of Cohn’s solution was inoculated with one fluid ounce of old decomposed 
Cohn’s solution. It was used for the following injections three weeks later, was then 
thoroughly decomposed, smelling strongly putrid and swarming with living bacteria. Each 
injection was sixteen minims taken from the upper stratum of the bottle. 
Experiment 106: 
June 13, 1876. Both thighs injected with the decomposed Cohn’s solution. 
June 20. Large flat area of thickening on right side about one inch in diameter. Left 
side, lump the size of a bean. 
June 27. The thickened patch on the right side has ulcerated; left side, no change. 
Animal thin and poorly. 
Experiment 107 : 
June 13, 1876. Both thighs injected with the decomposed Cohn’s solution. 
June 20. Nothing to be felt on either side. 
June 27. Nothing to be felt on either side. 48 
Experiment 108: 
June 13, 1876. Both thighs injected with the decomposed Cohn’s solution. 
June 20. Right side, very small thickening to be felt, like a string with a knot at the 
end. Left side, lump the size of a split pea. 
June 27. Right side, thickening disappeared. Left side, lump size of a small bean. 
Experiment 109 : 
June 30, 1876. Both thighs injected with the decomposed Cohn’s solution. 
June 20. Right side, considerable diffuse thickening to be felt, like a bean wrapped in 
cotton; left side, very little, if any thickening. 
June 27. Thickening on right side disappeared; cord of thickening, like a string, 
developed on left side. None of the animals of this series showed any impairment 
of their general health. 
g. Hypodermic injection of decomposed Cohn's solution after thorough aeration. 
Into the nozzle of a funnel a rubber cap, pierced with a small hole, was firmly wedged. 
Fluid poured in the funnel thus armed escaped through the hole in the rubber plug only drop 
by drop, and about as fast as in ordinary filtration. Some of the same Cohn’s solution used 
in was then passed through this plugged funnel nine successive times. After this 
aeration, the solution was of course as turbid as ever, but it had entirely lost the putrid smell 
it originally had, a slightly mawkish odor alone remaining. Sixteen minims of this aerated 
solution was used in each of the following injections : 
Experiment x 10: 
June 13, 1876. Both thighs of a rabbit injected with the aerated Cohn’s solution. 
June 20. Right side, nothing to be felt; left side, lump the size of a large pea. 
June 27. Right side, lump size of a shot; left side, lump size of a split pea. 
Experiment 111: 
June 13, 1876. Both thighs of a rabbit injected with the aerated Cohn’s solution. 
June 20. Right side, lump size of a marble; left side, lump size of a bean. 
June 27. Right side, lobular diffuse lump, size of a Lima bean ; left side, slight cord- 
like thickening. 
Experiment 112 : 
June 13, 1876. Both thighs of a rabbit injected with the aerated Cohn’s solution. 
June 20. Both sides, lumps size of a large pea. 
June 27. Both sides, lumps size of a large bean. 
Experiment 113 : 
June 13, 1876. Both thighs of a rabbit injected wdth the aerated Cohn’s solution. 
June 20. Right side, lump size of a large pea ; left side, lump size of a large bean. 
June 27. Both sides, lumps size of a large bean. These were pricked, and the usual 
yellowish creamy material exuded on pressure. None of the animals of this series 
suffered any impairment of their general health. 49 
IV.—INOCULATIONS OF PUTRID INFUSION OF CALF’S LIVER. 
a. Hypodermic injection of the putrid liver infusion. 
Pieces of calf’s liver were infused in water, and after a few days the infusion was used for 
experimentation. The fluid had an intensely strong putrid smell, and swarmed with living 
bacteria. , 
Experiment 114: 
November 12, 1875. One thigh of a rabbit injected with the putrid infusion. 
November 15. Small yellowish-white tumor found in the connective-tissue at site of 
injection, identical in appearance with those occurring after inoculation with diph- 
theritic membrane, tongue-scrapings, etc. 
b. Hypodermic injection of putrid liver-infusion after filtration through porous clay. 
Some of the infusion of “ a ” was drawn through the porous clay-cylinder. 
Experiment 115 : 
November 12, 1875. One thigh of a rabbit injected with the clear filtrate (three 
minims). 
November 15. No discoverable effect. 
c. Hypodermic injection of putrid liver-infusion after filtration through paper. 
Some of the infusion of “ a ” was filtered through a single paper filter, and some through 
a double filter, so managed that four thicknesses of paper were everywhere presented to the 
fluid. The filtrate in both cases showed no rod-bacteria, but multitudes of minute granules. 
Experiment 116 : 
November 12, 1875. One thigh of a rabbit injected with the filtrate from the single 
filter. 
November 15. No discoverable effect. 
Experiment 117 : 
November 12, 1875. One thigh of a rabbit injected with the filtrate from the double 
filter. 
November 15. No discoverable effect. 
d. Inoculation of solid residue from putrid liver-infusion, after evaporation by boiling, and 
treatment with boiling absolute alcohol. 
About nine fluid ounces of the unfiltered infusion was boiled continuously until it was 
nearly evaporated to dryness. The solid residue was then further boiled with absolute alcohol, 50 
and the alcohol then removed by filtration. The moist residue on the filter was used for inocu- 
lation in one case, and, mixed with water, for hypodermic injection, in another case. 
Experiment 118 : 
November 12, 1875. One thigh of a rabbit inoculated with the moist residue. 
November 15. Large lump at site of inoculation, of the usual character. 
Experiment 119 : 
November 12, 1875. One thigh of a rabbit injected with the moist residue mixed with 
water. 
November 15. Large lump, as in foregoing. 
e. Hypodermic injection of alcoholic solution frotn residue of evaporated putrid liver- 
infusion. 
The alcoholic filtrate of “ d” was used for the injection. 
Experiment 120: 
November 12, 1875. One thigh of a rabbit injected with the alcoholic filtrate. 
November 15. No discoverable effect. 
V.—INOCULATIONS OF SAND. 
Sand was roasted, and allowed to cool. Some was used for inoculation, dry; some, 
after mixing into a pulp with a little of Liebig’s extract of meat. 
Experiment 121 : 
May 15, 1875. Both thighs of a rabbit inoculated with dry sand. 
May 19. Animal in good condition; nothing to be felt. Legs explored; sand 
found encapsuled, each grain by itself. No yellow tumor ; no signs of inflammation. 
May 27. Legs explored ; thin line of slightly thickened connective-tissue, studded with 
sand particles connecting skin and muscle-wound. No yellow tumor; no leuco- 
cytes ; no bacteria. 
May 31. Animal perfectly well. 
Experiment 122 : 
May 15, 1875. Same procedure, history and results as in Experiment 121. 
Experiment 123 : 
May 15, 1875. Both thighs of a rabbit inoculated with the mixture of sand and meat 
extract. Subsequent history and results same as in Experiment 121. 
Experiment 124 : 
May 15, 1875. Same procedure, history and results as in Experiment 123. 51 
VI.—INOCULATIONS OF SALICYLIC ACID. 
a. Inoculation of muscular tissue with solid salicylic acid. 
Some of Squibb’s unbleached salicylic acid was mixed into a paste with water, and inocu- 
lated in the usual way. 
Experiment 125 : 
May 28, 1875. Both thighs of a rabbit inoculated with the paste of salicylic acid. 
June 4. Animal in excellent condition. Skin-wounds closed by scabs, without thick- 
ening. Left side : A depression felt over site of muscle-wound. Skin snipped, and 
parts explored. No sign whatever of any lesion in the connective-tissue. The hole 
made by the original muscle-wound remained as an excavation inch in diameter, 
presenting a brown-colored surface. The material of this brown layer of tissue con- 
sisted of connective-tissue elements, blood-corpuscles, and muscular fibres in a state 
of unnatural stiffness, shrinkage, and brownish discoloration—an appearance similar 
to what is produced in muscle by the action of weak chromic acid. No sign of pus, 
or of irritation of tissues in the neighborhood of the muscle-wound. 
July 1. Skin and muscle-wounds completely healed. Animal in good condition. 
July 10. Animal in good condition. 
Experiment 126 : 
May 28, 1875. Both thighs of a rabbit inoculated with the paste of salicylic acid. 
June 4. Skin of thighs snipped and parts explored. Appearances identical with those 
of foregoing experiment at -same date. 
June 24. Animal well; nothing to be felt from without ; legs not explored. 
July 10. Animal in good condition. 
Experiment 127 : 
May 28. 1875. Both thighs of a rabbit inoculated with the paste of salicylic acid. 
June 4. Animal in excellent condition. No thickening to be felt, and on opening skin, 
no lesion in the neighborhood of the skin-wound. 
June 12. Animal in excellent health. 
b. Hypodermic injection of salicylic acid dissolved in aqueous solution of sodic phosphate. 
The rabbits used in this and the following series of experiments were all of the same 
litter ; small animals, weighing 1% pounds each. 
Experiment 128: 
March 12, 1875. Injected following solution : Salicylic acid, 5 grains ; sodic sulphate, 
15 grains; water, 25 minims. For the following two hours animal somewhat stupid 
and quiet ; respirations quickened. No interference with any of the functions. 
March 13. Animal lively, apparently as well as ever. 52 
Experiment 129: 
March 15, 1875. Injected following solution by six different punctures over back and 
belly: Salicylic acid, 10 grains; sodic sulphate, 30 grains; water, 100 minims. 
The injecting occupied seven minutes. Immediate effects generally similar to those 
in Experiment 128, but shock-like condition more pronounced. 
March 16. Animal lively and well. 
Experiment 130: 
March 16, 1875. Injected into same rabbit used in Experiment 129, the following 
solution by three different punctures : Salicylic acid, 10 grains; sodic phosphate, 30 
grains ; water, 50 minims. Animal became soon quiet and dull, but in half an hour 
brightened somewhat. One hour after the injections, fell upon side, unable to stand. 
Fifteen minutes later opisthotonic convulsions set in, and for the following hour the 
condition remained the same. 
March 17, 8 o’clock A. M. Found dead and stiff. Autopsy at 3.15 o’clock p. M., just 
twenty-four hours after the injections ; Connective-tissue under skin of thorax and 
abdomen infiltrated with serum, striking a violet color on addition of solution of 
ferric chloride (a test for salicylic acid). Ecchymoses at sites of injection. On 
opening abdominal cavity it was found that the needle of the injecting syringe 
had pierced the thin wall of the abdomen and wounded the peritoneum and liver. 
No other lesion, traumatic or otherwise, of any abdominal or thoracic organ, nor of 
the central nervous system. Bladder distended with four fluid drachms of clear urine, 
giving deep violet color on addition of the ferric salt. Contents of small intestine 
and alcoholic extract of the brain also give the reaction with iron, but not the con- 
tents of the stomach, nor the cut surface of the lungs, nor blood taken from the 
heart. 
Experiment 131 : 
March 17, 1875. Same procedure as in Experiment 130, care being taken not to pierce 
the abdominal walls. Animal quiet and dull shortly after the injections, but not 
seriously affected. 
March 18. Animal lively and well. 
Experiment 132 : 
March 17, 1875. Injected following solution by seven different punctures. Salicylic 
acid, 15 grains ; sodic phosphate, 45 grains; water, 75 minims. Immediate effects 
similar to those of Experiment 131. 
March 18. Animal lively and well. 
Experiment 133: 
March 19, 1875. Injected following solution by ten separate punctures. Salicylic acid, 
24 grains; sodic phosphate, 72 grains ; water, 160 minims. The injecting occupied 
half an hour. Animal after last injection quiet and dull with increased rapidity of 
respiration. Fifteen minutes later, fell over on side with legs rigid and head thrown 53 
back, and in seven minutes thereafter died. Autopsy immediately: Connective- 
tissue at most points of injection infiltrated with blood, with many ecchymoses in 
the neighborhood; coagula of a dirty reddish-brown on the edges. No lesions of 
any organ, traumatic or otherwise. Urine in bladder, faeces in small intestine, 
blood in heart, aqueous humor, alcoholic decoction of brain, all give a violet color 
on addition of solution of ferric chloride. 
c. Hypodermic injection of simple aqueous solution of sodic phosphate, and of pure water 
as control experiments to the foregoing. 
Experiment 134: 
March 15, 1875. Injected the following solution by six separate punctures: sodic phos- 
phate, 30 grains; water, 100 minims (same solution as in Experiment 129, only 
minus the salicylic acid). Results same as in Experiment 129. • 
Experiment 135 : 
March 16, 1875. Same procedure and results as in Experiment 134. 
Experiment 136: 
March 16, 1875. Injected by six separate punctures 100 minims of pure water. Animal 
quiet and dull for fifteen minutes. 
March 17. Animal lively and well 
Experiment 137 : 
March 24, 1875. Injected the following solution by ten separate punctures : sodic phos- 
phate, 72 grains ; water 160 minims (same solution as in experiment 133 only minus 
the salicylic acid). Animal became immediately quiet and dull, breathing fast. 
Two hours later fell over on side in convulsions ; legs rigid and twitching, but in 
seven minutes regained the feet and remained sitting quietly. 
March 25 (twenty-four hours after last observation). Has eaten nothing ; sits heavy and 
dull with eyes half closed ; on pushing gently, fell over on side and pawed ineffectu- 
ally to regain feet. Died quietly half an hour later. 
March 26. Autopsy : No lesion of any organ, traumatic or otherwise, but subcutaneous 
connective-tissue deeply and generally infiltrated with serum. 
VII.—INOCULATIONS OF SALICYLIC ACID AND VACCINE VIRUS. 
Through the kindness of Dr. Frank P. Foster these inoculations were made at his estab- 
lishment by Dr. W. F. Cushman and Mr. H. Lawrence, in presence of Dr. Satterthwaite. 
Solutions were prepared as follows: I. Salicylic acid, 1 part; distilled water, 500 parts. 
II. Salicylic acid, 1 part ; sodic phosphate, 3 parts; distilled water, 250 parts. III. Sodic 
phosphate, 3 parts ; distilled water, 250 parts. All being in readiness for the vaccinations, 
three portions of pure fresh vaccine lymph were mixed with equal volumes of these solutions, 
respectively, and the mixtures allowed to stand one minute before being used for vaccination. 54 
Experiment 138 : 
March 28, 1876. A heifer was inoculated as follows: At four points with pure fresh 
vaccine lymph ; at four points with lymph mixed with solution I. (making a dilute 
lymph impregnated with salicylic acid in the proportion of 1 to 1,000 parts); at 
four points with lymph mixed with solution II. (making lymph containing salicylic 
acid 1 part to 500) ; and at five points with lymph mixed with solution III. 
April 4. Fine pocks produced in two out of the four inoculations of pure lymph ; in all 
four inoculations of lymph with salicylic acid 1 to 1,000; in all four inoculations of 
lymph with salicylic acid 1 to 500; and in four out of the five inoculations of lymph 
with simple sodic sulphate solution. In all cases the pocks produced by the adul- 
terated lymph were as fine as those produced by the pure, and were in all respects 
typical vaccine pocks. 
VIII.—EXPERIMENTS TO TEST THE POWER OF SALICYLIC ACID TO 
PREVENT THE DEVELOPMENT OF BACTERIA IN PUTREFIABLE 
FLUIDS. 
In these experiments equal volumes of clear putrefiable fluids in test-tubes were charged 
with various percentages of the substances whose power was to be tested, and the tubes were 
then inspected from day to day, or as often as occasion permitted, and the first appearance 
of cloudiness of the contained fluid noted as a sign that bacterial development was 
beginning. 
Experiments 139 to 175 inclusive: 
March 21, 1875. Thirty-six test-tubes of Cohn’s solution were charged with different 
proportions of salicylic acid of different makers, as in the following table. A test- 
tube of Cohn’s solution, unadulterated, was placed among the rest. The whole 
were set upon a warm mantel-piece, and watched for twelve days. The lines indicate 
the number of days that the contents of the tubes remained clear. 55 
Cohn's Solution and Salicylic Acid. 56 
Experiments 176 to 186 inclusive : 
March 22, 1875. Fresh human urine was boiled, filtered, and then charged with Squibb’s 
unbleached salicylic acid, as per table below. The test tubes were observed for 
eight days. 
Experiments 187 to 196 inclusive 
April 29, 1875. The following aqueous solutions of Squibb’s pure salicylic acid were 
made by the addition of a little sodic phosphate: 1: 100, 250, 500, 750, 1,000, 
1,500, 2,500, 3,000, 5,000, 10,000. Two fluid drachms of each solution were put 
into a test-tube, and then the test-tubes were severally inoculated with one drop 
each of a strong, putrid beef-decoction. The tubes were finally loosely stopped with 
cotton. The tubes were examined at intervals, with the following result: All the 
solutions weaker than 1-1500 began to turn cloudy almost immediately. The fol- 
lowing solutions were found cloudy on the days stated respectively : 1 -1500 on the 
6th day, 1-1000 on the 28th; 1-750 and 1-500 were clear on the 28th day, but were 
found cloudy on the 74th, no observation having been made between these dates ; 
1-250 and 1-100 remained clear during the whole 78 days of the observation. 
Experiments 197 to 206 inclusive : 
April 29, 1875. Precisely similar experiments were made with Squibb’s unbleached 
salicylic acid. The results were practically identical with the foregoing.