BACTERIOLOGIC EXAMINATION IN THE DIAG- 
NOSIS OF DIPHTHERIA. 
BY 
WILLIAM G. BISSELL, M.D., 
BACTERIOLOGIST, DEPARTMENT OF HEALTH, BUFFALO, N. Y. 
FROM 
THE MEDICAL NEWS, 
February 2, 1895. [Reprinted from The Medical News, February 2, 1895.) 
BACTERIOLOGIC EXAMINATION IN THE DIAG- 
NOSIS OF DIPHTHERIA.1 
By WILLIAM G. BISSELL, M.D., 
BACTERIOLOGIST, DEPARTMENT OF HEALTH, BUFFALO, N. Y. 
A great deal has been said on the laboratory- 
technique and the value of bacteriologic examina- 
tion in the diagnosis of diphtheria, yet the subject 
is of sufficient interest and importance to warrant a 
further explanation of the methods used in cul- 
ture examinations. It would seem unnecessary to 
go into the history of the organism further than to 
state that it was discovered by Klebs in 1883, and 
first artificially cultivated by Loeffler in the year fol- 
lowing. The germ is generally known as the Klebs- 
Loeffler diphtheria-bacillus, and it has been fully 
proved to be the etiologic factor in the production 
of the disease. 
It is generally admitted by all clinicians of ex- 
perience that it is often impossible to make an accu- 
rate diagnosis of diphtheria, either from the clinical 
history, the anatomic lesion, or from both. There 
are no constant differences that separate the simple 
non-contagious forms of inflammation from the 
1 Paper read before the semi-annual meeting of the Erie County 
Medical Society, January 8, 1895. 2 
diphtheric type, and it is in but a very small propor- 
tion of cases that an early and reliable diagnosis can 
be reached from any data obtainable. This was 
conclusively demonstrated in the examination of the 
cases of suspected diphtheria under treatment at the 
Willard Parker Hospital in New York, where the diag- 
noses were made by department diagnosticians and 
confirmed by the best medical talent. Subsequent 
bacteriologic examination in these cases revealed 
that from 30 to 50 per cent, were not diphtheria, but 
maladies of non-infectious character. Appreciate 
sending a case of follicular tonsillitis to a hospital 
and having it placed in a ward with diphtheric pa- 
tients ! This person, with a reduced vitality, is 
exposed to one of the most dreaded diseases when 
suffering from a malady to which hardly one in 
twenty-five succumbs. 
a. It is in just such cases that an early bacterio- 
logic examination is of the greatest sanitary im- 
portance. 
Furthermore, it has been proved that in cases in 
which the Klebs-Loeffler bacillus has been found 
virulent diphtheria has been transmitted to others, 
whereas the person carrying the contagion was 
hardly in a condition to be called ill. 
b. It is the systematic bacteriologic examination 
of such cases that plays a most important part in the 
advancement of preventive medicine. 
Again it has been fully demonstrated that the 
bacillus of diphtheria remains a varied length of 
time in the throats of persons convalescing from the 
disease,' even after the disappearance of all mem- 
brane and constitutional symptoms. Such persons, 3 
if allowed to go at large, are a constant source of 
infection, transmitting the disease to others, and in 
this way increasing the prevalence of the malady. 
c. It is from the bacteriologic examination in 
such cases that the greatest practical benefits can be 
derived. 
Early in January, 1893, through the energetic 
efforts of Dr. Herman M. Biggs, of New York, a 
communication was addressed to the Health Board 
of that city, recommending systematic bacterio- 
logic examination for the diagnosis of diphtheria. 
After some unavoidable delay, bacteriologists were 
appointed in accordance with the recommendation, 
with such assistance as was necessary thoroughly to 
carry on the work. From the time of the inaugura- 
tion of this scheme those in charge had very little 
doubt of its ultimate success, but there were a few 
who doubted its practical efficiency. Several depots 
were established in various parts of the city, where 
culture-sets could be procured, and every step was 
taken to increase the facilities for early examination. 
Circulars were printed and sent to physicians, 
asking their hearty co operation in the movement, 
and explaining the great necessity for such examina- 
tions. During the first few weeks the cases exam- 
ined were comparatively small, but they have con- 
tinually increased in number until at the present 
time the practice is universal. 
In order to make clear the methods used in such 
examination it will be necessary to describe briefly 
the materials required. The culture-medium most 
suited for this work is a preparation of blood-serum 
after the formula of Loeffler. Pure serum is obtained 4 
by allowing the freshly drawn blood of a young sheep 
or ox, contained in a wide-mouthed sterile jar, to re- 
main twenty-four hours in an ice chest. By that 
time the fluid part of the blood has separated from 
the solid portion, and is easily drawn off by means 
of a pipet. This serum is mixed in proportions of 
three to one of a 50 per cent, neutral sterile beef- 
infusion, containing 1 per cent, peptone, 1 per cent, 
dextrose, and y2 per cent of common salt. This 
mixture is then poured into small sterile tubes (put- 
ting about 2 c.c. in each tube) and carefully steril- 
ized in a blood-serum sterilizer, at a temperature of 
about 1600 F. The tube is put at an angle during 
the process of sterilization so that after the coagula- 
tion of the blood-serum mixture one will have the 
greatest possible surface for the planting of bacteria. 
After these tub<s have been rendered absolutely 
sterile they are ready for use. A sterilized tube 
containing a swab goes with each culture-tube, and 
these tubes compose the so-called culture-set. 
How to make the primary inoculation is explained 
by a printed slip accompanying each culture-set. 
The slip, as supplied by the Department of Health 
in this city, reads as follows : 
DIRECTIONS FOR MAKING CULTURES IN CASES OF SUS- 
PECTED DIPHTHERIA. 
The patient should be placed in a good light, and, if a 
child, properly held. In cases where it is possible to get 
a good view of the throat, depress the tongue and rub 
the cotton swab gently, but freely, against any visible 
exudate. 
In other cases, including those in which the exudate is 
confined to the larynx, avoiding the tongue, pass the 
swab far back, and rub it freely against the mucous mem- 5 
brane of the pharnyx and tonsils. Without laying the 
swab down, withdraw the cotton plug from the culture- 
tube, insert the swab, and rub that portion of it which 
has touched the exudate gently but thoroughly back and 
forth over all the surface of the blood-serum. Do not 
push the sw'ab into the blood-serum, nor break the sur- 
face in any way. Then replace the swab in its own tube, 
plug both tubes with the cotton plugs, and return the cul- 
ture-set at once to the Health Department. 
A report will be given by telephone the following morn- 
ing, or can be obtained after 12 noon. 
As soon as the tubes arrive at the department, that 
containing the inoculated blood serum is placed in 
the incubator at a temperature of 36° C., and 
allowed to remain until sufficient growth appears to 
permit of examination. This requires a period 
of from twelve to twenty-four hours. After the 
proper time has elapsed a cover-glass preparation 
is made from the growth in the tube, stained with 
alkaline methylene-blue solution, and mounted in the 
usual way with Canada balsam. On microscopic 
examination of such a preparation with a 1-12 oil- 
immersion lens-if it is from a case of true diph- 
theria-the following picture will present itself: A 
large number of rod-shaped organisms stained blue, 
varying somewhat in length, about the same size as 
the tubercle-bacillus, but twice as thick, frequently 
clubbed and spindle-shaped, and almost invariably 
irregularly stained, presenting heavily stained seg- 
ments in a lightly stained background. This is the 
bacillus of diphtheria. 
It is claimed by some that the recognition of the 
bacillus is extremely difficult, owing to its resem- 
blance to the germ described by Hoffman. It is 
the general opinion, however, that with thorough 
* 6 
examination of the bacterial growth, as described, 
which has been carefully inoculated according to 
the rules given, and kept at the temperature of 
the body, the microscopic examination can be, 
without doubt, relied upon. Of course, the major- 
ity of tubes sent in for examination will not reveal 
any bacilli, containing simply the common cocci 
found in the throat. In such cases a positive diag- 
nosis is extremely easy. It was thought at one time 
that the size of the germ had to do with its virulence, 
but this is now generally conceded not to be the fact. 
Much has been said by some authors as to an 
organism that by its culture-peculiarities and mi- 
croscopic appearance is identical with the Loeffler 
germ, yet does not possess pathogenic properties 
when injected into susceptible animals. The name 
given to the organism is the " pseudo-diphtheric 
bacillus," and, as expressed by Abbott and other 
prominent workers in this field, it seems certainly 
to be a misnomer, for it is most possible and proba- 
ble that this organism is none other than the true 
diphtheric germ, attenuated in virulence, or, as cir- 
cumstances have rendered it, a non-virulent diph- 
theric bacillus. 
It would appear to be far better to term " pseudo- 
diphtheric " those organisms that are enough like 
the true bacillus to attract attention, yet are distin- 
guishable from it by certain morphologic and cultural 
peculiarities aside from the question of virulence. 
It is a well-known fact that many pathogenic 
organisms, conspicuous among them being the mi- 
crococcus of sputum-septicemia and the so-called 
hemorrhagic septicemic group, undergo marked 7 
variations in the degree of their pathogenic proper- 
ties, yet such organisms are not designated as 
" pseudo," but simply the organisms themselves, 
having become attenuated in virulence. 
Loeffler, himself, found the Klebs-Loeffler bacillus 
present in the case of a perfectly healthy child. Was 
the germ found virulent? Was it the germ of true 
diphtheria, or was it the resistance on the part of the 
patient, the healthy secretions and conditions of the 
mucous membrane, which lessened its virulence in 
this particular case ? It seems most reasonable that 
it was the latter. If not, why is it that frequently, 
on examination of cultures made from the throats of 
persons taking care of diphtheric patients, one finds 
the germ present, and yet these persons present no 
constitutional disturbance. It is because the proper 
conditions are not present for the development of 
the disease, whereas others receiving the infection 
from an attendant might contract the disease in its 
most deadly form. 
There happens in a certain number of cases which 
clinically are diphtheria, that the first inoculation 
will fail to show any diphtheria-bacilli. These 
cases, however, are exceptional, and it is not usually 
the fault of the bacteriologist that such is the case, 
because there are certain factors that should be taken 
into consideration : 
1. The inoculation may have been made at so late 
a period in the disease that it is possible that the 
diphtheria-bacilli, though now absent, were present 
at an earlier time. 
2. The growth on the culture medium may be so 
scanty that it is probable that the inoculation was 8 
not properly made, or that some antiseptic had been 
applied to the throat shortly before obtaining the 
material for inoculating the tube, sufficient of the an- 
tiseptic remaining to prevent growth on the serum. 
3. The culture-medium may be badly contam- 
inated and a positive recognition of any one 
organism be impossible. 
4. The serum in the tube may have become too 
dry before the inoculation to permit of the growth 
of the diphtheria-bacilli. 
In all these cases a second inoculation should be 
requested by the bacteriologist before giving a defi- 
nite opinion as to the diagnosis. 
Of the primary cultures examined at the Health 
Department in this city since the 1st of March, 1894, 
sixty-five were found to contain the Klebs-Loeffler 
bacillus and ninety-nine were found not to contain 
that organism. This makesashowing that about 39 
per cent, of the cases were true diphtheria. 
One case examined during that time of peculiar 
interest was that in the practice of Dr. Eugene 
Smith, of this city. Dr. Smith on the 18th of De- 
cember, 1894, inoculated a tube from the pharynx 
of a child suffering with a malady characterized by 
great toxic disturbance, and, as he thought, possibly 
diphtheria. The tube was sent immediately to the 
Health Department and placed in the incubator. 
After twenty-four hours there was no apparent growth 
on the blood serum, and a second inoculation was 
requested. The result of the second inoculation was 
like that of the first, there being no apparent growth 
after the twenty-four hours in the incubator. On 
microscopic examination of the swab it was found to contain a considerable amount of a glistening 
whitish exudate. Two side-mountings were made 
of this exudate, one being stained with methylene- 
blue, and the other by Gram's method. On micro- 
scopic examination of these slides there was found 
to be present an organism having a distinct encap- 
sulation and resembling morphologically the organ- 
ism described by Fraenkel as being the etiologic 
factor in the production of pneumonia. The result 
of the examination was communicated to Dr. Smith. 
That night the child developed characteristic pneu- 
monic symptoms, and is now convalescing from the 
disease. The primarily inoculated tubes were left in 
the incubator, and after the fifth day showed a 
growth consisting of several small clusters of iso- 
lated fine points, growing closely side by side, like 
those of the diplococcus of pneumonia. Some of 
the exudate from the swab was inoculated into sev- 
eral mice, and the animals died invariably inside of 
forty-eight hours of an acute septicemia. 
All these points tend to show that the organisms 
found corresp md with the Fraenkel germ, and it is 
of interest in that it fully explained why develop- 
ment did not occur on the blood-serum during the 
first twenty-four hours. 
As to the points, in conclusion, which may be 
definitely stated, in relation to the bacteriologic 
examination in the diagnosis of diphtheria, there 
are the following: 
i. A positive diagnosis of diphtheria can be 
reached in the majority of all suspicious cases by 
careful examining of the primary culture, inoculated 
as described, inside of twenty-four hours. 
9 10 
2. In cases in which the bacillus is not found 
there is little chance of infection. 
3. In those cases in which the diphtheria-bacilli 
are found the cases should not be considered free 
from infective powers until subsequent bacterio- 
logic examinations fail to reveal the organism. 
Note.-During the discussion of this paper by 
various members of the Society an elderly and 
trusted physician brought up the old-time question 
as to whether diphtheria is primarily a local or a 
constitutional disease. He stated that he "believed 
that primarily it was always a constitutional disease, 
with perhaps sometimes coincident local manifesta- 
tions," i. e., it was always the result of etiologic fac- 
tors from within the body, and not a primary local 
affection. 
With others I strongly contested this idea, and a 
heated argument ensued. Bacteriologists and men 
of experience should discuss this question in the cur- 
rent literature. 
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