STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
BY J. H. WRIGHT, M D. STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
1 
[Reprinted from the Boston Medical and Surgical Journal 
of October 4 and 11,1894.] 
tion, is as to whether the organism loses its virulence by 
long-continued residence in the air-passages of diphthe- 
ria patients. 
It has been well shown by the work of Janson,10 
Heubner,11 Tobiesen 12 and Morse13 that the bacillus 
may persist in the throat long after convalescence has 
been established. The virulence of the organism in 
these cases of recovery has been found by Loftier,14 
Heubner,11 Tobiesen 12 and Escherich 2 to be undimin- 
ished, while, on the other hand, Roux and Yersin 1 have 
stated that a progressive diminution of virulence may 
be observed the longer the organism remains in the 
throat. The last named have recorded five cases in 
which virulent bacilli gradually gave place to non- 
virulent forms in the course of days and weeks. 
A more extensive piece of work on this point is that 
of Tobiesen,12 in which the bacilli from 19 convales- 
cent diphtheria cases, after their discharge from the 
hospital, were inoculated into guinea-pigs. Of the 19 
cases, 16 were found to still furnish bacilli which 
killed the guinea-pigs, with the characteristic lesions 
at the autopsy ; while on the animals inoculated from 
the 3 remaining cases, 2 eventually died and 1 de- 
veloped paralysis. 
From all the foregoing statements it would appear 
that in general no constant relationship can be made 
out between the virulence of the bacillus towards ani- 
mals, and the symptoms presented by the cases from 
which they were derived. Having at our disposal a 
large number of cases of diphtheria in the contagious 
wards of the Boston City Hospital, it was determined 
to again test this question of the virulence of the ba- 
cillus in the different types of cases above referred to. 
With this end in view two series of cases have been 
studied. The first and larger series comprises 59 
cases of various degrees of severity from which the 
cultures have been taken at an early period of the dis- 
ease when the organism could have been considered as 
not having been present a long time in the throat. 
These cultures have been inoculated into 114 guinea- 
pigs to determine their virulence. The second series 
comprises 23 cases in which the bacilli had been pres- 
ent in the throat or nose for a longer or shorter period 
of time when inoculated into guinea-pigs. In this se- 
ries, 35 animals were used. 
The methods used in the investigation have been as 
follows: The material for cultures has been obtained, 
in most of the cases, by touching the mucous mem- 
brane of the nose or throat with sterilized cotton 
“ swabs.” The “ swab ” consists of a piece of stiff 
wire, about five inches long, upon one end of which a 
small pledget of absorbent cotton has been wound. 
The swab thus made is placed in a test-tube, which is 
stoppered with cotton and the whole sterilized by heat 
before using. A large number of swabs prepared in 
this way are kept at hand in the hospital laboratory 
ready for use, and may be carried to and from the 
wards without danger of contamination. 
The culture medium used for growing discrete colo- 
nies of the organism from which to prepare pure cult- 
ures has been coagulated beef-blood serum, containing 
one part in four of one-per-cent, sugar bouillon (Lof- 
fier’s mixture). The mixture of blood serum and 
bouillon is coagulated in test-tubes by dry heat so as 
to form “ slants,” and then sterilized by steam in three 
successive days, as is done with ordinary media. On 
this culture medium the diphtheria bacilli grow vigor- 
ously and with characteristic morphology. 
STUDIES IN THE PATHOLOGY OF DIPH- 
THERIA. 
BY J. H. WRIGHT, M.D., 
Assistant in Pathology, Harvard Medical School. 
The bacteriological studies of diphtheria and the 
Klebs-Loftler bacillus, the results of which are here 
reported, have been carried on under the direction of 
Professor Councilman in the Sears Pathological Labo- 
ratory of the Harvard Medical School and to some 
extent, also, in the Pathological Laboratory of the 
Boston City Hospital. 
Practically all of the material for these studies has 
been derived from the contagious wards of the Boston 
City Hospital, for the most part during the terms of 
service of Dr. F. IT. Williams and Dr. E. M. Buck- 
ingham, or in the period extending from October, 
1893, to April, 1894. The work has comprised five 
main sections, as follows : 
I. A study of the virulence of the bacillus diphthe- 
ria in infections of the pharynx and air-passages of 
varying degrees of severity and duration. 
II. The results of autopsies on diphtheria cases. 
III. The pathological anatomy and bacteriology of 
experimental diphtheria. 
IV. Cases of infection of various ulcerated surfaces 
and other inflammatory processes with the Klebs-Lof- 
fler bacillus, as well as some other instances in which 
it has been isolated. 
V. On the morphology and biology of the Klebs- 
Loffler bacillus. 
I. 
A study of the virulence of the bacillus diphtherice in 
infections of the pharynx and air-passages of varying 
degrees of severity and duration. 
The question as to whether the bacilli obtained from 
mild cases of diphtheria are as virulent for guinea-pigs 
as those derived from severe cases has received special 
attention at the hands of but a few investigators. The 
most important work on this question is that of Roux 
and Yersin.1 These investigators inoculated guinea- 
pigs with cultures obtained from 40 fatal and 39 non- 
fatal cases, and compared the results. They concluded 
that, in general, there is a striking difference to be ob- 
served as to virulence toward guinea-pigs, between the 
bacilli from cases ending in death and those terminat- 
ing in recovery, although the latter often furnish or- 
ganisms of full virulence. 
Escherich 2 in his recent work confirmed these re- 
sults, finding that in general mild cases yield weakly 
virulent cultures, but that the exceptions to this rule 
are numerous. In an earlier publication,8 however, he 
had stated that he had noted no difference in the viru- 
lence of the bacilli from cases of different degrees of 
severity. The writers just quoted seem to be the only 
ones who have studied this point especially. On the 
other hand, the work of other investigators (Loffler,4 
v. Hofman,6 Zarniko,6 Beck,7 Spronk,8 Welch and 
Abbott,9 and others) who have inoculated guiuea-pigs 
with the Klebs-Loftler bacillus from numbers of diph- 
theria cases, seems to point to no such relationship 
between the clinical character of the case and the 
effect of the bacillus derived f-rom it upon the animal. 
Another aspect of the question of the virulence of 
the bacillus diphtheriae, which has received more atteu- 2 
STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
To obtain colonies of the bacillus from which to 
prepare pure cultures, some of the material secured on 
the swab in the manner above described, is applied 
over the surface of two or more tubes, and these are 
then placed in the incubator for development. After 
about eighteen to forty-eight hours, from the discrete 
colonies which have appeared, pure cultures may be 
made for inoculation. The cultures used and the 
methods employed in the inoculations have varied. 
With-a few exceptions, which will be found duly noted 
in the detailed reports of the work, bouillon and agar 
cultures have been inoculated which have been about 
twenty-four hours in the incubator. In the majority 
of cases cultures in one-per-cent, grape-sugar bouillon 
of neutral to slightly alkaline reaction and colored 
with litmus, have been injected into the tissues of the 
abdominal wall in quantities less than one cubic centi- 
metre by means of a hypodermatic syringe. 
Iu the same manner other fluid cultures and suspen- 
sions in sterilized water of the twenty-four hours’ 
growth of agar cultures have been injected. The 
method of direct inoculation from agar cultures by 
the platinum loop or wire through a wound on the 
abdominal wall has also been used in some instances. 
Iu order to avoid as much as possible any attenuation 
of the organism by cultivation, the cultures used for 
inoculation iu the first mentioned series of 59 cases, 
have all, with few exceptions, been of the second gen- 
eration from the patient. 
The limitation of the cultures used in these cases 
for testing the virulence to those of the second gener- 
ation, has greatly increased the necessary labor of the 
investigation, on account of the numerous failures to 
obtain pure cultures within so few generations. In 
the second series of 23 cases, however, the cultures 
used have not been so limited as to the number of 
generations. The usual number has been three ; and, 
unless otherwise specified iu the tables of results, the 
cultures used have been that number of generations 
from the patient. In both series of cases two animals, 
as a rule, have been inoculated from the same case, 
and in many cases with the same culture. Proceeding 
iu the manner above described, the study of two series 
of cases before mentioned has been carried out. 
In the subjoined tables are given some of the de- 
tails and the results of the work. In them are to be 
found the name and the age of the patient, the size 
and weight of the animals when known, the manner 
of inoculation, its results, and any remarks thought 
necessary. In addition to these, in the table of the 
second series, comprising those cases in which the ba- 
cillus has been present for a longer or shorter time, 
the number of days which have passed since it was 
first demonstrated to be present, up to the date of the 
taking of the cultures for inoculation, is given for each 
case in a separate column. 
The first series of 59 cases has been divided into 
four groups comprising the fatal, severe, medium 
severe, and mild cases. Iu comparing and discussing 
the results obtained from the study of both series of 
cases, we shall consider as indicating the virulence of 
the organism derived from the case, only the shortest 
time in which an animal died in those cases where more 
than one was inoculated. The sooner that death fol- 
lowed the inoculation, the more virulent will be con- 
sidered the culture which was inoculated. 
In general it may be said that a full virulent culture 
is one which causes the death of a guinea-pig within 
three days or less, a culture of medium virulence one 
which causes the death of the animal in from three to 
five days. Cultures which only produce local ne- 
crosis and ulceration or death after a greater number 
of days may be considered as of slight virulence. The 
death of the animal within forty-eight hours or less 
after the inoculation, indicates a very high degree of 
virulence in the culture inoculated. 
This is based upon the assumption which nearly all 
writers on experimental diphtheria seem to have 
adopted, namely, that the susceptibility of the guinea- 
pig to infection with the bacillus diphtherias is a nearly 
constant quantity. If this be true, and if the rapidity 
of the death of the animal be a measure of the degree 
of virulence of the organism, we should find that the 
two or more animals inoculated simultaneously in the 
abdominal wall with the bacilli from a given case would 
die very nearly at the same time. In many cases this 
has been observed, but a glance at the tabulated re- 
sults will show in this and other instances considerable 
variation in the times of the survival of animals inoc- 
ulated from the same case as well as from similar 
cases; so that we are inclined to doubt the entire cor- 
rectness of the assumption of a constant susceptibility. 
These variations are apparently more frequent in 
thost? cases in which different modes of inoculation 
have been used than iu those in which both animals 
have been inoculated from the same culture and in the 
same manner, but this circumstance does not suffice to 
explain all the differences in results observed. 
To account for these differences, it does not seem 
unreasonable to suppose that guinea-pigs vary in 
their degree of susceptibility to this infection, and 
this variation in susceptibility may be considered as 
playing a considerable part in the production of the 
irregularities in the effects of the inoculation. 
It would appear, however, that in general, inocula- 
tion with bouillon cultures is more rapidly fatal than 
the inoculation of the growth from agar cultures. 
The observations on this point are not altogether sat- 
isfactory, for it is very probable that in some few 
cases confusion has arisen as to which animal was in- 
oculated with the bouillon culture and which from the 
agar, but the evidence is on the whole sufficiently 
trustworthy to warrant the general statement. The 
difference between the forms of culture is especially 
apparent in those cases in which the animal, after re- 
covery from an inoculation with an agar culture, has 
rapidly succumbed to a bouillon culture of the same 
origin as the first, notwithstanding the fact that the 
organism had been in cultivation for a considerable 
time and so subject to the chances of attenuation. 
It is not probable that this greater virulence of the 
bouillon culture is due to an intoxication with the 
poisonous products of the bacillus preformed in the 
culture and in solution. That in cultures so young as 
those here employed and injected in the quantities 
noted, no effective amount of the poison of diphtheria 
exists is clearly shown by the results of those who 
have studied the toxalbumiu of the bacillus diphtherias. 
To determine for ourselves, however, whether the 
fluid of twenty-four-hour bouillon cultures contains any 
toxic substance, we have iu several instances injected 
the sterile filtrates of such cultures in the same manner 
as for bouillon cultures and in greater quantities, but 
never with any noticeable effect on the animal. Two 
of these experiments were essentially as follows : Two- 
tenths of a cubic centimetre of a twenty-four-hour sugar Name. 
Days 
Weight (grins.) 
1 
] 
■■ ■ ~ 
Age. 
K.-L. 
or size of guin 
Mode of Inoculation. 
Kesult of inoculation. 
Remarks. 
pres. 
pig- 
r. t. 
1 year 
42 
210 
.2 c. c. Sugar bouillon (litmus) 
Death, 72 hours 
K.-L. bacilli in nose, never in 
throat. Cult. 2d gen. Medium 
severity, but eventually fatal. 
P. H. 
3 years 
51 
200 
.2 c. c. Sugar bouillon (litmus) 
Death, 48 hours 
Main symptom coryza. Cult. 
48 hours. 
P. w. 
13 mos. 
49 
200 
.2 c. c. Sugar bouillon (litmus) 
Death, 6 days 
No lesions in animal at au- 
topsy. Medium severity. 
G. C. 
6 years 
30 
Well grown 
1. c.c. Glycerine bouillon 
Death, 36 hours 
F. B. 
4 years 
39 
290 
.3 c. c. Sugar bouillon (litmus) 
Death, 36 hours 
Culture 2d gen. 
P. K. 
5 years 
29 
330 
3. c. c. Sugar bouillon (litmus) 
Ulceration, killed 
Killed after 25 days. Med- 
ium severity. 
M. C. 
4 years 
25 
310 
.3 c. e. Sugar bouillon (litmus) 
Death, 38 days 
“ 81 days 
No lesions at autopsy. 
170 
.15 
T. H. 
23 years 
18 
225 
.5 c.c. Sugar bouillon (litmus) 
.2 
Death, 36 hours 
B. K. 
27 
33 
265 
" 36 hours 
Ltion. 
27 years 
Medium size 
.5 c. c. Water susp. agar cult. 
Recovered 
Death, 4$ days 
Survived a second inocula- 
Small 
1. c. c. Sugar bouillon (litmus) 
1. “ “ “ 
‘ ‘ 72 hours 
Large 
“ 48 hours 
M. McD. 
19 years 
21 
240 
.3 c. c. Sugar bouillon (litmus) 
Death, 4 days 
H. U. 
5 years 
26 
210 
.2 c. c. Sugar bouillon (litmus) 
Death, 6 days 
Dr. P. 
23 years 
21 
235 
.25 c. c. Sugar bouillon (litmus) 
Death, 36 hours 
220 
.2 “ “ 
“ 36 hours 
26 
310 
.25 “ “ 
“ 36 hours 
Dr. T. 
26 years 
12 
260 
.3 c.c. Sugar bouillon (litmus) 
Death, 33 days 
Severe case. 
W. T. 
21 years 
11 
290 
.3 c. c. Sugar bouillon (litmus) 
Death, 61 days 
W. M. 
21 years 
14 
180 
.2 c.c. Sugar bouillon (litmus) 
Death, 361 days 
No marked lesions at au- 
topsy. 
Dr. S. 
26 years 
t9 
280 
.2 c. c. Sugar bouillon (litmus) 
2 <• “ “ 
Death, 72 hours 
Med. severity. 1st animal, 
230 
29 days 
48 hrs., cult. 4th gen; 2d ani- 
mal, 24 hrs., cult. 5th gen. 
D. T. 
6 years 
15? 
280 
.3 c. c. Sugar bouillon (litmus) 
Death, 72 hours 
7 M. S. 
7 years 
14 
240 
Blood serum cult, loop 
Recovered 
Cult, of 2d gen. 
210 
.2 c. c. Sugar bouillon (litmus) 
Death, 4.) days 
No lesions at autopsy. Cul- 
ture of 3d gen. 
G. P. 
7 years 
10? 
270 
.2 c. c. Sugar bouillon (litmus) 
.3 
Death, 291 days 
310 
“ 19 days 
No lesions at autopsy. 
H. C. 
U years 
8 
280 
.3 c. c. Sugar bouillon (litmus) 
.2 
Death, 72 hours 
200 
“ 72 hours 
M E. 
6 years 
9 
210 
.2 c. c. Sugar bouillon (litmus) 
Death, 11 days 
I. McL. 
5 years 
35 
210 
.2 c. c. Sugar bouillon (litmus) 
Death, 8 weeks 
Severe ease. 
J. S. 
6 years 
9 
1. c. c. Water susp. agar cult. 
Death, 36 hours 
Med. severity. K.-L. bacilli 
Large 
1. 
“ 30 hours 
jresent for some weeks when 
;he culture was taken. 
Cases in which the Bacillus had been Present for a Longer or Shorter Period of Time. 
Name. 
Age. 
Weight (grins.) or 
size of guin. pigs. 
Mode of Inoculation. 
Result of Inoculation. 
Remarks. 
M. M. 
4 years 
225 
.2 c. c. Sugar bouillon (litmus) 
Death, 36 hours 
Dr. T. 
26 years 
250 
.2 c. c. Sugar bouillon (litmus) 
Death, 37 days 
No marked lesions at autopsy. 
Culture third gen. 
Dr. B. 
41 years 
230 
.3 c. c. Sugar bouillon (litmus) 
.2 
Death, 36 hours 
200 
“ 36 hours 
C. M. 
13 years 
230 
.2 c. c. Sugar bouillon (litmus) 
.2 “ “ 
Death, 3£ days 
230 
“ 4£ days 
Dr. S. 
28 years 
200 
.2 c. c. Sugar bouillon (litmus) 
,4 “ “ 
Death, 72 hours 
240 
“ 72 hours 
A. McD. 
20 years 
350 
.5 c. c. Sugar bouillon (litmus) 
Death, 36 hours 
250 
“ 36 hours 
L. 
8 years 
Well grown 
1. c. c. Glycerine bouillon 
1. 
Death, 36 hours 
A. C. 
Large 
Medium size 
“ 36 hours 
[brane of trachea. 
9 years 
1. c. c. Sugar bouillon (litmus) 
Death, 36 hours 
Culture from pseudo-mem- 
350 
Agar culture, loop 
Ulceration. Recov. Reinoc. 
Dr. Crowell’s case. 
Reinoculation 
.5 c.c. Sugar bouillon (litmus) 
Survived reinoculation. 
Cases of Medium Severity. 
F. G. 
4 years 
350 
.3 c.c. Sugar bouillon (litmus) 
.2 “ “ “ 
Death, 3 days 
Scarlet fever coincident. 
260 
“ 4J days 
C. G. 
5 years 
210 
.2 c. c. Sugar bouillon (litmus) 
.3 “ “ “ 
Death, 72 hours 
330 
“ 72 hours 
F. B. 
4 years 
350 
.5 c. c. Sugar bouillon (litmus) 
.2 
Death, 48 hours 
190 
“ 72 hours 
W. Mcl. 
6 years 
435 
.5 c. c. Sugar bouillon (litmus) 
Death, 6£ days 
240 
“ 72 hours 
P. W. 
11 months 
280 
.2 c. c. Sugar bouillon (litmus) 
.5 “ “ “ 
Death, 3 days 
Scarlet fever coincident. 
310 
“ 72 hours 
K.-L. bacilli in throat 10 days 
when this culture was taken. 
E. M. 
25 years 
290 
.5 c. c. Sugar bouillon (litmus) 
.5 “ “ “ 
Recovered 
250 
Death, 8| days 
E. G. 
10 years 
Medium size 
1. c. c. Water susp. agar cult. 
Death, 48 hours 
Medium size 
1. c.c. Glycerine bouillon 
“ 36 hours 
D. G. 
14 years 
Medium size 
Agar culture loop 
Recovered. Reinoculated 
Scarlet fever coincident. 
Medium size 
1. c.c. Glycerine bouillon 
Death, 48 hours 
Guin.-pig that recovered was 
Reinoculation 
.4 c.c. Sugar bouillon (litmus) 
Ulceration. Survived 
reinoculated after about 5 wks. 
M. K. 
17 years 
Medium size 
Agar culture loop 
Recovered. Reinoculated 
Both animals reinoculated 
Medium size 
Reinoculation 
1. c.c. Glycerine bouillon 
Death, 72 hours 
with 72 hours glycerine bouillon 
culture after 2 weeks. 
Reinoculation 
1. “ “ 
“ 72 hours 
E. D. 
3|.years 
Large 
Agar culture loop 
Death, 36 hours 
Small 
1. c.c. Sugar bouillon (litmus) 
“ 24 hours 
F. M. 
6J years 
Small 
1. c. c. Sugar bouillon (litmus) 
Death, 48 hours 
Scarlet fever coincident. 
Large 
Agar culture loop 
“ 48 hours 
M. S. 
6 years 
Large 
1. c. c. Bouillon 
Death. 36 hours 
Scarlet fever coincident. 
.5 “ 
“ 36 hours 
Agar culture loop 
“ 48 hours 
Mild Cases. 
W. N. 
32 years 
360 
.3 c. c. Sugar bouillon (litmus) 
Death, 17 days 
Culture in 3d gen. 
B. 
30 years 
Large 
Agar culture loop 
Ulceration. Recovered 
The animal which recovered 
Small 
1. c. c. Sugar bouillon (litmus) 
Death, 36 hours 
was reinoculated after 2 weeks. 
Reinoculation 
1. c. c. Glycerine bouillon 
Death, 72 hours 
S. M. 
50 years 
.5 c.c. Water susp. agar cult. 
.5 c.c. “ “ “ 
Death, 6& days 
Reinoculation 
Recovered. Reinoculated 
S. R. 
27 years 
1. c. c. Bouillon 
.5 c. c. Water susp. agar cult. 
,5 c. c. “ “ “ 
Death, 24 hours 
Culture 72 hrs. old. Reinocu- 
lated about 6 weeks after first 
Death, 48 hours 
Death, 48 hours 
[inoc. 
V. P. 
3 years 
.5 c. c. Water susp. agar cult. 
Death, 7i days 
Medium size 
Agar culture loop 
Death, 36 hours 
.5 c. c. Water susp. agar cult. 
Recovered. Reinoculated 
J.C. 
2 years 
Reinoculation 
240 
1. c. c. Bouillon 
Blood serum culture loop 
Death, 72 hours 
Second inoculation 27 days 
after the first. 
Death, 72 hours 
Convalescent from scarlet 
200 
.2 c. c. Sugar bouillon (litmus) 
Death, 36 hours 
[fever. 
W. M. 
2 years 
190 
Blood serum culture loop 
Death, 72 hours 
Scarlet fever convalescent. 
210 
.2 c. c. Sugar bouillon (litmus) 
Death, 48 hours 
No membrane. Coryza main 
ymptom. 
N. D. 
5 years 
Well grown 
Agar culture loop 
Recovered. Reinoculated 
2d inoculation after 13 days. 
Agar culture loop 
Recovered. Reinoculated 
Reinoculation 
1. c. c. Glycerine bouillon 
Death, 12-18 hours 
Bouillon cult. 72 hours old. 
Reinoculation 
I. c.c. “ “ 
Death, 72 hours 
W. P. 
7 years 
180 
.2 c. c. Sugar bouillon (litmus) 
Death, 7J days 
Convalescent from scarlet 
fever. Coryza the main sym p- 
tom. Cult. 3d gen. 
W. M. 
2J years 
270 
.2 c. c. Sugar bouillon (litmus) 
Recovered. Ulceration 
Scarlet fever convalescent. 
No membrane. Cult. 3d gen. 
J. L. 
6 years 
225 
.2 c. c. Sugar bouillon (litmus) 
.3 c. c. “ “ “ 
Death, 23 days 
[days. 
K.-L. bacilli already present 6 
285 
Recovered 
Cult. 3d gen. 
Severe Cases. 
Name. 
Age. 
Weight (grms ) or| 
size of guin. pigs, j 
Mode of Inoculation. 
Result of Inoculation. 
Remarks. 
T. M. 
6 years 
285 
.3 c. c. Sugar bouillon (litmus) 
.2 “ “ 
Death, 4£ days 
250 
“ 36 hours 
0. C. 
10 years 
200 
.2 c. c. Sugar bouillon (litmus) 
.4 “ “ 
Death, 7) days 
290 
Paralysis. Killed after 3 wks. 
,L M. 
3 years 
200 
.2 c. c. Sugar bouillon (litmus) 
Death, 3) days * 
370 
.5 
“ 48 hours 
H. W. 
2 years 
300 
.5 c. c. Sugar bouillon (litmus) 
.2 
Death, 48 hours 
220 
Recovered 
L. C. 
13 years 
330 
.5 c. c Sugar bouillon (litmus) 
2 
Death, 48 hours 
220 
“ 48 hours 
C. K. 
11 years 
290 
.3 c. c. Sugar bouillon (litmus) 
.3 “ “ “ 
Death, 36 hours 
290 
“ 36 hours 
K. T. 
2J years 
270 
.2 c. c. Sugar bouillon (litmus) 
.4 
Death, 36 hours 
275 
“ 36 hours 
E. K. 
2i years 
340 
.5 c. c. Sugar bouillon (litmus) 
.3 “ “ “ 
Death, 48 hours 
Measles coincident. 
275 
“ 48 hours 
K. B. 
3 years 
270 
.3 c.c. Sugar bouillon (litmus) 
.2 “ “ 
Death, 36 hours 
230 
“ 36 hours 
J. T. 
8 years 
240 
.2 c. c. Sugar bouillon (litmus) 
1.8 “ “ 
Recovered. Ulceration 
Bacillus under cultivation 
M. L. • 
4 years 
240 
240 
Death, 36 hours 
more than 4 weeks when inocu- 
lated into second guinea-pig. 
.2 c. c. Sugar bouillon (litmus) 
Death, 36 hours 
B. L. 
3 yrs. 3 mos. 
215 
.2 c.c. Sugar bouillon (litmus) 
.5 “ “ 
Recovered 
Scarlet fever coincident. 
300 
R. R. 
5 years 
Small 
1. c. c. Sugar bouillon (litmus) 
1. “ “ 
Death, 72 hours 
Scarlet fever coincident. 
Very large 
“ 72 hours 
M. D. 
2 years 
Small 
.5 c. c. Sugar bouillon (litmus) 
Death, 4 days 
“ 4j days 
Scarlet fever coincident. 
Large 
1. 
P. D. 
4 years 
Small 
.5 c. c. Sugar bouillon (litmus) 
1. “ “ “ 
Death, 36 hours 
Large 
“ 72 hours 
G. B. 
9 years 
Medium size 
1. c. c. Glycerine bouillon 
Death, 36 hours 
Medium size 
1. “ “ 
“ 36 hours 
A. G. 
2 years 
Medium size 
1. c. c. Glycerine bouillon 
1. “ 
Death, 48 hours 
Medium size 
“ 4) days 
L. G 
7 years 
280 
.25 c. c. Sugar bouillon (litmus) 
Death, 72 hours 
Culture from membrane cast 
of trachea and bronchi. 
M. C. 
4 years 
Well grown 
1. c. c. Glycerine bouillon 
Death, 36 hours 
Culture from tracheal pseudo- 
membrane. 
B. H. 
8J years 
Large 
Agar culture loop 
Death, 36 hours 
Small 
1. c. c. Sugar bouillon (litmus) 
“ 36 hours 
T. K. 
4 years 
Large 
Agar culture loop 
Death, 3£ days 
Small 
1. c.c. Sugar bouillon (litmus) 
“ 36 hours 
E. B. 
1 yr. 10 mos. 
Small 
l. c. c. Sugar bouillon (litmus) 
Death, 24 hours 
Medium size 
Agar culture loop 
“ 36 hours 
A. R. 
2J years 
.5 c. c. Water susp. agar cult. 
Death, 48 hours 
Two agar cultures used. 
Agar culture loop 
“ 3i days 
.5 c. c. Water susp. agar cult. 
1. “ “ “ 
“ 36 hours 
“ 36 hours 
E. H. 
3 yrs. 5 mos. 
.5 c.c. Water susp. agar cult. 
.5 “ 
Death, 3J days 
“ 6 days 
E. F. 
7 years 
.5 c. c. Water susp. agar cult. 
Death, 13-14 days 
Reinoculation 
.5 
Recovered and reinoculated 
1. cc. (?) Glycerine bouillon 
Survived. Reinoculation 
J. R. 
3 years 
.5 c. c. Water susp. agar cult. 
.5 “ “ 
Death, 4 days 
.... 
Killed after 10 days 
E. R. 
13 months 
.5 c. c. Water susp. agar cult. 
•5 44 44 4k 44 
Death, 8J days 
The bacillus had been in cul- 
Large 
Recovered. Reinoculated 
tures about 6 weeks when re- 
Reinoculation 
1. c. c. Glycerine bouillon 
Death, 48 hours 
inoculated. 
B. M. 
21 years 
275 
.2 c. c. Sugar bouillon (litmus) 
.4 “ “ “ 
Death, 36 hours 
Pneumonia. 
290 
“ 72 hours 
INOCULATIONS WITH THE BACILLUS DIPHTHERIA. 
Fatal Cases. STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
3 
bouillon culture, two generations removed from the case 
of diphtheria, was injected iu the usual manner into a 
guinea-pig to test the virulence. At the same time a 
portion of the culture was filtered through unglazed 
porcelain, and nine-tenths of a cubic centimetre of the 
sterile filtrate injected into another guinea-pig in the 
same way as the bouillon culture. Likewise the cul- 
ture from another case of diphtheria was tested and 
the filtrate injected, but in this second case the quan- 
tity of filtrate injected into the abdominal wall was one 
cubic centimetre, and an additional cubic centimetre 
was thrown into the peritoneal cavity. Both of the 
animals inoculated with the bouillon cultures died 
within thirty-six hours, while those in which the fil- 
trate had been injected in comparatively large quan- 
tities showed no ill effects. 
It is clearly evident, therefore, that the toxic effects 
of the bouillon cultures here used have little to do with 
their apparently greater activity. Furthermore, as to 
whether a more rapid death follows the injection of 
a larger than a smaller quantity of the same culture, 
we do not care to express a positive opinion from the 
data at hand, for it is uncertain in some cases which 
animal received the larger quantity. It may be said, 
however, considering the results of inoculation of 
bouillon cultures as a whole, that two-tenths of a cubic 
centimetre seems to be quite as effective as one cubic 
centimetre. 
The results of the study of the 59 cases of the first 
series may be briefly summarized as follows, if we ap- 
ply the criteria of virulence above alluded to : 
Of this number 28 were fatal and 31 ended in re- 
covery. 
Of the 28 fatal cases, cultures from 22, or 79 per cent., 
caused the death of at least one animal within three 
days, while of the 31 non-fatal cases, those of 23, or 
74 per cent., had this effect. What may be considered 
as full virulent cultures were therefore only a little 
less frequently found among the non-fatal cases than 
among the fatal cases. 
Of the 40 fatal cases of Roux and Yersin all, or 
100 per cent., killed the animal within four days. 
Comparing our 28 fatal cases with these, we find that 
25, or 89 per cent, of the cases gave cultures of this 
degree of virulence. The difference, therefore, be- 
tween the fatal cases of Roux and Yersin and the similar 
cases of our series is represented by 100 per cent, in 
the former and 89 per cent, in the latter, of fairly 
virulent organisms. Of the 39 non-fatal cases of the 
above-named writers, 17, or 44 per cent., killed the ani- 
mal in less than three days, while in our 31 cases, 23, 
or 74 per cent., had that result. 
It cannot be said, therefore, that our results are 
completely in harmony with the classic studies of Roux 
and Yersin on diphtheria. 
We may also combine the different groups of our 
first series in other ways, and institute comparisons. 
Comparing the fatal and severe cases together, on the 
one hand, with the mild and medium severe cases com- 
bined, on the other hand, we find that of the 36 fatal 
and severe cases, 28, or 78 per cent., killed a guinea- 
pig within three days, while of the 23 cases of mild 
and medium severity, 17, or 74 per cent., did so. The 
relation of the two combinations as to the frequency 
with which full virulent bacilli are found among them 
may therefore be expressed by the numbers 78 and 
74, apparently indicating that full virulent cultures are 
not more frequently obtained from fatal and severe 
cases than from the others. Again, considering the 
mild cases by themselves, it is found that 7 of the 11, 
or 64 per cent., as compared with 79 per cent, of the 
fatal cases, or 78 per cent, of the fatal and severe cases 
combined, gave cultures which caused the death of the 
animal within three days, and were therefore full 
virulent. 
In making these comparisons between the different 
classes of cases, we have of course based them upon 
the percentage among them of what may be considered 
as quite virulent organisms and have not considered 
the cases which have yielded cultures of medium or 
slight virulence. It has been thought best to limit 
the comparisons to this point in order to avoid confu- 
sion. 
The study of the second series of 23 cases, in which 
the bacilli have been present for from 8 to 51 days in 
the throat or nose, has been rendered possible by the 
rule of the Boston City Hospital, that no diphtheria 
case can be discharged until cultures from the throat 
and nose show the absence of Klebs-Loffler bacilli, al- 
though the case be otherwise fully recovered. By 
consulting the records of the routine bacteriological 
work on diphtheria carried on in the pathological labo- 
ratory in the same institution, we have been enabled 
to give in all of the cases, except one, the number of 
days which have passed from the date of the first dem- 
onstration of the organism by cultures in the throat or 
nose to the date of the taking of the culture for inocu- 
lation. 
In the-table of the results of our study of this series, 
the culture used is to be considered as of the third 
generation from the case, unless otherwise stated. 
These cases, moreover, unless otherwise specified, have 
been of mild character originally. The result of the 
work of this series may be briefly summarized as fol- 
lows: Of the 23 cases, cultures from 11 have caused 
the death of the guinea-pig in less than three days, 
or 48 per cent, gave full virulent bacilli. It is inter- 
esting to note that in 7 of the 11 virulent cases the 
preseuce of the bacilli in the throat or nose had first 
been shown by cultures 26 to 51 days before the time 
that the culture inoculated was taken. 
It will be seen, however, that this series, considered 
as a whole, shows fewer virulent cultures than the 
other series of 59 cases, the percentage in these per- 
sistent cases being 48 per cent, as compared with the 
78 per cent, of the fatal and severe cases or the 74 
per cent, of the mild and medium cases. 
Whether these differences are due to any attenua- 
tion of the organism in this second series of cases, ow- 
ing to the slightly greater number of generations 
through which it has been cultivated, we do not know. 
It seems, however, to be a generally accepted fact that 
the bacillus diphtherias is not easily attenuated by cul- 
tivation ; and the general tendency of our own experi- 
ence is in this direction, as will be seen from the re- 
sults in some of the instances before alluded to, in 
which reinoculations of an animal have been made 
with a fresh culture of the same bacillus as the first 
inoculation. In some of these instances the organism 
has proven virulent although it had been under culti- 
vation for some time. It may be mentioned that 
among the series of persistent cases are four cases, the 
bacilli from which had previously been inoculated and 
the results included in the first series. In two of these 
four cases the culture had been taken for inoculation 
respectively 33 and 39 days before the taking of the 4 
STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
culture of the present series. Both the earlier and 
later cultures in these two cases killed at least one 
animal in forty-eight hours, one of them with the later 
culture causing the death of the guinea-pig in thirty-six 
hours. Of the other two cases of the four above men- 
tioned, one seemed to show some diminution in the 
virulence of the bacillus, while the fourth case on both 
occasions seemed to yield organisms of little virulence. 
If the results of this study of the cases of the second 
series be compared with those of Roux and Yersin and 
of Tobiesen on the same class of cases, they will be 
found to be totally at variance with those of the for- 
mer, but to agree fairly well with those of the last 
mentioned. Tobiesen12 obtained from 16 out of 19 
convalescent cases cultures which caused the death of 
guinea-pigs with characteristic lesions, while Roux and 
Yersin 1 found that in similar cases the occurrence of 
virulent bacilli was the exception, and believed that a 
progressive attenuation of virulence took place. 
Considering now the whole number of 82 cases in 
both series, we find that but one case seemed to furnish 
organisms of complete innocuousness toward guinea- 
pigs. This case was a fatal one, and in neither of the 
two animals inoculated was any effect noted. There 
are, however, 12 cases which deserve special attention, 
as the bacilli from them showed little virulence. In 
2 of these cases the animal developed a characteristic 
induration and ulceration at the seat of inoculation. 
One was purposely killed, the other survived. In 6 
others of these cases the animals died from 4J to about 
54 days after inoculation, with very slight lesions (or 
none at all) observed at the autopsy. Cultures from 
the seat of inoculation were made at 5 of the autopsies, 
but in only one of them was the Klebs-Loffler bacillus 
recovered. This animal had been inoculated about 
38 days before. In the remaining 4 cases the animals 
died in from 14 to days with lesions of experimen- 
tal diphtheria, and the bacilli were removed from the 
seat of inoculation. Of one of these 4 cases it should 
be explained that lesions were present in one of the 
animals inoculated, but not in the other or only in 
slight degree, and that the bacilli were found at the 
seat of inoculation in the latter, but not in the former. 
The case has, therefore, been considered as one in 
which lesions were produced, and in which the bacilli 
were recovered from the seat of inoculation. 
It may be seen from this description that of these 
12 cases with bacilli of little virulence, 6 yielded 
bacilli which called forth more or less of a character- 
istic reaction in the animals, either locally or in the 
organs. 
Finally, we may state that, with the exception of 
these 12 cases, from all of the remaining cases bacilli 
were obtained which killed at least one guinea-pig 
within ten days. 
From our study of these 82 cases the following 
general statements may be made : 
(1) That there is practically no difference in viru- 
lence to be observed between the bacilli derived from 
severe and mild cases of diphtheria. 
(2) That cases in which the Klebs-Loffler bacilli 
have been present for a longer or shorter period of 
time, furnish fewer virulent cultures than cases of re- 
cent beginning. 
(3) That the Klebs-Loffler bacillus does not, as a 
rule, lose its virulence by long-continued residence in 
the pharynx and air-passages. 
(4) That the Klebs-Loffler bacillus exists with all 
degress of virulence down to innocuousness, as far as 
is shown by the results of its inoculation into guinea- 
pigs, and that the intensity of the reaction in the ani- 
mal bears no constant relation to the symptoms pre- 
sented by the case from which it was derived. 
(5) That there is no relation to be observed be- 
tween the age or sex of the patient and the virulence 
of the bacillus. 
In conclusion, the writer desires to express his 
acknowledgments to Dr. Francis H. Williams for 
valuable clinical data on some of the cases. The 
thanks of the writer are also due to Dr. W. H. Pres- 
cott for kind assistance in this and other portions of 
the work. 
II. 
The results of fourteen autopsies on diphtheria cases. 
The autopsies on cases of diphtheria which are here 
reported have been performed, unless otherwise spec- 
ified, at the Boston City Hospital during the period 
extending from November, 1893, to May, 1894. The 
chief interest in this work attaches to the results of 
the bacteriological examination of the organs. 
Up to the time of the well-known work of Frosch 15 
it was generally believed that the bacillus diphtheria 
never invaded the internal viscera, but was only to be 
found in the local inflammatory lesions. In 10 or 15 
autopsies on this disease, this investigator, by using 
large amounts of material for each culture, could dem- 
onstrate the presence of the bacillus either in the 
blood or in some of the organs. One of the most fre- 
quent places in which it was found was in the pneu- 
monic areas of the lungs. Previous to the work of 
Frosch but a very few instances are on record in which 
the Klebs-Loffler bacillus has been met with in these 
situations. One of these is its occurrence in the 
spleen in a case reported by Kolisko and Paltauf.16 
In the cervical lymph-glands the bacillus has been 
observed by Schmorl17 in 7 out of 10 cases. Recently, 
Booker 86 obtained it in cultures from the spleen, sub- 
maxillary gland, lung, and blood of heart in a case of 
diphtheria. 
In the broncho-pneumonia of diphtheria it seems to 
have escaped observation entirely in spite of the great 
amount of work which has been done on the bacteri- 
ology of this frequent complication of the disease, until 
Johnston 18 found in it a single case, and Strelitz 19 in 
one case among eight autopsies on diphtheria. In 
addition to these cases and those reported by Frosch, 
it has also been observed by Flexner 20 in one of the 
two cases examined by him. 
It is apparent, therefore, that the principal points to 
be considered in these autopsies are the occurrence of 
the organism in the viscera in general and in the 
broncho pneumonias in particular. 
METHODS. 
The medium generally employed for cultures at the 
autopsy has been the coagulated blood serum described 
in the first section of this report. The cultures were, 
in general, made by spreading over the surface of this 
coagulated serum in test-tubes as large an amount of 
the tissue or fluid as would adhere to the end of a 
piece of coarse,'flattened platinum wire, previously 
sterilized in the flame of a Bunsen burner. The Klebs- 
Loffler bacilli are usually present in very small num- 
bers in the organs, and we are inclined to attribute the 
comparative frequency with which we have met them STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
5 
both to the use of the culture medium above mentioned, 
upon which they grow much more vigorously than 
upon the ordinary glycerine agar or similar material, 
and to the large amount of the fluid or tissue spread 
over the culture media. In the detailed reports of 
the results of the study of these cases which will be 
found below, it has been thought sufficient to only in- 
dicate the lesions presented at each autopsy by giving 
the anatomical diagnosis and any necessary remarks. 
In the bacteriological diagnosis of the cases the pres- 
ence of the four organisms, the Klebs-Loffler bacillus, 
the streptococcus pyogenes, the pneumococcus (diplo- 
coccus lanceolatus) and the staphylococcus pyogenes 
aureus are in general the only organisms which have 
been considered. Other bacteria, including the bacillus 
coli communis, have developed iu the cultures more or 
less frequently ; but we have devoted little attention to 
these. 
Autopsy I. By Dr. Wright. Fred W.,aged eight 
years. Clinical diagnosis, scarlet fever. No bacterio- 
logical examination of throat during life, as there was 
no evidence of diphtheria. 
Anatomical diagnosis : Inflammation of pharynx, 
larynx and trachea with muco-purulent exudation; 
slight ulceration of epiglottis; broncho-pneumonia; 
congestion and edema of lungs; acute splenic tumor ; 
acute parenchymatous degeneration of liver and kid- 
neys ; acute lymphadenitis. 
Bacteriological diagnosis by cultures : Klebs-Loffler 
bacilli in exudation of pharynx and ulcer of epiglottis ; 
a culture from one lung contained a few Klebs-Loffler 
bacilli, also streptococci; pneumococci (diplococcus 
lanceolatus), staphylococci (aureus), and other organ- 
isms ; cultures from spleen, kidney, liver, mesenteric 
gland and blood of heart were negative. 
Two guinea-pigs were inoculated with .2 c. c. each 
of a twenty-four-hour bouillon culture of the third 
generation of the Klebs-Loffler bacilli from the pharynx. 
Both died in thirty-six hours, with the characteristic 
lesions at the autopsy. The bacilli were recovered by 
culture from the seat of inoculation in each animal, 
but from none of the organs. • 
A culture of the Klebs-Loffler bacilli found in the 
lung (sixth generation) was similarly inoculated into 
another guinea-pig in the quantity of .2 c. c. Death 
in seventy-two hours, with the characteristic lesions. 
Klebs-Loffler bacilli recovered by culture from seat of 
inoculation. Organs sterile. 
This case is of interest as affording an instance of a 
fatal diphtheria without the presence of pseudomem- 
brane. 
Autopsy II. By Dr. Councilman. Isabella N., 
aged nineteen. Cliuical diagnosis, typhoid fever and 
diphtheria. This case has been more fully reported 
by Dr. Morse in the reports of the Boston City Hospital. 
Anatomical diagnosis : Diphtheritic inflammation of 
pharynx; diphtheritic ulceration of tonsil and larynx ; 
tracheitis ; typhoid ulceration of ilium and colon ; heart 
thrombus ; renal infarction ; broncho-pneumonia, con- 
gestion, edema and bronchitis of lung ; acute splenic 
tumor ; acute lymphadenitis. 
Bacteriological diagnosis by cultures: Klebs-Loffler 
bacilli and streptococci in tonsil and trachea. Typhoid 
bacilli and streptococci in heart thrombus, kidney in- 
farction, liver and kidney. Typhoid bacilli iu spleen 
and in gall-bladder. Streptococci in pleura and lung. 
Bacillus coli communis in mesenteric gland. Cultures 
from blood of heart and tracheal lymph-gland negative. 
The Klebs-Loffler bacilli from the trachea were in- 
oculated into two guinea-pigs. One of the animals 
died in forty-eight hours, and exhibited the character- 
istic lesions of experimental diphtheria at the autopsy; 
.5 to .7 c. c. of a twenty-four-hour bouillon culture was 
used for the inoculation of this animal. The other 
animal recovered, having been inoculated from a cult- 
ure on agar. 
This case is an example of poly-infection with the 
Klebs-Loffler bacilli, the streptococcus pyogenes and 
the typhoid bacillus. The last-named organism was 
distinguished from the bacillus coli communis by not 
producing gas bubbles in solid media, by having no 
effect on litmus milk and by not growing visibly on 
potato ; also by the fact that it was actively motile and 
grew characteristically on gelatine. 
It should be stated, however, that in the case of the 
kidney infarction the typhoid bacilli did not satisfy the 
requirements as to the culture on litmus milk. 
Autopsy III. By Dr. Stokes. Sylvester P., 
aged ten months. 
Anatomical diagnosis: fibrinous exudation on epi- 
glottis ; acute laryngitis; broncho-pneumonia of both 
lungs; acute lymphadenitis of cervical, bronchial and 
mesenteric glands ; acute parenchymatous degeneration 
of liver and kidneys ; acute splenic tumor. 
Bacteriological diagnosis : Klebs-Loffler bacilli and 
streptococci in larynx, cervical lymph-gland and lung. 
Streptococci in heart-blood, spleen and bronchial lymph- 
gland. Streptococci and bacillus coli communis in kid- 
ney. Cultures from the liver and mesenteric lymph- 
gland negative. 
With an agar culture of the Klebs-Loffler bacilli 
from the cervical lymph-gland two guinea-pigs were 
inoculated. One of the animals died in forty-eight 
hours with the characteristic lesions. At the autopsy 
of the animal, cultures were made as usual from the 
seat of inoculation and the organs. In the culture 
from the kidney one colony of Klebs-Loffler bacilli 
developed, and in the culture from the seat of inocula- 
tion the colonies of the organisms were numerous. 
This case is an example of the frequent association 
which has been observed of a streptococcus septicemia 
along with the infection with the Klebs-Loffler bacil- 
lus. 
Autopsy IV. By Dr. Wright. Edith T., aged 
four years, three months. 
Anatomical diagnosis: diphtheritic inflammation of 
pharynx and larynx, acute lymphadenitis, acute splenic 
tumor, ecchymoses in right lung and in mucous mem- 
brane of stomach. 
Bacteriological diagnosis by cultures : Klebs-Loffler 
bacilli in pharynx, tonsil and epiglottis. Klebs Loftier 
bacilli and streptococci in right lung and in mucous 
membrane of stomach. Kidney, spleen, liver, right 
ventricle of heart, tracheal and mesenteric lymph-glands 
all sterile. 
A guinea-pig, weight 210 gms. received .2 c. c. sub- 
cutaneously of a twenty-four-hour bouillon culture of 
the Klebs-Loffler bacilli from the lung. Death in 
forty-eight hours with the characteristic lesions, and 
the bacillus was recovered from the seat of inoculation, 
but found nowhere else. 
The finding of Klebs-Loffler bacilli in the stomach 
has been reported by Loftier21 in a single case. In 
his case a diphtheritic inflammation of the stomach was 
observed, which was not apparent in the one here 
reported. 6 
STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
Autopsy V. By Dr. Councilman. Fred B., aged 
four years. 
Anatomical diagnosis: Diphtheria and septicemia ; 
erythema of skin ; diphtheritic inflammation of pendu- 
lous palate and tonsils ; acute lymphadenitis, with necro- 
tic foci in cervical lymph-glands; edema of posterior 
tracheal tissues ; acute nephritis. 
Bacteriological diagnosis by cultures : Klebs-Loffler 
bacilli and streptococci in spleen, liver and cervical 
lymph-glands. Streptococci in kidney and blood of 
heart. 
A guinea-pig weighing 210 gms. received subcutane- 
ously .2 c. c. of a twenty-four-hour bouillon culture of 
the Klebs-Loffler bacillus found in the lung. Death in 
thirty-six hours with the usual lesions. In addition 
to being recovered from the seat of inoculation, the 
bacilli were also found in the cultures made from the 
blood, liver and spleen of the animal. The kidney 
was negative. 
Autopsy VI. By Dr. Wright. Beatrice B., aged 
nine years. 
Anatomical diagnosis : Diphtheritic inflammation of 
pharynx, tonsils, epiglottis aud larynx; tracheitis; 
broncho-pneumonia of both lungs ; acute lymphadenitis ; 
acute splenic tumor ; arterio-sclerosis of beginning 
of ascending aorta. 
Bacteriological diagnosis: Klebs-Loffler bacilli in 
larynx, tonsil aud lung; spleen, liver, kidney, heart- 
blood aud mesenteric lymph-gland negative for either 
Klebs-Loffler bacilli or streptococci. 
A twenty-four-hour bouillon culture of the bacillus 
from the tonsil aud also from the lung killed two 
guinea-pigs in thirty-six hours, with the usual lesions. 
The quantity injected in each case was .2 c. c. 
Autopsy VII. By Dr. Councilman. Charles Van 
G., aged three years. 
Anatomical diagnosis : Diphtheritic inflammation of 
mucous membrane of nose and pharynx ; slight 
broncho-pneumonia in both lungs; excoriations on 
face and nose. 
Bacteriological diagnosis by cultures : Klebs-Lofller 
bacilli aud streptococci in pharynx, nasal cavity and 
lung. Streptococci in blood of heart, in liver aud in 
spleen. Kidney and cervical lymph-glaud sterile. In 
mesenteric gland Klebs-Loffler bacilli. The culture 
from the excoriation on face showed the presence of 
Klebs-Loffler bacilli, and that from the nose Klebs- 
Loffler bacilli and streptococci. 
Autopsy VIII. By Dr. Councilman Child. Fe- 
male. 
Anatomical diagnosis: Operation wound • of hip; 
abscess about hip-joint, involving ilium and retroperi- 
toneal tissues ; metastatic abscesses in kidney, lungs 
and left shoulder; diphtheritic inflammation of larynx, 
with necrosis and erosion of epiglottis ; broncho-pneu- 
monia and bronchitis. 
Bacteriological diagnosis by cultures: Staphylo- 
coccus aureus in primary abscess, kidney, spleen aud 
liver. Klebs-Loffler bacilli aud staphylococcus pyo- 
genes aureus in cultures from tonsil, epiglottis and lung 
(areas of broucho-pneumouia). 
Autopsy IX. By Dr. Wright. Irene T., colored, 
aged one year. In contagious wards more than two 
months. Klebs-Loffler bacilli in cultures made from 
nose during seven or eight weeks. Never found in 
throat during life. Vide first case in the second series 
of preceding section. 
Anatomical diagnosis : Extensive diphtheritic ulcer- 
ation of epiglottis and larynx ; diphtheritic inflamma- 
tion of inferior turbinate bones and nasal cavities with 
fibrinous exudation ; infarction of lung; acute de- 
generation of liver and kidneys ; acute lymphadenitis ; 
acute splenic tumor ; slight pericardial effusion ; otitis 
media and abscess of both mastoid bones. 
Bacteriological diagnosis by cultures : Klebs-Loffler 
bacilli in ulcer of larynx and epiglottis and in bronchial 
lymph-glaud. In lung iufarction fairly numerous 
Klebs-Loffler bacilli and other bacteria. Streptococci 
in liver and spleen. Pneumococci in kidney (diplo- 
coccus lanceolatus). In blood of heart a few bacilli 
(coli communis ?). Klebs-Loffler bacilli and strepto- 
cocci in pus of mastoid abscess. 
This case will again be referred to in a following 
section. 
Autopsy X. By Dr. Councilman. Charles R., 
aged four years. 
Anatomical diagnosis: Diphtheritic ulceration of 
pharynx and larynx ; edema of pharynx ; tracheitis ; 
bronchitis ; extensive broncho pneumonia of right lung ; 
erythema of skin ; purulent conjunctivitis and keratitis. 
Bacteriological diagnosis by cultures: Epiglottis, 
Klebs-Lofller bacilli aud streptococci. Conjunctival 
pus, Klebs-Loffler bacilli and other bacteria. Lung, 
Klebs-Loffler bacilli, streptococci, pneumococci and 
other organisms. Edematous tissue with necrotic foci, 
behind left tonsil, streptococci. Kidney, streptococci, 
one colony; two to three colonies of staphylococcus 
aureus and other organisms. Spleen, very numerous 
colonies of various organisms, among them pneumococci. 
Autopsy XI. By Dr. Councilman. Thomas M., 
aged six years. Vide list of fatal cases, Section 1. 
The bacilli taken from the throat during life were 
found virulent. This case will be referred to in an- 
other section. 
Anatomical diagnosis : Diphtheritic inflammation of 
tonsils ; diphtheritic inflammation and ulceration of 
trachea ; broncho-pneumonia and edema of both lungs ; 
acute parenchymatous degeneration ; acute lymph- 
adenitis ; paronychia of toe. 
Bacteriological diagnosis by cultures: In tonsil, 
trachea and bronchial lymph-gland, Klebs-Loffler bacilli, 
streptococci and staphylococcus pyogenes aureus. In 
lungs, Klebs-Loffler bacilli, streptococci, pneumococci, 
and in one lung also staphylococci pyogenes aureus. 
In liver, Klebs-Loffler bacilli and streptococci. 
Streptococci in blood of heart and liver. Klebs-Loffler 
bacilli and staphylococcus pyogenes aureus in pus from 
paronychia. Right kidney, spleen and tracheal lymph- 
gland sterile; left kidney a very few colonies of the 
bacillus coli communis. 
A guinea-pig weighing 200 gms. was inoculated with 
.2 c. c. of a twenty-four-hour bouillon culture of the 
Klebs-Loffler bacilli from the bronchial lymph-gland. 
No reaction was noted in the animal. 
This case is noteworthy as being a fatal diphtheria 
in which a non-virulent organism was found at the 
autopsy while the Klebs-Loffler bacilli found during life 
were virulent. 
Autopsy XII. By Dr. Wright. Annie O’T., 
aged one year. 
Anatomical diagnosis : Diphtheritic inflammation of 
pharynx, larynx and trachea; broncho-pneumonia; 
ulceration of base of tongue; necrosis and ulceration 
of epiglottis ; acute lymphadenitis ; acute splenic tumor. 
Bacteriological diagnosis by cultures : Klebs-Loffler 
bacilli in larynx, pharynx, bronchi of both lungs and in STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
7 
small superficial ulcer on left hand. Streptococci and 
Klebs-Lofiler bacilli in cervical lymph-gland and in one 
lung. Streptococci in the other lung. Kidney, spleen, 
liver and blood of heart, sterile. 
A guinea-pig weighing 280 gms. was inoculated with 
.3 c. c. of a twenty four-hour bouillon culture of the 
bacilli from a bronchus. Death after six days. The 
organism was found by culture in the blood and spleen 
of the animal, in addition to being found at the seat of 
inoculation. 
Another guinea-pig weighing 245 gms. received .2 
c. c. of a twenty-four-hour bouillon culture of the 
bacilli found in the lung. The animal survived for 
thirty-six days, with an ulceration at the seat of inocu- 
lation. The bacilli were recovered by cultures from 
the ulcerated tissue, but from none of the organs. 
Autopsy XIII. By Dr. Councilman. Edward B. 
Clinical diagnosis, scarlet fever. The case had been 
recently operated upon for congenital hernia before 
entry into hospital. 
Anatomical diagnosis : Erythema of skin ; operation 
wound in abdominal wall with abscess formation ; 
diphtheritic ulceration of pharynx and larynx ; bronchi- 
tis ; broncho-pneumonia of both lungs; acute degener- 
ation of parenchymatous organs. 
Bacteriological diagnosis by cultures : Larynx and 
pharynx, Klebs-Lofiler bacilli. In one of these cultures 
also streptococci. Kidney, numerous streptococci. 
Spleen, numerous Klebs-Lofiler bacilli and a few strep- 
tococci. Other organisms present. Liver, a few 
streptococci. 
Autopsy XIV. By Dr. Wright. Sarah F., aged 
two and a half years. 
Anatomical diagnosis : Diphtheritic inflammation of 
epiglottis, trachea and bronchi; pseudomembrane pres- 
ent in trachea about bifurcation and extending into 
bronchi; bronchitis; congestion of lungs ; ecchymoses 
in mucous membrane of stomach ; acute lymphadenitis ; 
acute splenic tumor; acute parenchymatous degener- 
ation ; excoriations on leg and about anus and vulva. 
Bacteriological diagnosis by cultures : Klebs-Lofiler 
bacilli and staphylococcus pyogenes aureus in trachea, 
lung, liver, kidney, heart-blood, and in excoriations on 
leg. In addition to these the lung culture contained a 
few streptococci and pneumococci (?) ; that of the 
liver, streptococci; and the kidney culture, pneumo- 
cocci. The spleen was sterile. In culture from 
tracheal lymph-gland a few streptococci and one colony 
of Klebs-Lofiler bacilli. In the culture from mesen- 
teric lymph-gland a very few Klebs-Lofiler bacilli and 
other organisms. 
The important points in the foregoing may be briefly 
summed up as follows : 
Of the 14 autopsies the Klebs Loffler bacillus has 
been found in the lung in 13 cases, in the liver in 3, 
in the spleen 2, in the cervical or bronchial lymph- 
glands in 5, in the kidney in 1, in the blood of heart 
in 1, and in the mesenteric lymph-glands in 2. It has 
been cultivated from the mucous membrane of the 
stomach in two instances and once from the edematous 
tissue behind the esophagus. In 7 of the 12 cases, or 
nearly 60 per cent., there has been a streptococcus 
septicemia in addition to the diphtheritic infection. 
This seems to be another confirmation of the view 
of Barbier 33 and others that there are two distinct 
kinds of diphtheria: one, the toxic form or a monoin- 
fection with the Klebs-Lofiler bacillus ; the other a poly- 
infection with that organism and the streptococcus. 
In two other cases there has also been a similar poly- 
infection in which the staphylococcus pyogenes aureus 
has taken the place of the streptococcus in the septi- 
cemia. One of these septicemias, however, might be 
considered as having an infection-atrium in an abscess 
about the hip-joint, and one of the streptococcus septi- 
cemias an infection-atrium in an operation wound for 
congenital hernia. 
In addition to these there is one other case of poly- 
infection (Autopsy X), in which there seems to have 
been a general infection with several organisms ; but, 
owing to the lack of cultures from the liver and heart 
blood, nothing very definite can be said about it. 
Broncho-pneumonia was present in 10 of the 14 
autopsies. The occurrence of the Klebs-Loftier bacillus 
in the lung seems to be independent of the coincidence 
of this lesion, for it has been absent in one broncho- 
pneumonia and yet present where no pneumonic condi- 
tion was made out. 
In fact, we are inclined to explain the frequent 
occurrence of the bacillus in the cultures from the 
lung in the cases to its being present in the smaller 
bronchi — an explanation which Strelitz has made use 
of in the case reported by him. The organism most 
frequently found associated with the Klebs-Lofiler 
bacillus in the lungs has been the streptococcus. These 
two have been present together in 9 cases, in 7 of 
which there was a pneumonic condition. The strep- 
tococcus has also been noted in pure culture in one 
pneumonia. 
The pneumococcus (diplococcus lanceolatus) has 
been found much less frequently, appearing in but 3 
or probably 4 of the 10 cases of pneumonia, and then 
always along with the streptococcus and Klebs-Lofiler 
bacillus. 
The staphylococcus pyogenes aureus has been ob- 
served in four of the pneumonias, once with the Klebs- 
Lofiler bacillus alone, and three times in company with 
all three of the above mentioned organisms. 
III. 
The pathological anatomy and bacteriology of experi- 
mental diphtheria. 
In the course of all of this work autopsies on guinea- 
pigs which have died after inoculation with the Klebs- 
Lofiler bacillus have been made in 160 instances. 
This number includes two animals inoculated from an 
autopsy not included in our list. In the majority of 
cases notes have been taken with reference to the con- 
dition of the tissues at the seat of inoculation, the 
presence or absence of the characteristic subcutaneous 
edema, the presence of the acute lymphadenitis, of fluid 
in the thoracic cavity, of edema of the lungs, of necro- 
tic foci in the liver and the condition of the suprarenal 
capsules. 
These points embrace nearly all of the gross lesions 
which are observed in experimental diphtheria, and it 
has been considered worth while to give here in a 
statistical manner the results of our observations. 
Local reaction of varying intensity has been observed 
in 145 of the 160 guinea-pigs. This has consisted in 
general of injection, edema, hemorrhage, fibrinous ex- 
udation and necrosis of the tissues of the abdominal 
wall. Great variation is observed in the character of 
the lesions at this point. In some cases nothing more 
than slight injection and a few slight hemorrhages are 
seen, while in others all of the conditions mentioned 8 
STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
may be present. In animals which have survived for 
some days the tissues at this point may be sometimes 
observed to be infiltrated with a dense, tough, brawny 
material extending over a considerable area and greatly 
increasing the thickness of the belly wall. Over this 
area the skin is tightly adherent. In 13 animals, in- 
cluding 2 which did not succumb to the effects of the 
inoculation, ulceration has been observed. In some 
of these the eschar has been several centimetres in 
diameter. The sloughing process seems to be preceded 
by an induration of the character above described in 
the tissues beneath. 
The occurrence of the typical gelatinous edema of 
the subcutaneous tissues of the abdomen and thorax 
has been observed in a certain number of cases. 
Local edema, more or less extensive, may be said to 
have been present in about 100 of the 160 animals. 
Enlargement and usually more or less congestion of 
some or all of the lymph-glands of the inguinal and 
axillary regions has been noted in 142 of the autopsies. 
In some cases the glands have presented a hemorrhagic 
condition. 
The lymph-glands at the brim of the pelvis in the 
retroperitoneal region and those of the mesentery 
seem to be usually affected along with the superficial 
ones. Congestion of the mesenteric glands is much 
less frequent, however, than in the case of the retro- 
peritoneal glands. 
Effusion of fluid into the thoracic cavity has been 
noted in 68 of the 160 autopsies. The fluid was almost 
invariably colorless and varied greatly in amount. 
In some instances the thorax has apparently been 
completely filled. Edema of the lungs is recorded in 
82 cases. 
A characteristic lesion of experimental diphtheria is 
the presence of necrotic areas, visible to the naked 
eye in the liver. These lesions are not common, as 
will be apparent from the fact that they have occurred 
in about 49 instances, or 31 per ceut., and their occur- 
rence seems to bear no relation to the duration of the 
infection in the animal. 
A more striking phenomenon, however, than any of 
the preceding, is the congestion or hemorrhagic condi- 
tion of the suprarenal capsules. These have been 
found more or less red in color, instead of the normal 
bright yellow, in 130 of the 160 cases. This condition 
is almost invariably present in animals which have 
succumbed within a few days after inoculation ; and 
i n general it may be said that the combination of all 
the lesions enumerated above is to be more frequently 
observed in acute cases. 
But one of the auimals inoculated in the course of 
the work developed paralysis. This is in accord with 
the experience of Loftier, who seems to have observed 
it very rarely in the guinea-pigs inoculated by him. 
In our study of the bacteriology of experimental 
diphtheria the main purpose has been to determine the 
frequency with which the Klebs-Loffler bacillus invaded 
the blood and internal organs of the inoculated animal. 
In this disease in the guinea-pig as well as in diphtheria 
in man, the bacillus has been considered as remaining 
at the point of invasion. The only observations of its 
occurrence in the organs of an inoculated guinea-pig 
which we have found in the literature is that of Zar- 
niko,p who mentions having met with it in a necrotic 
focus in the liver, and that of Abbott,22 who has re- 
ported the presence of the bacillus in certain lymphatic 
structures in the mesentery. 
At the autopsies of the animals, as a matter of rou- 
tine, cultures have been made from the seat of inocu- 
lation, blood of heart, liver, spleen and kidney. The 
coagulated blood serum previously described has been 
the medium employed for the purpose, with a few ex- 
ceptions. As in the autopsies on man, rather more 
material than is usual has been commonly taken for 
each culture, by means of the coarse flattened platinum 
wire. In a few cases cultures have either not been 
made at all or only from some of the above-named 
situations. The numbers given below include cultures 
from three animals which have not died spontaneously, 
but have been purposely killed. The results may be 
briefly summarized as follows : among 155 livers from 
which cultures were made, 19 yielded Klebs-Loffler 
bacilli. Of 152 cultures from as many spleens, 15 
contained the same organism. The bacillus also grew 
in 4 of 151 cultures from the kidney, and in 7 of 153 
cultures from the blood of the heart. 
The organism has therefore occurred most frequently 
in the liver and spleen, being found in the former in 
12 per cent, of the autopsies. The colonies which de- 
velop in these cultures are, as a rule, few in number, 
rarely exceeding a half-dozen in a tube. The presence 
of the Klebs-Loffler bacilli in the viscera does not seem 
to bear any relation to the length of time the animal 
survived nor to the clinical character of the case from 
which the culture used for inoculation was derived, 
nor is its presence in the liver definitely associated 
with the occurrence of the foci of necrosis alluded to 
above. Moreover, we have no reason to believe that 
its presence in any internal organ has any effect per se 
upon the tissue of that organ. 
The cultures from the seat of inoculation in 161 
animals have contained the bacillus inoculated in all 
but 20. It may also be mentioned that from three 
guinea-pigs the organism has been recovered by culture 
from the seat of inoculation about thirty-one to thirty- 
eight days after the date of inoculation. The persis- 
tence of the bacillus in the tissues for this length of 
time is interesting. 
IV. 
Cases of infection of various ulcerated surfaces and in- 
flammatory processes with the Klebs-Loffler bacillus, 
as well as some other instances in which it has been 
observed. 
The occurrence of the Klebs-Loffler bacillus in other 
pathogenic processes than diphtheria has been rarely 
noted in the literature. The only other condition in 
which its presence has been repeatedly demonstrated 
is rhinitis fibrinosa. That this disease, however, is 
really to be regarded as a form of diphtheria of the 
nasal mucous membrane, has been shown by the work 
of Concetti,28 Stamm,24 Bagiusky 26 and Abbott.28 
In the edematous tissue about tracheotomy wounds 
in diphtheria, Sprouk27 has found the Klebs-Loffler 
bacillus in three cases. It has also been observed by 
Neisser 28 in a superficial ulceration about the anus of 
a diphtheria case, and was proven virulent. Brunner 29 
isolated it from three cases of inflammation of the 
fingers and from a case of phlegmon of the scrotum. 
The bacilli from only two of these were found to be 
virulent, the others yielding organisms of a mild degree 
of virulence. In all four cases the bacillus was accom- 
panied by pus organisms. 
In diphtheritic conjunctivitis its presence has been 
reported by Escherieh,2 by Babes,80 by Elschig 81 and STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
by Kolisko and Paltauf 16 in single cases. In the case 
of the first mentioned the organism was non-virulent. 
Three cases of the occurrence of the Klebs-Loffler 
bacillus in the pus of otitis media have been reported 
by Councilman,32 and one case of its occurrence in 
diphtheritis of the stomach by Loffler.21 Finally, 
Howard 88 has found it in pure cultures and in large 
number in the valvular vegetations, spleen and kidney 
of a case of ulcerative endocarditis. The organism 
here was found to be non-virulent. The writers named 
seem to comprise all those who have observed this organ- 
ism iu inflammatory processes other than diphtheria. 
It will be noted that in most of the few cases in 
which inoculations have been made, the bacillus from 
these sources has exhibited little or no viruleucg. This 
lack of virulence, or rather the manifestation of only 
slight virulence, towards guinea-pigs has also been ob- 
served in some of our cases, as will be seen below. 
In the study of cases of the occurrence of the Klebs- 
Loffler bacillus which are reported in this section, the 
same methods as those described iu the preceding sec- 
tion have been employed. 
When convenient the sterilized cotton “swabs” 
have been used for obtaining the material for culture. 
The bouillon cultures used for inoculation have been 
made in the one-per-ceut. sugar bouillon colored with 
litmus, which was mentioned in the first section of this 
work. Cultures, have, as in the other cases, been 
made from the organs and the seat of inoculation of 
the animals which succumbed. 
Some of the cases have been observed at the au- 
topsies reported in the second section, but it has been 
thought better to reserve the special descriptions for 
this section. 
Case I. Fistula in ano. Wm. B., aged eighteen 
years. Admitted to contagious wards of the Boston 
City Hospital with diphtheria. Klebs-Loffler bacilli 
were fouud in throat by culture. The patient also had 
a fistula in auo. Some two weeks after admission 
active inflammation developed in this. The ulcerated 
surface exposed between the nates was about one inch 
square. The tissue about it was inflamed for two or 
three inches. The ulcer was covered with a greenish, 
slimy exudation, and eventually a false membrane was 
formed. This acute condition subsided after a time, 
and the patient also recovered from the diphtheria. 
A culture from the ulcer showed the presence of 
Klebs-Loffler bacilli. These were found very virulent, 
the two animals dying in thirty-six and seventy-two 
hours respectively after inoculation with .2 c. c. and 
.4 c. c. of a twenty-four-hour bouillon culture. They 
exhibited the characteristic lesions, and the organism 
was recovered by culture from the seat of inoculation 
in both. 
Case II. Ulcer on arm. Winifred C., aged 
ten months. Vide Case X. Admitted to contagious 
wards with diphtheria. Klebs-Loffler bacilli demon- 
strated by culture. Some time after admission, from a 
vaccine pustule on left arm a deep gangrenous ulcer 
formed. A culture from this showed the presence of 
Klebs-Loffler bacilli. 
Two guinea-pigs, weighing 210 and 260 gms. re- 
ceived respectively .2 and 1 c. c. of a twenty-four-hour 
bouillon culture of the third generation. Both survived 
from seven to eight days. At their autopsy, exudation 
and infiltration was found at the seat of inoculation. 
The only other marked lesion was edema of the lungs 
in each, as well as some glandular enlargement. 
The Klebs-Loffler bacilli were recovered bj cultures 
from the seat of inoculation in both, and from the liver, 
spleen and kidney of one. The cultures from the other 
animal showed the presence of the bacillus in the 
spleen alone. 
Case III. Excoriations on face. Charles Van G., 
aged three years. Vide Autopsy VII. 
At the autopsy of this case, excoriations on face and 
nose were noted and cultures made from them. In 
the culture from the excoriation on the nose Klebs- 
Loffler bacilli and streptococci developed and in that 
of the excoriations on the face, Klebs-Loffler bacilli. 
No animal inoculations were made. 
Case IV. Excoriation on lip. Fred B., aged four 
years. Vide Autopsy V. 
At the autopsy of this case a number of small ex- 
coriations were observed on the lip below the nasal 
opening. Klebs-Loffler bacilli developed in the cult- 
ure made from these. 
A guinea-pig weighing 425 gms. received .3 c. c. of 
a twenty-four-hour bouillon culture (third generation) 
of this bacillus subcutaneously. The animal died in 
forty-eight hours. 
The usual appearances of acute experimental diph- 
theria were noted at the autopsy. The bacillus was 
recovered by culture from the seat of inoculation, but 
from nowhere else. 
Case V. Excoriation on leg. Sarah F., aged two 
and a half years. Vide Autopsy XIV. 
At the autopsy of this case a slight excoriation over 
anterior aspect of the left tibia was observed and a 
culture was made from it. Klebs-Loffler bacilli and 
staphylococcus pyogenes aureus developed in this. No 
animal inoculation. 
Case VI. Ulcer on leg. Idella McL., aged five 
years. Vide Section I, Series 2. Admitted to the 
contagious wards with diphtheria. Klebs-Loffler bacilli 
present by culture. Some time after admission a deep 
undermining ulcer with brawny edges and grayish 
base formed on right leg. It was supposed to have 
originated from a puncture with a hypodermatic 
syringe. 
A culture on two occasions, several days apart, 
showed the presence of Klebs-Loffler bacilli. The 
bacilli obtained from the first culture did not cause the 
death of the animal until about twenty-nine days after 
inoculation. The guinea-pig weighed 190 gms. No 
definite lesions were found at the autopsy, and the 
bacillus was not recovered from the seat of inoculation. 
The second culture, however, furnished a virulent 
organism, killing a guinea-pig (weight 210 gms.) in 
thirty-six hours. The quantity inoculated in each 
case was .2 c. c. and a bouillon culture was used. The 
first animal was inoculated with a culture of the fifth 
generation and the second animal with one of the third 
generation. It may be possible that the discrepancy 
in the results of the inoculation may have been due 
to some attenuation of the organism which was ob- 
tained in the first instance. It may be added that the 
second animal showed characteristic lesions and the 
bacilli were recovered from the seat of inoculation but 
from none of the organs. 
It is worthy of note that the bacilli from the nose 
of this case showed little or no virulence. 
Case VIT. Ulcer on hand. Annie O’T., aged one 
year. Vide Autopsy XII. 
At the autopsy of this case a small superficial ulcer 
less than one centimetre in diameter was observed on 10 
STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
the left hand. A culture made from this yielded Kleb- 
Loffler bacilli as well as other organisms. 
A guinea-pig weighing 220 gms. was inoculated sub- 
cutaneously with .3 to .5 c. c. of a twenty-four-hour 
bouillon culture of the third generation. The animal 
survived four and a half days. Characteristic lesions 
were found at the autopsy. The blood of the heart 
as well as the seat of inoculation was found to contain 
Klebs-Loffler bacilli. In the culture from the former 
but one colony developed. The other organs were 
negative. It may be mentioned that the bacilli from 
the bronchus and from the lung of this case were also 
found to be of medium or slight virulence toward the 
guinea-pig. as may be seen by Autopsy XII. 
Case VIII. Otitis media. Pus from ear. Irene 
T., aged one year. Vide Autopsy IX and Case IX. 
The otitis media with perforation of the tympanum 
occurred in the course of diphtheria. Klebs-Loffler 
bacilli had been present, by culture, iu the nose for 
some time (vide Section 1, Series 2). A moderate 
number of Klebs-Loffler bacilli and numerous strepto- 
cocci were found to be present iu cultures made during 
life from the thin but abundant purulent discharge from 
the ear. 
A guinea-pig weighing 190 gms. was inoculated sub- 
cutaneously with .2 c. c. of a twenty-four-hour bouillon 
culture (third generation) of the Klebs-Loffler bacilli 
thus obtained. The animal died in thirty-six hours 
with characteristic lesions. The bacillus was recovered 
only from the seat of inoculation. 
Case IX. Mastoid abscess. Irene T. Vide Au- 
topsy IX, et supra. 
At the section the calvarium and brain were removed 
and the mastoid cells on both sides broken open. Both 
were found to contain creamy pus. A culture was 
made from the pus of the mastoid bone of the side 
opposite to that of the ear from which the culture 
mentioned above was taken (intra vitam). On this 
there developed Klebs-Loffler bacilli and streptococci. 
No animal inoculated. 
Case X. Otitis media (?) . Pus from ear. Win- 
ifred C., aged ten months. Vide Case II. 
Admitted to contagious wards with diphtheria. The 
presence of Klebs-Loffler bacilli was demonstrated by 
cultures. In the course of her stay in the ward a dis- 
charge from the ear was observed. This was found 
to contain Klebs-Loffler bacilli along with other organ- 
isms. 
A guinea-pig of 340 gms. weight, which was inocu- 
lated with .3 c. c. of a twenty-four-hour bouillou cult- 
ure, fifth generation, survived eleven and one-half 
days. Some of the lesions of experimental diphtheria 
were found at the autopsy, principally shown by a red 
color of the suprareuals. The bacillus was recovered 
fiom the seat of inoculation. As in the case of the 
bacillus from the ulceration on the arm of this same 
patient, this bacillus from the ear showed only a slight 
degree of virulence. Some attenuation by cultivation 
may have taken place, however, as it was inoculated 
in the fifth generation. 
Case XI. Purulent conjunctivitis with keratitis. 
Charles R., aged four years. Vide Autopsy X. 
At the autopsy the right eye was observed to con- 
tain a considerable amount of pus beneath the lids, 
and the cornea was cloudy. A culture from the pus 
showed the presence of Klebs-Loffler bacilli in addition 
to pus organisms. No inoculation of an animal was 
*made. 
Case XII. Thomas M., aged six years. Vide 
Autopsy XI. 
At the autopsy a culture was taken from the par- 
onychia of the toe. In this there developed Klebs- 
Loffler bacilli and numerous colonies of staphylococcus 
pyogenes aureus. 
A guinea-pig weighing 200 gms. was inoculated 
with .2 c. c. of a twenty-four-hour bouillon culture, 
third generation, of the Klebs-Loffler bacilli thus ob- 
tained. The animal survived, as did also the guinea- 
pig inoculated with the Klebs-Loffler bacillus found in 
the bronchial glaud. As has been stated elsewhere, 
virulent bacilli were found in the throat of this case 
during life. The organism inoculated was studied in 
cultures ou blood serum, agar and litmus bouillon, and 
was found both in cultures and in morphology to agree 
with the typical Klebs-Loffler bacillus. The litmus 
bouillon was rapidly colored red, which, according to 
Escherich,2 is only seen in cultures of the virulent 
bacillus. 
Case XIII. Acute ulcerative endocarditis. Au- 
topsy by Dr. Councilman. Body, that of a young 
man. 
Anatomical diagnosis. Acute ulcerative endocardi- 
tis, acute iutracapillary glomerulo-nephritis. 
Bacteriological diagnosis by cultures : Spleen, liver, 
both lungs, wall of left ventricle of heart, all negative. 
Aortic valve (vegetation), Klebs-Loffler bacilli (a half- 
dozen colonies), pneumococci, staphylococci, bacillus 
coli communis. Tricuspid valve, Klebs-Loffler bacilli 
(a half-dozen colonies), pneumococci and other organ- 
isms. Kidney, two typical colonies of typical Klebs- 
Loffler bacilli developed in the culture. 
The Klebs-Loffler bacilli from the valves of each 
side of the heart were inoculated in bouillon cultures 
into four guinea-pigs. The quantities used varied from 
.3 or .4 c. c. to 1 c. c. All of the animals survived, 
the inoculation having little or no effect upon them 
even locally. The bacilli from each valve were also 
carefully studied in their growth in bouillon, agar, 
blood serum, potato and gelatine as well as in their 
morphology. In all these respects they were found to 
be identical with the Klebs-Loffler bacillus. 
It may be mentioned also that they produced a rapid 
reddening of the litmus-sugar bouillon, which Escherich 
states is only done by virulent Klebs-Loffler bacilli. 
This red color was rapidly produced and persisted in- 
definitely. The agar cultures were never observed to 
show the brown color which the same writer regards 
as characteristic of the so-called pseudo-diphtheritic 
bacillus. The bacillus fouud in the kidney also changed 
the bouillon to a red color. 
This case resembles very much the case of ulcerative 
endocarditis reported by Howard, which we have 
before alluded to. Iu that case a uon-virulent Klebs- 
Loffler bacillus was found in the valvular vegetations 
and iu the organs. Its identity with the true Klebs- 
Loffler bacillus in all respects except virulence was 
fully demonstrated. 
Case XIV. From a tuberculous lung. John S., 
aged twenty-six years. Autopsy by Dr. Councilman. 
The case was essentially one of disseminated tuber- 
culosis. The lungs were most affected. At the au- 
topsy cultures were made as usual from all the organs. 
There was a tuberculous ulcer of the trachea from 
which no cultures were made. In the culture from 
the lung numerous colonies of Klebs-Loffler bacilli and 
streptococci developed, in addition to a few colonies of STUDIES IN THE ELVTIIOLOGY OF DIPHTHERIA. 
11 
staphylococcus aureus and other organisms. Strepto- 
cocci and other organisms were present in the culture 
made from the spleen, while the kidney and liver were 
sterile. 
The Klebs-Loffler bacillus from the culture from the 
lung was inoculated into a guinea-pig weighing 200 
gms.; .5 c. c. of a twenty-four-hour bouillon culture of 
the second generation was used. Death in thirty-six 
hours, with the lesions of experimental diphtheria. 
The bacillus was recovered from the seat of inoculation, 
but from none of the organs. 
Case XV. From a lung in the stage of gray he- 
patization of a lobar pneumonia. Annie K., £ged 
fifty-eight years. Autopsy by Dr. Wright. 
At the autopsy portions of both lungs were found 
to be consolidated, and were in the stage of gray hepa- 
tization. One lung had partly broken down, and a 
thoracentesis had been performed. The culture from 
a consolidated area in one of the lungs developed Klebs- 
Loffler bacilli (not numerous) in addition to numerous 
colonies of streptococci and a few of the staphylococcus 
pyogenes aureus, as well as other bacteria. The cult- 
ures from the blood, kidney, spleen and liver showed 
the presence of a streptococcus septicemia. The throat 
was not examined. A guinea-pig inoculated in the 
usual manner with the Klebs-Loffler bacillus died after 
about three weeks. No lesions found. 
The organism agreed with the Klebs-Loffler bacillus 
in its morphology and in its growth in blood serum 
and in litmus bouillon, which latter was reddened. 
The production of acid thus indicated is said by Es- 
cherich, as before noted, to occur only with the virulent 
form of the organism. The bacillus has not proven 
virulent and constitutes another exception to that 
writer’s statement. 
Case XVI. From the skin of the thigh of a diph- 
theria patient. Beatrice C., admitted to hospital with 
diphtheria. As the child lay in its crib with one 
thigh exposed, a culture was obtained by rubbing a 
swab over the skin where a dark reddish area was ob- 
served. This area of discoloration was evidently due 
to a puncture with a hypodermatic syringe. There 
was no ulceration. A moderately large number of 
colonies developed, among which was a fair proportion 
of colonies of the Klebs-Loffler bacillus. This, when 
inoculated into a guinea-pig, caused its death in seven 
and a half days. The animal weighed 230 gms., and 
received .2 c. c. of a twenty-four-hour bouillon culture 
of the second generation. At the autopsy of the ani- 
mal dense induration and exudation in the tissues 
about the point of inoculation were found, together 
with lymphadenitis and some edema of the lungs. 
The spleen was large and soft. The suprarenals nor- 
mal. The bacillus was recovered from the seat of 
inoculation, but from nowhere else. 
Case XVII. Unsuspected diphtheria. Autopsy by 
Dr. Wentworth on the body of a child. 
The child had been wearing a trecheotomy tube for 
some two months, in consequence of an operation on 
the larynx for a new growth. Death occurred rather 
suddenly after a slight rise of temperature lasting 
about three days. The organs were brought to the 
laboratory for examination. Diphtheritic membrane 
was found in the trachea about the bifurcation. No 
broncho-pneumonia. A culture from this showed the 
presence of Klebs-Loffler bacilli and streptococci. No 
animal inoculations were made. 
The striking thing about the Klebs-Loffler bacilli 
obtained in these cases is the large proportion of mildly 
virulent or non-virulent forms among them. Out of 
12 cases in which the virulence has been tested, three 
have yielded bacilli of little or no pathogenic effect on 
the animals, and in three other cases the guinea-pigs 
inoculated from them have survived, seven and a half 
days and longer. It may therefore be said that a mild 
degree of virulence, or none at all, has been observed 
in oO per cent, of the organisms so obtained which 
have been tested. 
These results are in accord with the experience of 
others, as noted in the first part of this section. 
Whether this lack of virulence of the bacillus is due to 
attenuation by long continued residence away from its 
normal habitation, the air-passages, future study may 
decide. 
From a consideration of certain of the above cases, 
we think that we are justified in holding that any 
excoriated or ulcerated surface on the body of a 
diphtheria patient will usually be found to be infected 
with the bacillus of that disease. 
V. 
On the morphology and biology of the Klebs-LbJJler 
bacillus. 
The Klebs-Loffler bacillus in its morphology is the 
most characteristic and at the same time the most 
variable organism known to bacteriologists. As has 
best been, shown by Abbott,34,26 its morphology varies 
markedly with the medium upon which it is grown, so 
that the bacilli from a blood-serum culture will have 
little resemblance in form to their descendants grow- 
ing on an agar culture. 
The bacilli from bouillon cultures are also different 
in morphology from these. Its microscopic appear- 
ance, when taken from a blood-serum culture and 
stained with Loffler’s methylene blue, is so character- 
istic that the practised eye can identify the organism 
by this alone. The gross appearances of the same cult- 
ure are also of importance, though less characteristic. 
In our work we have based ihe recognition of the 
Klebs-Loffler bacillus mainly upon its macroscopic and 
microscopic peculiarities when growing on this medium 
and, in most cases, in bouillon as well. 
After a large experience in the study of the mor- 
phology of the Klebs-Loffler bacillus under these con- 
ditions, we think that at least two forms of it may be 
recognized. One of these is the more common, and is 
distinguished by being a more or less segmented rod 
with intensely staining ends and perhaps one or two 
sharply defined intensely staining areas between. The 
other form is much longer and much more segmented, 
often somewhat constricted at the middle and gently 
swelling toward either end, one of which may be some- 
what thicker than the other. This long form presents 
usually two slight curve?. Aside from its greater 
length, it is also distinguished from the shorter form 
by not commonly presenting the sharply defined, deeply 
staining points in its protoplasm; but it is irregularly 
segmented, each segment staining fairly deeply and 
uniformly, and being separated from the next by a 
narrow, faintly-stained interval. 
We have satisfied ourselves that these long forms 
are a distinct type of the bacillus, and are not the pro- 
duct of special conditions. They have occurred only 
in a small proportion of the cases. This form does not 
change in successive cultures. 12 
STUDIES IN THE PATHOLOGY OF DIPHTHERIA. 
The existence of the different forms of the Klebs- 
Loffier bacillus has been noted by others. Martin 85 
recognizes three — a short, a medium and a long form, 
and claims that there is definite relation between the 
severity of the case and the form or type of bacillus 
found in it. In our cases, however, we have not made 
out any constant association of either one of the two 
forms alluded to, with either mild or severe cases of 
diphtheria. Moreover, we have not observed any con- 
stant differences between these two forms as to their 
virulence towards guinea-pigs. 
As to the much-mooted question of the pseudo-diph- 
theritic bacillus, we are of the opinion of Roux and 
Yersin,1 Abbott87 and others, that in many instances 
the organism to which this name has been applied is 
nothing but a non-virulent form of the Klebs Lbffler 
bacillus. The work of these investigators and others, 
as well as our own results, shows that there are Klebs- 
Loffier bacilli which are not virulent towards guinea- 
pigs, but which are otherwise identical with virulent 
bacilli. This is not surprising in view of what we 
know of the great variation of other bacteria in their 
effects upon animals. 
These non-virulent forms of the diphtheria bacillus 
seem to have been called pseudo-diphtheria bacilli by 
some writers, owing to the apparently deeply rooted 
conviction that all true Klebs-Loffier bacilli are viru- 
lent towards guinea-pigs, which is not true. The term 
“pseudo” should no more be applied to a non-virulent 
diphtheria bacillus than to a non-virulent specimen of 
the frequently deadly staphylococcus pyogenes aureus. 
In addition to these non-virulent forms of the true 
Klebs-Loffier bacillus, v. Hofman,6 Zarniko,6 Esche- 
rich 2 and others have described more or less clearly 
other non-virulent bacilli which resemble the Klebs- 
Loffier bacillus very much, and yet differ from it in cer- 
tain respects. These they have also named “ pseudo- 
diphtheria” bacilli. In the course of our studies on 
diphtheria and other pathological processes, we have 
not infrequently met with organisms of this character; 
but their morphological and cultural peculiarities 
sufficed after careful study to exclude them from con- 
sideration. We consider that these and the so-called 
pseudo-diphtheria bacilli of Escherieh and others have 
nothing more to do with the causation of diphtheria 
than scores of other bacilli which may be found in the 
pharynx. 
In conclusion, a point in the biology of the 
Klebs-Loffier bacillus deserves attention. As is well 
known, the diphtheria bacillus in its growth in neutral 
or slightly alkaline bouillon, produces an acid reaction 
in the medium. This has been regarded by some 
writers as one of the characteristics by which it may 
by distinguished from the so-called “ pseudo-diphtheria ” 
bacillus. Escherieh,2 moreover, considers that the 
production of the acid reaction, as shown by the chang- 
ing of the violet color of the litmus-sugar bouillon to 
a red, is a reliable sign of virulence in the culture, 
lie has recently stated that he has never seen an in- 
stance where an acid-forming culture has failed to kill 
the animal. This has not been our experience, for we 
have tested the acid production by the bacilli of most 
of our cases in the same way that Escherieh did. We 
have found that the bacilli from all sources which have 
shown little or no virulence, have all without ex- 
ception turned the litmus bouillon red. These cases 
which have furnished organisms of little or no virulence 
have been 21 in number, in 20 of which the culture 
used for testing the acid production was also used for 
iuoculatiou. We cannot, therefore, agree with Es- 
cherich as to the significance of a reddening of the 
litmus bouillon cultures. 
Lastly, it may be said that we have never observed 
a satisfactory instance of a change of the red color of 
the bouillon back again to violet or blue in any of the 
large number of bouillon cultures which we have kept 
for a long time. This is surprising in view of the re- 
sults of others, who have found that the acid reaction 
which first appears, later gives place to an alkaline re- 
action, so that we should expect to have the color of 
the litmus change accordingly. 
All of the studies contained in this paper have been 
carried on under the direction of Professor Council- 
man of the Harvard Medical School, to whom the 
writer is under many obligations for good suggestions 
and active interest in the work. 
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