tive, to exclude any possible doubt in the matter,
cultures should then be made on chest-serum-agar,
poured in dishes, as proposed by Heiman, also, if with
negative results, on three consecutive days. His
method of procuring the urine in chronic urethritis
is to allow the patient to void his urine either imme-
diately into two sterilized centrifugal tubes or first
into two sterile bottles. The first tube will contain
threads of the anterior urethra; the second tube will
be likely to contain secretion from the posterior urethra
and from the prostate gland if, while urinating, the
patient's prostate be pressed upon with the finger.
Tubes containing such urine are placed in the centri-
fuge and whirled for three minutes at twelve hundred




532 BACTERIOLOGY.

or more revolutions per mintue; the threads are thrown
down. The " centrifuged" sediment will be found
to contain most of the bacteria present, epithelial cells,
and at times spermatozoa. Normal urine on being
" centrifuged" at this velocity will be found at times
slightly turbid at the bottom of the tube. This tur-
bidity will be found, on microscopical examination, to
consist of epithelial cells, a few leucocytes, and some
bacteria.

Heiman looks upon the decolorization by Gram's
method as the only reliable criterion, so far as known,
for the gonococcus in discharges from the mucous mem-
branes, and it is of material help, also, in determining
whether a culture is or is not that of the gonococcus.
The careful examination of gonorrhoeal threads with
cover-glass by Gram's method is a very tedious affair,
as in every instance no less than three cover-glass prep-
arations should be looked over before the absence of
the gonococcus is proved. It would require many
hours upon each and every specimen, especially if the
gonococci are present in very small number, before a
reliable and conscientious opinion could be rendered.
If, after all, a negative opinion is ventured, we still
are under the necessity of proving that because the
threads which we fished out for the cover-glass exami-
nation were free from gonococci the remaining ones
were also. For this reason the culture medium is more
sensitive for bacteria than is the cover-glass, for we are
able to plant each and every thread of the sediment in
the centrifugal tube. Fiirbringer, in his work, men-
tions the fact that in certain cases the absence of the
gonococcus in many examinations of cover-glass prep-
arations is not a positive proof that the gonococcus is



MICEOCOCCUS GONORRHCEJE. 533

not present. Heiman was able to confirm the correct-
ness of the above allusion, for on one occasion, in
examining threads, when he could not demonstrate the
gonococcus in cover-glass preparations, he succeeded in
growing it on chest-serum-agar plates, while in all
instances in which he found the gonococcus in threads
in cover-glass preparations he invariably succeeded in
growing it on chest-serum-agar plates. The culture
methods, of course, presuppose that one has the facili-
ties and knowledge to carry them out successfully, other-
wise the microscopical methods are to be used alone.

In acute cases the specimen for examination may be
collected, when the patient is before one, by passing a
sterilized platinum wire loop as far up into the urethra
as possible and withdrawing some of the secretion.
This is a far less satisfactory method than that sug-
gested by Heiman, by " centrifuging," except when
the pus is abundant.

The Frequency with which Gonococci are Found in
Smears or Cultures in Cases of Chronic Urethritis.
Heiman found in 61 cases 14 by cultures and 13 by
smears. The following results were obtained by other
observers by cover-glass preparations : Goll, accord-
ing to his elaborate article, examined 1046 cases of
chronic urethritis varying in duration between four
weeks to six years or more, finding gonococci in 178
cases, the remainder giving negative results. Neisser,
out of 143 cases varying in duration between two
months and eight years, found gonococci in 80 cases.
Weinrich, out of 25 similar cases, obtained 2 positive
results. E. Noeggerath, in 1887, deplored the fact
that on account of the lack of culture media for
the gonococcus we cannot always demonstrate them.



534 BACTERIOLOGY.

Brose, in 1893, stated that the culture medium is the
only reliable agent for the detection of the gonococcus.
This latter statement is certainly applicable to chronic
urethritis of the male. Neisser, in 1893, stated that
in chronic urethritis with slight discharge the examina-
tion with a culture medium for gonococci will replace
the cover-glass.



CHAPTER XXXI.

BACILLUS PYOCYANEUS (BACILLUS OF GREEN AND
OF BLUE PUS) BACILLUS PROTEUS VULGARIS
BACILLUS OF MALIGNANT (EDEMA BACILLUS
AEROGENES CAPSULATUS.

BACILLUS PYOCYANEUS.

THE blue and green coloration which is occasionally
found to accompany the purulent discharges from open
wounds is usually due to the action of the bacillus
pyocyaneus. According to recent investigations this
bacillus appears to be very widely distributed.

Morphology. Slender rods from 0.3// to 1/j. broad and
from 2/^ to 6// long; frequently united in pairs or in
chains of four to six elements; occasionally growing
out into long filaments and twisted spirals. The bacil-
lus is actively motile, a single flagellum being attached
to one end. Does not form spores. Stains with the
ordinary aniline colors; does not stain with Gram's
solution.

Biological Characters. An aerobic, liquefying, motile
bacillus. Capable also of an anaerobic existence, but
then produces no pigment. Grows readily on all arti-
ficial culture media at the room-temperature, though
best at 37 C., and gives to some of them a bright
green color in the presence of oxygen. In gelatin
plate cultures the colonies are rapidly developed,
imparting to the medium a fluorescent green color;
liquefaction begins at the end of two or three days,



536 BACTERIOLOGY.

and by the fifth day the gelatin is usually all lique-
fied. The deep colonies, before liquefaction sets in,
appear as round, granular masses with scalloped mar-
gins, having a yellowish-green color; the surface col-
onies have a darker green centre, surrounded by a
delicate, radiating zone. In stick cultures in gelatin
liquefaction occurs at first near the surface, in the form
of a small funnel, and gradually extends downward;
later the liquefied gelatin is separated from the solid
part of the medium by a horizontal plane, a greenish-
yellow color being imparted to that portion which is
in contact with the air. On agar a wrinkled, moist,
greenish-white layer is developed, while the surround-
ing medium is bright green; this subsequently becomes
darker in color, changing to blue-green or almost black.
In bouillon the green color is produced, and the growth
appears as a delicate, flocculent sediment. Milk is coag-
ulated with coincident acid reaction.

There is some difference of opinion with regard to
the pigments produced by the bacillus pyocyaneus.
Gessard's view is that two pigments are produced by
this bacillus one of a fluorescent green and the other
(pyocyanin) of a blue color. Pyocyanin is soluble in
chloroform, and may be obtained from pure solution
in long, blue needles. This pigment, which is thus
extracted by chloroform, distinguishes the bacillus
pyocyaneus from other fluorescing bacteria.

Pathogenesis. This bacillus is very widely distributed
in nature; it is found on the healthy skin of man,
in purulent discharges and in serous wound secre-
tions. Its presence in wounds greatly delays the
process of repair and may give rise to a general
depression of the vital powers from the absorption of



BACILLUS PYOCTANEUS. 537

its toxic products. Its pathogenic effects on animals
have been carefully studied. It is pathogenic for
guinea-pigs and rabbits. Subcutaneous or intra-peri-
toneal injections of not too small quantities of a
recent culture 1 c.c. or more of a bouillon culture
usually cause the death of the animal in from twenty-
four to thirty-six hours. Subcutaneous inoculations pro-
duce an extensive inflammatory oedema and purulent
infiltration of the tissues; a serofibrinous or purulent
peritonitis is induced by the introduction of the bacillus
into the peritoneal cavity. The bacilli multipy in the
body, and may be found in the serous or purulent fluid
in the subcutaneous tissues or abdominal cavity as well
as in the blood and various organs. When smaller
quantities are injected subcutaneously the animal usu-
ally recovers, only a local inflammatory reaction being
set up (abscess), and it is subsequently immune against
a second inoculation with doses which would prove
fatal to an unprotected animal. Immunity may also
be secured by the injection of a considerable amount
of a sterilized culture. It is interesting to note that
Bouchard, Charrin, and Guignard have shown that in
rabbits which have been inoculated with a culture of
the bacillus anthracis a fatal result may be prevented
by inoculating the same animal soon after with a pure
culture of the bacillus pyocyaneus. Similar results
have been obtained by Wood head and Wood by the
injection of sterilized cultures of this bacillus, made
immediately after injection with the anthrax bacillus.
Loew and Emmerich have shown that the enzymes
produced in the pyocyaneus cultures are capable of de-
stroying many forms of bacteria in the test-tube, and
have slight protecting value in the body.



538 BACTERIOLOGY.

Our knowledge of the pathogenic importance of the
bacillus pyocyaneus in human diseases has been much
increased by recent investigations. Thus cases have
been reported in which this bacillus has been obtained
in pure culture from pus derived from the tympanic
cavity in disease of the middle ear, from cases of oph-
thalmia and bronchopneumonia. Kruse and Pasquale
have found the same micro-organism in three cases of
idiopathic abscess of the liver, in two of them in immense
numbers and in pure culture. Ernst and Schiirmayer
report the presence of the bacillus pyocyaneus in serous
inflammation of the pericardial sac and of the knee-
joint. Ehlers gives the history of a disease in two
sisters who were attacked simultaneously with fever,
albuminuria, and paralysis. It was thought that they
would turn out to be typhoid fever or meningitis, but
on the twelfth day there was an eruption of blisters,
from the contents of which the bacillus pyocyaneus was
isolated. Jadkewitsch reports the case of a patient
suffering from eczema of the lower extremities, in
whom three times during a period of ten years there
was eruption of boils containing blue pus, with accom-
panying symptoms of poisoning, emaciation, prostra-
tion, diarrhoea, and paresis. Krambals refers to seven
cases in which a general pyocyaneous infection occurred,
and adds an eighth from his own experience. In this
the bacillus pyocyaneus was obtained post-mortem from
green pus in the pleural cavity, from serum in the peri-
cardial sac, and from the spleen in pure culture.
Schimmelbush states that a physician injected 0.5 c.c.
of sterilized (by heat) culture into his forearm. As a
result of this injection, after a few hours he had a
slight chill, followed by fever, which at the end of



BACILLUS PROTEUS VULGARIS. 539

twelve hours reached 38.8 C.; an erysipelatous-like
swelling of the forearm occurred, and the glands in
the axilla were swollen and painful. Neumann has
obtained the bacillus pyocyaneus in pure culture in
two cases of hsematemesis and melsena of the new-born
from the blood and other organs. Lartigau found it
in well-water, and in great abundance in the intestinal
discharges of a number of cases made ill by drinking
the water.

We may, therefore, conclude from these facts that
the bacillus pyocyaneus, although ordinarily non-patho-
genic for man, may under certain conditions become a
dangerous source of infection. Children would seem
to be particularly susceptible to this infection.

The differential diagnosis of the pyocyaneus from
other fluorescing bacteria is easy enough as long as it
retains its pigment-producing property. When an agar
culture is agitated with chloroform a blue coloration
demonstrates the presence of this bacillus. When the
pyocyanin is no longer formed, however, the diagnosis
is by no means easy, particularly when the pathogenic
properties are also gone.

BACILLUS PROTEUS VULGAEIS.

This bacillus, which is one of the most common and
widely distributed putrefactive bacteria, was discovered
by Hauser (1885) along with other species of proteus
in putrefying substances. These bacteria were formerly
included under the name a bacterium termo " by previous
observers, who applied this name to any minute motile
bacilli found in putrefying infusions.

Morphology. Bacilli varying greatly in size; most
commonly occurring 0.6// broad and 1.2/* long, but



540 BACTERIOLOGY.

shorter and longer forms may also be seen, even grow-
ing out into flexible filaments, which are sometimes
more or less wavy or twisted like braids of hair. The
bacillus does not form spores, and stains readily with
f uchsin or gentian-violet.

Biological Characters. An aerobic, facultative anaer-
obic, liquefying, motile bacillus. Grows rapidly in
the usual culture media at the room-temperature.

Growth on Gelatin. The growth upon gelatin plates
containing 5 per cent, of gelatin is very characteristic.
At the end of ten or twelve hours at room-tem-
perature small round depressions in the gelatin are
observed, which contain liquefied gelatin and a whitish
mass consisting of bacilli in the centre. Under a low-
power lens these depressions are seen to be surrounded
by a radiating zone composed of two or more layers,
outside of which is a zone of a single layer, from which
amoeba-like processes extend upon the surface of the
gelatin. These processes are constantly undergoing
changes in their form and position. The young colo-
nies deep down in the gelatin are somewhat more
compact, and rounded or humpbacked; later they
are covered with soft down; then they form irregular,
radiating masses and simulate the superficial colonies.
But it is difficult to describe all the forms which the
proteus vulgaris takes on in all the stages of its growth
on gelatin plates. When the consistency of the medium
is more solid, as in 10 per cent, gelatin, the liquefac-
tion and migration of surface colonies are more or less
retarded. In gelatin stick cultures the growth is less
characteristic liquefaction takes place rapidly along
the line of puncture, and soon the entire contents of
the tube are liquefied.



BACILLUS PROTEUS VULGARIS. 541

Upon nutrient agar a rapidly spreading, moist, thin,
grayish-white layer appears, and migration of the col-
onies also occurs. Milk is coagulated, with the pro-
duction of acid.

The cultures in media containing albumin or gelatin
have a disagreeable, putrefactive odor, and become alka-
line in reaction. Growth is most luxuriant at a tem-
perature of 24 C., but is plentiful also at 37 C. It
is a facultative anaerobe and grows also in the presence
of oxygen, but the proteus then loses its power of lique-
fying gelatin. It produces indol and phenol from pep-
tone solutions. The proteus develops fairly well in
urine, and decomposes urea into carbonate of ammonia.

Pathogenesis. This bacillus is pathogenic for rabbits
and guinea-pigs when injected in large quantities into
the circulation, into the abdominal cavity, or subcuta-
neously, producing death of the animals with symptoms
of poisoning. Hauser has obtained the bacillus proteus
vulgaris from a case of purulent peritonitis, from puru-
lent puerperal endometritis, and from a phlegmonous
inflammation of the hand. Brunner also reports simi-
lar infections in which this organism was found asso-
ciated with pus cocci, and Charrin describes a case of
pleuritis during pregnancy in which the proteus was
present and a foul-smelling secretion was produced.
Death in this case, which ensued without further com-
plication, is said to have been due probably to the
poisonous products of the proteus.

An interesting example of pure toxaemia resulting
from the toxin of the proteus is reported by Levy :
While conducting some experiments on this organism
he had an opportunity of making a bacteriological ex-
amination in the case of a man who died after a short



542 BACTERIOLOGY.

attack of cholera morbus. From the vomited material
and the stools he obtained a pure culture of the pro-
teus; but the blood, collected at the autopsy, was sterile.
In the meantime seventeen other persons who had eaten
at the same restaurant were taken sick in the same
way. Upon examination at the restaurant it was found
that the bottom of the ice-chest in which the meat was
kept was covered with a slimy, brown layer, which
gave off a disagreeable odor. Cultures from this gave
the proteus as the principal organism present. Injec-
tions into animals of the pure cultures produced similar
symptoms as occurred in the human subjects. Levy
concludes that in so-called " flesh-poisoning " bacteria
of this group are chiefly concerned, and that the patho-
genic effects are due to toxic products evolved during
their development.

Booker, from his extended researches into this sub-
ject, concludes that the proteus plays an important part
in the production of the morbid symptoms which char-
acterize cholera infantuna. Proteus vulgaris was found
in the al vine discharge in a large proportion of the cases
examined by him, but was not found in the feces of
healthy infants. "The prominent symptoms in the
cases of cholera infantum in which the proteus bacteria
were found were drowsiness, stupor, emaciation, and
great reduction in flesh, more or less collapse, frequent
vomiting and purging, with watery and generally offen-
sive stools."

Next to the bacillus coli communis the proteus vul-
garis appears to be the micro-organism most frequently
concerned in the etiology of pyelonephritis. In cases
of cystitis and of pyelonephritis this bacillus is often
found in pure cultures or associated with other bac-
teria. It probably gets into the bladder chiefly through



BACILLUS OF MALIGNANT (EDEMA. 543

catheterization. From the animal experiments of the
authors above mentioned, simple injection of pure cul-
tures of proteus into the bladder, without artificial sup-
pression of urine, invariably produces severe cystitis.
The fact that this organism grows in urine is sufficient
to account for the extension of the purulent process
finally to the kidneys.

The proteus vulgaris is, however, a harmles parasite
when located in the mucous membrane of the nasal
cavities. Here it only decomposes the secretions, with
the production of a putrefactive odor. On the whole,
considering the very wide distribution of this organism
in nature, it is remarkable how few diseases are pro-
duced by it.

BACILLUS OF MALIGNANT (EDEMA.

This bacillus is widely distributed, being found in
the superficial layers of the soil, in putrefying sub-
stances, in the blood of animals which have been suffo-
cated (by invasion from the intestine), in foul water,
etc. It was discovered (1877) by Pasteur in animals
after injections of putrefying liquids, and named by
him " vibrion septique." He recognized its anaerobic
nature, but did not obtain it in pure culture. Koch
(1881) carefully studied this micro-organism, described
it in detail, and gave it the name " bacillus cedematous
maligni." It was isolated first in pure culture by
Liborius.

Morphology. The oedema bacillus is a rod of from
0.8/2 to \IJL in width, and of very varying length, from
2// to 10// or more, according to .the conditions of its
cultivation and growth. It is usually found in pairs,
joined end to end, but may occur in chains or long
filaments. It forms spores, and these are situated



544 BACTERIOLOGY.

in or near the middle of the body of the rods. The
spores vary in length and are oval in form, being often
of greater diameter than the bacilli, to which they give
a more or less oval or spindle shape.

The bacilli stain readily by the usual aniline colors
employed, but are decolorized by Gram's method.

Biological Characters. A strictly anaerobic, liquefy-
ing, motile bacillus. Forms spores. It grows, however,
in all the usual culture media in the absence of oxygen.
Development takes place at the room-temperature, but
more rapidly and abundantly at 37 C.

Growth in Gelatin. This bacillus may be cultivated
in ordinary nutrient gelatin, but the growth is more
abundant in glucose-gelatin containing 1 or 2 per cent,
of glucose. Gas-bubbles are formed and the gelatin
liquefies.

Growth on Agar. On agar plates the colonies appear
as dull, whitish points, irregular in outline, and when
examined under a low-power lens are seen to be com-
posed of a dense network of interlacing threads radi-
ating irregularly from the centre toward the periphery.

Blood-serum is rapidly liquefied, with the production
of gas. Cultures of the malignant oedema bacillus give
off a peculiar, disagreeable odor.

Pathogenesis. The bacillus of malignant oedema is
especially pathogenic for mice, guinea-pigs, and rabbits,
although man, horses, dogs, goats, sheep, calves, pigs,
chickens, and pigeons are also susceptible. A small
quantity of a pure culture injected beneath the skin of
a susceptible animal gives rise to an extensive hemor-
rhagic oedema of the subcutaneous connective tissue,
which extends over the entire surface of the abdomen
and thorax, causing hypersemia and redness of the
superficial muscles. There is no odor developed, and



BACILLUS AEROGENES CAPSULATUS. 545

little, if any, production of gas. In infection with
garden earth, owing to the presence of associated ba-
cilli, the effused serum is frothy from the development
of gas, and possesses a putrefactive odor. The disease,
in natural infection caused by the contamination of
wounds with earth or feces, runs the course above de-
scribed. Simple abrasion of the skin is not sufficient
to produce infection; owing to the bacillus being capable
only of an anaerobic existence, the poison must pene-
trate deep into the tissues. Malignant oedema is con-
fined mostly to the domestic animals, but cases have
also been reported in man.

Animals which recover from malignant oedema are
subsequently immune. Artificial immunity may be
induced in guinea-pigs by injecting filtered cultures of
the malignant oedema bacillus in harmless quantities.

BACILLUS AEROGENES OAPSULATUS.

Found by Welch in the bloodvessels of a patient
suffering with aortic aneurism; on autopsy, made in
cool weather eight hours after death, the vessels were
observed to be full of gas-bubbles. Since then it has
been found in a number of cases in which gas has de-
veloped from within sixty hours of death until some
hours after death. These cases are, as a rule, marked by
delirium, rapid pulse, high temperature, and the devel-
opment of emphysema and discoloration of the diseased
area, or of marked abdominal distention when the peri-
toneal cavity is involved.

Morphology. Straight or slightly curved rods, with
rounded or sometimes square-cut ends; somewhat
thicker than the anthrax bacilli and varying in length ;
occasionally long threads and chains are seen. The

35



546 BACTERIOLOGY.

bacilli in the animal body, and sometimes in cultures,
are enclosed in a transparent capsule.

Biological Characters. An anaerobic, non-motile, non-
liquefying bacillus. Does not form spores. Grows at
the room-temperature, but more rapidly at 37 C., in
the usual culture media in the absence of oxygen, and
is accompanied by the production of gas. Nutrient
gelatin is not liquefied by the growth of this bacillus,
but it is gradually peptonized. In agar colonies are
developed which are from 1 to 2 mm. or more in
diameter, grayish-white in color, and in the form of
flattened spheres, ovals, or irregular masses, beset with
hair-like projections. Bouillon is diffusely clouded, and
a white sediment is formed. Milk is rapidly coagulated.

Pathogenesis. Usually non-pathogenic in healthy
animals, although Dunham found that the bacillus taken
freshly from human infection is sometimes very virulent.
When quantities up to 2.5 c.c. of fresh bouillon cultures
are injected into the circulation of rabbits and the ani-
mals killed shortly after the injection, the bacilli de-
velop rapidly, with an abundant formation of gas in
the bloodvessels and organs, especially the liver. The
following is one of the best methods of obtaining