monic infection has been used for the treatment of the
disease, although considerable, is still too few to warrant
the expression of any definite opinion as to the final
value of this as a therapeutic agent. In the cases we
have observed there has been in some a slight imme-
diate lowering of the temperature ; in others no ap-
parent change. As a rule, the cases did rather better
than was expected, but certainly no striking curative
effects were apparent. The cases did not develop
pnetimococcus blood infection, and it seems probable
that the serum may be able to prevent a general infec-
tion from taking place from the diseased lung, even
though it may fail to influence the local process. It
has also been shown that these injections of antipneu-
motoxic serum are practically harmless.



CHAPTER XXIX.

DIPLOCOCCUS INTRACELLULARIS MENINGITIDIS.

IN the description of the micrococcus lanceolatus
reference was made to this organism as the most fre-
quent cause of meningitis, especially when it compli-
cated pneumonia. In 1887, Weichselbaum discovered
another micrococcus in the exudate of cerebro-spinal
meningitis in six cases, two of which were not com-
plicated by pneumonia. He obtained it in pure cultures,
studied its characteristics, and showed that this organism
was clearly distinguishable from the micrococcus lanceo-
latus, and especially by its usual presence in the interior
of pus-cells, on which account he called it diplococcus
intracellularis meningitidis. The frequency of the occur-
rence of this diplococcus in meningitis and its restric-
tion to this disease affords sufficient evidence for the
assumption that it was concerned in its production.
In 1895, Jaeger and Scheurer found it in the nasal
secretions of eighteen living persons suffering from this
disease during an epidemic.

Morphology. This organism occurs as biscuit-shaped
micrococci, usually united in pairs, but also in groups
of four and in small masses; sometimes solitary and
smaller degenerated forms are found. In the exuda-
tion, like the gonococcus, to which it bears a close re-
semblance in form and arrangement, it is distinguished
by its presence within the polynuclear leucocytes. It



INTRA CELL ULARIS MENINGITIDIS. 517

never appears within the nucleus and rarely within
other cells (Fig. 67).

It stains with all the ordinary aniline colors, but best
with Loffler's methylene-blue. According to Weich-
selbaum, it is decolorized by Gram's solution; Jaeger
states that this is not constantly the case.

FIG. 67.




Diplococcus intracellularis meningitidis. X 1100 diameters.

Biological Characters. It does not grow at room-tem-
perature, but only in the incubator, and its develop-
ment is usually scanty on the surface of agar, but some-
times a few colonies grow quite vigorously. It does
not grow at all or scarcely any in bouillon, and very
scantily in bouillon plus one-third blood-serum. It
grows comparatively well on Loffler's blood-serum
medium as used for diphtheria cultures.

When grown on nutrient agar or glycerin-agar, at
the end of forty-eight hours in the incubator, there
develops a tolerably good growth, appearing as a flat
layer of colonies, about one-eighth of an inch in diam-



518 BACTERIOLOGY.

eter, grayish- white in color, viscid and non-confluent
unless very close together. On Loffler's blood-serum
the growth forms round, whitish, shining viscid-look-
ing colonies, with smooth and sharply-defined outlines,
and may attain diameters of one-eighth to one-sixteenth
of an inch in twenty-four hours. The colonies tend to
become confluent and do not liquefy the serum. In
acute cases, where the organisms are apt to be more
abundant, a great many minute colonies may develop
instead of a few larger ones. On agar plates the
deep-lying colonies are almost invisible to the naked
eye; somewhat magnified they appear as finely granular
colonies, with a dentated border. On the surface they
are larger, appearing as pale disks, almost transparent
at the edges, but more compact toward the centres,
which are yellowish -gray in color. Cultivated in arti-
ficial media it soon loses its vitality within six days
and requires, therefore, to be transplanted to fresh
material at short intervals at least every two days.

Pathogenesis. This organism does not show much
pathogenic power for animals. It is most pathogenic
for mice and guinea-pigs, less so for rabbits and dogs.
Subcutaneous injections in animals give negative re-
sults; intrapleural or intraperitoneal inoculations in
mice and guinea-pigs, when given in large doses, are
usually successful. Intravenous injections in rabbits
have caused the death of the animal, but no diplococci
or pathological changes have been found as a result of
the injections.

When mice are inoculated into the pleural or peri-
toneal cavities they usually fall sick and die within
thirty-six to forty-eight hours, showing slight fibrino-
purulent exudation. In the blood and enlarged spleen



INTRA CELL ULARIS MENINGITIDIS. 519

. diplococci are found in small numbers and mostly free;
in the pleuritic exudation they are present in consider-
able quantities, less so in the peritoneal fluid, but then
occurring in the interior of pus-cells.

Certain experiments made by Weichselbaum on dogs,
though not entirely successful, are interesting as showing
the similarity of the disease produced in them artificially
with meningitis as occurring in man. The three dogs,
trephined and inoculated subdurally with 0.5 to 2 c.c. of
afresh culture, all died: No. 1 within twelve hours, No.
2 in three days, and No. 3 in twelve days. In Nos. 1
and 2 there were found hypersemia of the meninges, with
inflammatory softening of the brain at the point of inocu-
lation, which on nearer inspection proved to be a true
encephalitic process. In dog No. 2, in which the dis-
ease was of longer duration, these changes were the
most pronounced. Numerous diplococci were observed
in the sections removed, for the most part free, but
some few within the pus-cells. In dog No. 3, in
which the disease lasted twelve days, between the dura
mater and the brain, at the point of inoculation, was
found a thick, reddish, purulent liquid; in the brain
itself an abscess had formed, about the size of a hazel-
nut, filled with tough, yellow pus, while the abscess
walls consisted of softened brain -substance infiltrated
with numerous hemorrhagic deposits, and simultane-
ously the ventricles on that side contained a cloudy,
reddish fluid, with flocks of pus; but no diplococci
could be demonstrated in the blood or exudations.
Weichselbaum suggests that under natural conditions
the diplococci gain access to the brain and meninges by
way of the nose, ear, and upper air-passages. Cerebro-
spinal meningitis, as is well known, is often accorn-



520 BACTERIOLOGY.

panied by rhinitis and purulent inflammation of the
mucous membranes of the nose. In one of his six cases
Weichselbaum succeeded in obtaining in pure culture
diplococci from the nasal secretion. Scheurer, in his
eighteen cases, found the diplococci in the nasal secre-
tions of all of them during life. In fifty healthy in-
dividuals examined they were found in the nasal secre-
tions of only two of them, one being a man suffering
at the time from a severe cold. This man, it is inter-
esting to note, had been engaged in disinfecting a room
which had previously been occupied by a patient with
cerebro-spinal meningitis.

Bacteriological Diagnosis. By means of lumbar punc-
ture fluid can be readily obtained without danger from
the spinal canal. The microscopical examination will
frequently reveal numerous cells crowded with diplo-
cocci. When considerable quantities are inoculated
upon Loffler's blood-serum mixture or upon glycerin
agar, as a rule, a greater or less number of colonies
having the characteristics already described will de-
velop. The value, clinically, of the examination is
that about 40 per cent, of the cases due to this coccus
recover, while almost all of those due to the pneumo-
coccus and streptococcus die. In fifty-five cases exam-
ined by Councilman, Mallory, and Wright, diplococci
were found in the fluid removed by lumbar puncture in
thirty-eight, either by microscopical examination or
cultures.

The longest time after the onset of the disease in which
positive results were obtained by culture was twenty-
nine days. In a number of cases examined by us for
Northrup a rather smaller percentage of the cases were
found to be due to this diplococcus. In many cases



INTEA CELL ULAEIS MENINGITIDIS. 521

there are very few diplococci present in the spinal fluid,
so that a failure to find them in a microscopical examina-
tion should not be taken to prove that the disease was
not due to this organism. For cultures a considerable
amount of fluid must be used, for we have found, as
described by Councilman and others, that there may
be very few living diplococci even in 1 c.c. of fluid.

To obtain the fluid the patient should lie on the right
side with the knees drawn up and the left shoulder de-
pressed. The skin of the patient's back, the hands of
the operator, and the large antitoxin syringe should be
sterile. The needle should be 4 cm. in length, with a
diameter of 1 mm. for children, and longer for adults.

The puncture is generally made between the third
and fourth lumbar vertebrae. The thumb of the left
hand is pressed between the spinous processes, and the
point of the needle is entered about 1 cm. to the right
of the median line and on a level with the thumb-nail,
and directed slightly upward and inward toward the
median line. At a depth of 3 or 4 cm. in children and
7 or 8 cm. in adults the needle enters the subarachnoid
space, and the fluid flows out in drops or in a stream.
If the needle meets a bony obstruction withdraw and
thrust again rather than make lateral movements.
Any blood obscures the microscopical examination.
The fluid is allowed to drop into absolutely sterile test-
tubes or vials with sterile stoppers. From 5 to 15 c.c.
should be withdrawn. No ill effects have been ob-
served from the operations.



CHAPTER XXX.

MICROCOCCUS GONORRHOEA (OONOCOCCUS NEISSER).

THIS micrococcus was first observed by Neisser, in
1879, in gonorrhoeal discharges, and described by him
under the name of " gonococcus"; but though several
attempted to discover a medium upon which it might
be cultivated, it was reserved for Bumm, in 1885, to
obtain it in pure culture and isolate it and then prove
its infective virulence by inoculation into man. Since
that time the gonococcus has been cultivated on various
media, which, though modifications of Bumm's, are an
improvement on his original method, and as the result
of various inoculation experiments there now remains
no doubt that this organism is the specific cause of
gonorrhoea in man.

Morphological Characters. Micrococci, occurring
mostly in the form of diplococci that is, in pairs or
in groups of four. The bodies of the diplococci are
elongated, and, as shown in stained preparations, have
an unstained division or interspace between two flat-
tened surfaces facing one another, which gives them
their characteristic " coffee-bean " or "biscuit" shape
(Fig. 68). The diameter of an associated pair of cells
varies from 0.8// to 1.6// in the long diameter
average about 1.25// by 0.6/* to 0.8/u. in the cross
diameter. In gonorrhoea gonococci are found mostly
in small, irregular groups in or upon the pus-cells, and



MIGROGOGGUS GONORRHCEJE. 523

generally extranuclear. When found in other portions
of the field this is mostly due to the mechanical effect
of smearing the pus on cover-glass slides, and should
not be considered as characteristic. That the gonococci
really lie within the protoplasm of the cells is proved
by the fact that in carefully made preparations they
are usually not found outside of the pus-cells. They
appear usually as diplococci, in groups of two or four,

FIG. 68.




Smear from pure culture of gonococcus on agar. (HEIMAN.)

but at times they occur as round, single, and undivided
cells. Others, again, are irregular in shape or granular
in appearance, involution forms, particularly in older
cultures and in chronic urethritis of long standing.
The pus-cells containing gonococci are most numerous
in the later or purulent stage of the disease, not so fre-
quent in the beginning of infection, or as long as the
discharge is of a serous character (Fig. 69).

The gonococcus stains readily with the basic aniline
colors, especially with methyl-violet, gentian-violet, and



524 BACTERIOLOGY.

fuchsin; not so readily with methylene-blue, which is,
however, one of the best staining agents for demon-
strating its presence in pus. Beautiful double-stained
preparations may be made from gonorrhoeal pus by
treating cover-glass smears with methyl-violet and
eosin. Gonococci are decolorized by Gram's solution;
but this cannot be depended upon alone to absolutely
distinguish the gonococcus from all other diplococci

FIG. 69.




Gonococcus in pus-cells. X 1100 diameters.

found in the urethra and vulvo-vaginal tract, for espe-
cially in the female other diplococci are occasionally
found which are also not stained by Gram's method.
It serves, however, to distinguish this micrococcus from
the common pyogenic cocci, which retain their color
when treated in the same way, and in the male urethra
it is practically certain, as no organism has been found
in that location which in morphology and staining is
identical with the gonococcus. It is certainly the most
distinctive characteristic of the staining properties of



MICROCOCCUS GONORRHCEjE. 525

the gonococcus, and it is a test that should never be
neglected in differentiating this organism from others
which are morphologically similar.

Biological Characters. The elaborate experiments of
Bumm and others have shown that at the ordinary room-
temperature no growth of the specific micrococcus occurs
on the culture media. Apparently positive results which
have been reported are found to be due to other diplo-
cocci morphologically almost identical with the gono-
coccus.

Since Bumm's experiments a number of culture
methods have been proposed for the gonococcus which
are an improvement on Bumm's, partly because the
growth produced is more constant and luxuriant and
partly because the media employed are more readily
prepared. Wertheim (1892) succeeded in developing
luxuriant and virulent cultures to many generations on
a mixture of placenta blood-serum and 2 per cent, pep-
tone-agar. His method is briefly as follows : Several
loops of gonorrhoeal pus are diffused through liquid
blood-serum warmed to 40 C. contained in a test-
tube. Two dilutions are made from this, and an
equal quantity of melted 2 per cent, agar cooled to
40 C. is added to the three tubes, and the contents,
after thorough mixing, poured into Petri dishes. The
Petri dishes are placed in an incubating oven at a
temperature of 36 to 37 C. At the end of twenty-
four hours there will have developed on at least one of
the plates distinct colonies; these are translucent, finely
granular, with scalloped margin. By transferring such
a colony to slant-cultures of serum-agar, pure cultures
of the gonococcus are obtained; these are somewhat
shining in appearance and of a grayish-white color.



526 BACTERIOLOGY.

Wertheim demonstrated that the addition of peptone
to the culture medium was an important factor in the
cultivation of gonococci. Kiefer (1895) proposed a cul-
ture medium consisting of one part of hydrothorax or
ascitic fluid and one part of a fluid containing 3.5 per
cent, agar, 5 per cent, peptone, 2 per cent, glycerin, and
0.5 per cent. salt.

Simultaneously with Kiefer, Heiman recommended
a medium made from hydrocele fluid, or from " chest-
serum" obtained from a patient suffering from hy-
drocele or hydrothorax or acute pleurisy. Having
experimented with all the various culture media here-
tofore prepared for the cultivation of the gonococctis,
Heiman believes this medium to be superior to placenta -
serum, sheep blood-serum, or ascitic fluid, because of
the large amount of serum albumin which it contains.
The medium consists of a 2 per cent, agar, plus 2 per
cent, peptone, plus 0.5 per cent, salt and 2 per cent,
glucose. Of this mixture two parts are added to one
part of chest -serum, which is, if necessary, fractionally
sterilized between 65 and 70 C. for one hour for seven
consecutive days. The chest-serum-agar should have a
neutral reaction. The growth on this medium is thus
described : In plate cultures streaked on the surface,
growth abundant, colonies circular in shape, edges
somewhat irregular, shading off into yellowish- white;
texture finely granular in periphery, presenting punc-
tated spots of higher refraction in and around the
centre of yellowish color (Fig. 70).

The gonococcus has but little resistant power against
outside influences. It is killed by weak disinfecting
solutions and by desiccation in thin layers. In com-
paratively thick layers, however, as when gonorrhoeal



MICROCOCCUS GONORRHCE^E.



527



pus is smeared on linen, it has lived for forty-nine
days, and dried on glass for twenty-nine days (Heiman).
No development takes place below 25 C. or above
39 C.; it is killed at a temperature over 42 C.

Pathogenesis. Non-transmissible to dogs, monkeys,
horses, and rabbits, whether inoculations be made into
the urethral, vaginal, or congenital mucous membranes.
According to Wertheim, purulent peritonitis, not caus-

FIG. 70.




Colonies of gonococci on pleuritic fluid agar. (HEIMAN.)

ing death, is produced in certain animals by the intro-
duction of pieces of serum-agar containing colonies of
the gonococcus. This effect was produced constantly in
mice, occasionally in guinea-pigs, and rarely, if ever,
in dogs, rats, and rabbits.

Though animal inoculations are thus followed by
negative results, the etiological relation of the gono-
coccus to human gonorrhoea has been demonstrated
beyond question by the infection of healthy men with
the disease by inoculation. Thus, Bumm has produced



528 BACTERIOLOGY.

gonorrhoea in normal urethral mucous membranes by
inoculation of a pure culture on blood-serum in the
second generation; Wertheim, in the thirtieth; Kiefer,
in the sixth, and Heiman in the fifth generation. At
the same time the distinctive morphological, staining and
biological characters of the organism were carefully noted
and confirmed to be those of the gonococcus by these ob-
servers; the typical incubation and symptoms of the dis-
ease resulted in all cases in the subjects experimented on.
According to the observations of the most reliable
investigators and those most familiar with the various
forms of micrococci which are likely to be mistaken
for the gonococcus, affections due to this organism are
usually restricted to the mucous membranes of the
urethra, conjunctiva, bladder, cervix uteri, and rectum.
It rarely, if ever, produces a vaginitis in adults; but
occasionally a vulvo- vaginitis in young children. For-
merly the presence of gonococci could only be deter-
mined microscopically; but since the introduction of the
serum-agar the culture method has rendered the diag-
nosis of gonorrhoea much more reliable. This method
of investigation, moreover, has given valuable infor-
mation with regard to the nature of many infections
complicating or resulting from gonorrhoea, particularly
in affections of the uterus and joints, about which there
was heretofore considerable doubt, though the micro-
cocci often found in these organs were morphologically
identical with the gonococcus. It has now been shown
by the culture method that gonococci may occur in the
joints in gonorrhoeal arthritis, in the Fallopian tubes in
salpingitis, and in ovarian abscesses; and Wertheim
asserts that he has found them in the infiltrated con-
nective tissue in parametritis.



MICROCOCCUS GONOREHCEjE. 529

Worthy of special notice in this connection are the
cases of endocarditis accompanying gonorrhoea, and some-
times terminating fatally, when they are known as en-
docarditis gonorrhoeica maligna. The question naturally
arises in these cases whether it is the gonococcus or
some other coccus or diplococcus which has infected
the endocardium. Here, again, it is only by means of
the culture method that this question can be settled
definitely. Fliigge draws attention to this matter, and
states that but few cases have been recorded in which
the information given as to the cause of the disease can
be unhesitatingly accepted. Weichselbaum mentions
a case of endocarditis accompanying gonorrhoea which
was shown by the culture method to be due to strepto-
coccus infection, proving that so-called gonorrhoeal
endocarditis may be a secondary infection. Other cases
are recorded by Leyden, Hiss, Councilman, and Wilms
which are said to have been most probably of gonor-
rhoeal origin; but in these only microscopical examina-
tions were made and no culture experiments, or only
cultures on gelatin plates, etc., which were inadequate.
Welch also reports a case of endocarditis with general
septicaemia following gonorrhoea, in which he demon-
strated the gonococcus in the blood of a living person
in cover-glass and culture medium. No other patho-
genic bacteria were found.

Immunizing Serum. As animals are not infected by
the gonococcus they are not very suitable for injections
with the cultures for the purpose of producing an anti-
toxic or bactericidal serum. Their insusceptibility pre-
sents also an almost insurmountable obstacle to the
testing of the blood of animals under treatment, so that
although it may be possible to bring about an artificial

34



530 BACTERIOLOGY.

immunity against gonorrhoeal infection, information on
this subject is at present wanting. Immunity in man
seems to be similar to that produced after infection with
the other pyogenic cocci that is, only slight in amount
and for a short period. It is known that a urethra or
cervix may contain gonococci which lie dormant and
may be innocuous in that person for years, but which
may at any time excite an acute gonorrhoea in the one
carrying the infection or in another person.

The Bacteriological Diagnosis of Gonorrhoea. In view
of the fact that several non-specific forms of urethritis
exist, and also that micrococci morphologically similar
to the gonococcus Neisser are often found in the normal
urethral and vulvo-vaginal tract, it becomes a matter
of great importance to be able to detect gonococci when
present and to differentiate these from the non-specific
organisms. The gonococci also which occur in old cul-
tures and in chronic urethritis of long standing often
take on a very diversified appearance sometimes
nothing but an irregular, granular mass being seen,
which renders their detection difficult. From a medico-
legal and social stand-point, therefore, the differential
diagnosis of the gonococcus has in certain cases a very
practical significance.

There are two methods of differential diagnosis now
available the microscopical and the cultural. Animal
inoculations are of little value, as they are not sus-
ceptible, and, of course, human inoculations are, except
in extremely important cases, generally impossible. In
the microscopical diagnosis it should be borne in mind
that the specific gonococci in carefully made prepara-
tions are found always largely within the pus-cells.
Diplococci morphologically similar to gonococci occur-



MICROCOCCUS GONOERHCE^. 531

ring in other portions of the field and outside of the
pus-cells should not be considered specific by this test
only. It should also be remembered that thegonococci
are decolorized by Gram's method, while other similar
micrococci which occur in the urethra are, as a rule,
at least, not so decolorized. Organisms having these
characteristics can for all practical purposes be con-
sidered as certainly gonococci if obtained from the
urethra. From the vulvo-vaginal tract the certainty is
not so great; here cultures should also be made. Burnm,
Heiman and others have shown that other diplococci
are occasionally found in gouorrhoeal pus from the vulvo-
vaginal tract, and very rarely, indeed, from the urethra,
which do not stain by this method. Cover-glass prepa-
rations from subacute or chronic cases should be ex-
amined, if possible, with a microscope provided with a
mechanical stage, and should always be stained by
Gram's method and the examination repeated on three
consecutive days. Should these specimens prove nega-