Bacteriological Diagnosis. From the above it is appar-
ent that fully developed characteristic cases of diph-
theria are readily diagnosticated, but that many of the
less marked, or at an early period undeveloped, cases
are difficult to differentiate the one from the other.
In these cases cultures are of the utmost value, since
they enable us to isolate those in which the bacilli are
found, and to give preventive injections of antitoxin
to both the sick and those in contact with them, if this
has not already been done. As a rule, cultures do not
give us as much information as to the gravity of the
case as the clinical appearances, for by the end of
twenty-four to forty-eight hours the extent of the dis-
ease is usually easy of determination. The reported
absence of bacilli in a culture must be given weight in
proportion to the skill with which the culture was made,
the suitableness of the media, and the knowledge and
experience of the one who examined it.
Diphtheria does not occur without the presence of
the diphtheria bacilli; but there have been many cases
of diphtheria in which for one or another reason no
bacilli were found in the cultures by the examiner.
In many of these cases later cultures revealed them.
In a convalescent case the absence of bacilli in any one
culture indicates that there are certainly not many
bacilli left in the throat. Only repeated cultures can
prove their total absence.
TECHNIQUE OF THE BACTERIOLOGICAL DIAGNOSIS.
Collection of the Blood-serum and its Preparation for Use
in Cultures. A covered glass jar which has been thor-
378 BACTERIOLOGY.
oughly cleansed with hot water is taken to the slaughter-
house and filled with freshly-shed blood from a calf or
sheep. The blood is received directly in the jar as it
spurts from the cut in the throat of the animal. After
the edge of the jar has been wiped it is covered with
the lid and set aside, where it may stand quietly until
the blood has thoroughly clotted. The jar is then car-
ried to the laboratory and placed in an ice-chest. If
the jar containing the blood is carried about before the
latter has clotted, very imperfect separation of the serum
will take place. It is well to inspect the blood in the
jar after it has been standing a few hours, and if the
clot is found adhering to the sides, to separate it by a
rod. The blood is allowed to remain twenty -four hours
on the ice, and then the serum which surrounds the clot
is siphoned off by a rubber tube and mixed with one-
third its quantity of nutrient beef broth, to which 1
per cent, glucose has been added. This constitutes the
Loffler blood-serum mixture. This is poured into tubes,
which should be about four inches in length and two-
thirds of an inch in diameter, having been previously
plugged witli cotton and sterilized by dry heat at
150 C. for one hour. Care should be taken in filling
the tubes to avoid the formation of air-bubbles, as they
leave a permanently uneven surface when the strum
has been coagulated by heat. To prevent this the end
of the pipette or funnel which contains the serum should
be inserted well into the test-tube. About 2 c.c. are
sufficient for each tube. The tubes, having been filled
to the required height, are now to be coagulated and
sterilized. They are placed slanted at the proper angle
and then kept for two hours at a temperature just below
95 C. For this purpose a Koch serum coagulator
DIPHTHERIA BACILLUS. 379
(Fig. 22) or a double boiler serves best, though a steam
sterilizer will suffice. If the latter is used a wire
frame must be arranged to hold the tubes at the proper
inclination, and the degree of heat must be carefully
watched, as otherwise the temperature may go too
high, and if the serum is actually boiled the culture
medium will be spoiled. After sterilization by this
process the tubes containing the sterile, solidified blood-
serum can be placed in covered tin boxes or stopped
with sterile corks and kept for months. The serum thus
prepared is quite opaque and firm. A mixture of blood-
cells renders the serum darker, but it is not less useful.
The Swab for Inoculating Culture Tubes. The swab
to inoculate the serum is made as follows : A stiff, thin
iron rod, six inches in length, is roughened at one end
by a few blows of a hammer, and about this end a little
absorbent cotton is firmly wound. Each swab is then
placed in a separate glass tube, and the mouths of the
tubes are plugged with cotton. The tubes and rods are
then sterilizt d by dry heat at about 150 C. for one
hour, and stored for future use. Th<se cotton swabs
have proved much more serviceable for making inocu-
lations than platinum wire needles, especially in young
children and in laryngeal cases. It is easier to use the
cotton swab in such cases, and it gathers up so much
more material for the inoculation that it has seemed
more reliable.
For convenience and safety in transportation a " cul-
ture outfit' 7 has been devised, which consists of a small
wooden box containing a tube of blood-serum, a tube
holding a swab, and a record blank. These " culture
outfits " may be carried or sent by messenger or ex-
press to any place desired.
380 BACTERIOLOGY.
Directions for Inoculating Culture Tubes with the Ex-
udate. The patient is placed in a good light, and, if a
child, properly held. The swab is removed from its
tube, and, while the tongue is depressed with a spoon,
is passed into the pharynx (if possible, without touch-
ing the tongue or other parts of the mouth) and is
rubbed gently but firmly against any visible membrane
on the tonsils or in the pharynx, and then, without
being laid down, the swab is immediately inserted in
the blood-serum tube, and the portion which has pre-
viously been in contact with the exudate is rubbed a
number of times back and forth over the whole sur-
face of the serum. This should be done thoroughly,
but it is to be gently done, so as not to break the sur-
face of the serum. The swab should then be placed
in its tube, and both tubes, thin cotton plugs having
been inserted, are reserved for examination or sent to
the laboratory or collecting station (as in New York
City). If sent to the health department laboratories
for examination the blank forms of report which usu-
ally accompany each "outfit" should be filled out and
forwarded with the tubes.
Where there is no visible membrane (it may be
present in the nose or larynx) the swab should be
thoroughly rubbed over the mucous membrane of the
pharynx and tonsils, and in the nasal cavities, and a
culture made from these. In very young children care
should be taken not to use the swab when the throat
contains food or vomited matter, as then the bacterio-
logical examination is rendered more difficult. Under
no conditions should any attempt be made to collect
the material shortly after the application of strong
DIPHTHERIA BACILLUS. 381
disinfectants (especially solutions of corrosive subli-
mate) to the throat.
Examination of Cultures. The culture tubes which
have been inoculated, as described above, are kept in
an incubator at 37 C. for twelve hours, and are then
ready for examination. When great haste is required,
even five hours will often suffice for a sufficient growth
of bacteria for a skilled examiner to decide as to the
presence or absence of the bacilli. On inspection it
will be seen that the surface of the blood-serum is
dotted with numerous colonies, which are just visible.
No diagnosis can be made from simple inspection ; if,
however, the serum is found to be liquefied or shows
other evidences of contamination the examination will
probably be unsatisfactory.
In order to make a microscopical preparation a clean
platinum needle is inserted in the tube and quite a
large number of colonies are swept with it from the
surface of the culture medium, a part being selected
where small colonies only are found. A sufficient
amount of the bacteria adherent to the needle are
washed off in the drop of water previously placed on
the cover-glass and smeared over its surface. The
bacteria on the glass are then allowed to dry in the
air. The cover-glass is then passed quickly through
the flame of a Bunsen burner or alcohol lamp, three
times in the usual way, covered with a few drops of
Loffler's solution of alkaline methylene-blue, and left
without heating for ten minutes. It is then rinsed off
in clear water, dried, and mounted in balsam. When
other methods of staining are desired they are carried
out in the proper way.
In the great majority of cases one of two pictures
382 BACTERIOLOGY.
will be seen with the 1/12 oil immersion lens either
an enormous number of characteristic Loffler-ba( illi,
with a moderate number of cocci, or a pure culture of
cocci, mostly in pairs or short chains (see Streptococ-
cus). In a few cases there will be an approximately
even mixture of Loffler bacilli and cocci, and in others
a great excess of cocci. Beside these, there will be
occasionally met preparations in which, with the cocci,
there are mingled bacilli more or less resembling the
Loffler bacilli. These bacilli, which are usually of
the pseudodiphtheria type of bacilli (see Fig. 46), are
especially frequent in cultures from the nose.
In not more than one case in twenty will there be
any serious difficulty in making the diagnosis, if the
serum in the tube was moist and had been properly
inoculated. In such a case another culture must be
made or the bacilli plated out and tested in pure
culture.
Direct Microscopical Examination of the Exudate.
An immediate diagnosis without the use of cultures
is often possible from a microscopical examination of
the exudate. This is made by smearing a slide or
cover-^lass with a little of the exudate from the swab,
drying, heating, staining, and examining it microscop-
ically. This examination, however, is much more diffi-
cult, and the results are more uncertain than when the
covers are prepared from cultures. The bacilli from
the membrane are usually less typical in appearance
than those found in cultures, and they are mixed with
fibrin, pus, and epithelial cells. They may also be
very few in number in the parts reached by the swab,
or bacilli may be met with which closely resemble the
Loffier bacilli in appearance, but which differ greatly
DIPHTHERIA BACILLUS. 383
in growth and in other characteristics, and have abso-
lutely no connection with them. When in a smear
containing mostly cocci a few of these doubtful bacilli
are present, it is impossible either to exclude or to make
the diagnosis of diphtheria with certainty. Although
in some cases this immediate examination may be of
the greatest value, it is not a method suitable for gen-
eral use, and should always be controlled by cultures.
Animal Inoculation as a Test of Virulence. If the
determination of the virulence of the bacilli found is
of importance, animal inoculations must be made. Ex-
periments on animals form the only method of deter-
mining with certainty the virulence of the diphtheria
bacillus. For this purpose, alkaline broth cultures of
forty-eight hours 7 growth should be used for the sub-
cutaneous inoculation of guinea-pigs. The amount
injected should not be more than one-fifth per cent, of
the body-weight of the animal inoculated unless con-
trols with antitoxin are made. In the large majority
of cases, when the bacilli are virulent, this amount
causes death within seventy-two hours. At the autopsy
the characteristic lesions already described are found.
Bacilli which in cultures and in animal experiments
have shown themselves to be characteristic may be
regarded for practical purposes as certainly true diph-
theria bacilli, and as capable of producing diphtheria
in man under favorable conditions.
For an absolute test of specific virulence antitoxin
must be used. A guinea-pig is injected with antitoxin,
and then this and a control animal, with double the
fatal dose of a broth culture of the bacilli to be tested;
if the guinea-pig which received the antitoxin lives,
while the control dies, it was surely a diphtheria bacil-
384 BACTERIOLOGY.
lus which killed by means of diphtheria toxin or, in
other words, not simply a virulent bacillus, but a viru-
lent diphtheria bacillus. When the bacilli to be tested
grow poorly in the simple nutrient bouillon they should
be grown in bouillon to which one-third its quantity of
ascitic fluid has been added. Quite a number of bacilli
have been met with which killed 250 gramme guinea-
pigs in doses of 2 to 15 c.c., and yet were unaffected
by antitoxin. These bacilli, though slightly virulent
to guinea-pigs, produce no diphtheria toxin, and so
cannot, to the best of our belief, produce diphtheria in
man.
CHAPTER XXII.
THE BACILLUS OF TETANUS.
IN 1884, Nicolaier, a student in Fliigge's Institute,
produced tetanus in mice and rabbits by the subcutaneous
inoculation of particles of garden earth, and showed that
the disease was transmissible by inoculation from these
animals to others. Carle and Rattoiie, in 1884, demon-
strated the infectious nature of tetanus as it occurs in
man. Finally, Kitasato, in 1889, obtained the bacillus
of tetanus in pure culture and described his method of
obtaining it and its biological characters.
The tetanus bacillus occurs in nature as a common
inhabitant of the soil, at least in places where manure
has been thrown, being abundant in many localities,
not only in the superficial layers, but also at the depth
of several feet. It has been found in many different
substances and places in hay-dust, in horse and cow
manure, in the mortar of old masonry, in the dust from
horses' hair, in the dust in rooms of houses, barracks,
and hospitals, in the air, and in the arrow poison of
certain savages in the New Hebrides, who obtained it
by smearing the arrow-heads with dirt from crab holes
in the swamps.
Morphology. Motile, slender rods, with rounded ends,
0.3// to 0.5/jt. in diameter by 2/j. to 4fj. in length, usually
occurring singly, but, especially in old cultures-, often
growing in long threads. They form round spores,
25
386 BACTERIOLOGY.
thicker than the cell (from I ft to 1.5// in diameter),
occupying one of its extremities and giving to the rods
the appearance of small pins (Fig. 48). It is stained
with the ordinary aniline dyes, and is not decolorized
by Gram's method. The spores may be demonstrated
by double-staining with ZiehPs method.
FIG. 48.
Tetanus bacilli with spores in distended ends. X 1100 diameters.
Biology. An anaerobic, liquefying, motile (though not
very actively motile) bacillus. Forms spores, and in
the spore stage it is not motile. It does not grow at
temperatures below 14 C., but grows slowly at tem-
peratures from 20 to 24 C., and best at 37 C., when
it rapidly forms spores. It will not grow in the pres-
ence of oxygen or carbon dioxide gas, but grows well in
an atmosphere of pure hydrogen.
The bacillus of tetanus grows in ordinary nutrient
gelatin and agar of a slightly alkaline reaction. The
addition to the media of 1.5 per cent, of glucose causes
the development to be more rapid and abundant. It
also grows abundantly in alkaline bouillon under an
atmosphere of hydrogen.
THE BACILLUS OF TETANUS. 387
Its growth in the animal organism is comparatively
scanty, and is usually associated with other bacteria;
hence, it is difficult to obtain it in pure culture. The
method of procedure proposed by Kitasato, which, how-
ever, is not always successful, consists in inoculating an
agar tube with the tetanus-bearing material (pus from
the inoculation wound), keeping this for twenty-four to
forty-eight hours at a temperature of 37 C., and, after
the tetanus spores have formed, heating it for about an
hour at 80 C., to destroy the associated bacteria. The
spores of the tetanus bacillus being able to survive this
exposure, anaerobic cultures are then made in the usual
way, and the tetanus colonies thus isolated. The fur-
ther development is unattended with difficulty. On
gelatin plates the colonies develop slowly; they resemble
somewhat the colonies of the bacillus subtilis, and have
a dense, opaque centre surrounded by fine, diverging
rays. Liquefaction takes place more slowly, however,
than with the bacillus subtilis, and the resemblance to
these colonies is soon lost. In old cultures the entire
mass is made iip of a number of fine threads, and the
colonies are not unlike those of the common mould.
The colonies on agar are quite characteristic (San-
felice). To the naked eye they present the appearance
of light, fleecy clouds; under the microscope, a tangle
of fine threads. The extreme fineness of the threads
enables them to be distinguished from the colonies of
-other anaerobes.
The stab cultures in gelatin exhibit the appearance of
a cloudy, linear mass, with prolongations radiating into
the gelatin from all sides. Liquefaction takes place
slowly, generally with the production of gas. In stab
cultures in agar a growth occurs not unlike in structure
388 BACTERIOLOGY.
that of a miniature pine-tree. Alkaline bouillon is ren-
dered somewhat turbid by the growth of the tetanus
bacillus. In all cases a production of gas results, accom-
panied by a characteristic and very disagreeable empy-
reumatic odor. It also grows in acid culture media,
but of itself produces no acid. It develops in milk
without coagulating it, and starch is not hydrated by
it in its growth (Sanfelice).
The spores of the tetanus bacillus are very resistant
to outside influences; they retain their vitality for
months and years in a desiccated condition, and are
not destroyed in two and a half months when present in
putrefying material (Turco). They withstand an ex-
posure of one hour to 80 C., but are killed by an ex-
posure of five minutes to 100 C. in the steam sterilizer.
They resist the action of 5 per cent, carbolic acid for
ten hours, but succumb when exposed to it for fifteen
hours. A 5 per cent, solution of carbolic acid, how-
ever, to which 0.5 per cent, of hydrochloric acid has
been added, destroys them in two hours. When acted
upon for three hours by bichloride of mercury (1 : 1000)
they are killed, and in thirty minutes when 0.5 per cent.
HC1 is added to the solution. If the solution contains
1 : 1000 bichloride, with 5 per cent, carbolic and a 0.5
per cent. HC1, the spores are killed in ten minutes.
Silver nitrate solutions destroy the spores in one minute
in 1 per cent, solution and in five minutes in 1 : 1000
solution.
Pathogenesis. In mice, guinea-pigs, rabbits, rats,
horses, goats, and a number of other animals inocula-
tions of pure cultures of the tetanus bacillus cause typi-
cal .tetanus after an incubation of from one to three
days. A mere trace only as much as remains cling-
THE BACILLUS OF TETANUS. 389
ing to a platinum needle of an old culture is often
sufficient to kill very susceptible animals like mice and
guinea-pigs. Other animals require a larger amount.
Birds are but little susceptible, and fowls scarcely at
all. It is a remarkable fact that an amount of toxin
sufficient to kill a hen would suffice to kill 500 horses.
On the inoculation of less than a fatal dose in test-
animals a local tetanus may be produced, which lasts
for days and weeks and then ends in recovery. On
killing the animal there is found at autopsy, just at the
point of inoculation, a hemorrhagic spot, and no changes
here or in the interior organs other than these. A few
tetanus bacilli may be detected locally with great diffi-
culty, often none at all; possibly a few may be found
in the region of the lymphatic glands. From this
scanty occurrence of bacilli the conclusion has been
reached that the bacilli of tetanus, when inoculated in
pure culture, do not multiply in the living body, but
only produce lesions through the absorption of the
poison which they produce at the point of infec-
tion. These authors also found that pure cultures of
tetanus, after the germs had sporulated and the toxins
had been destroyed by heat, could be injected into
animals without producing tetanus. Even one or two
millions of spores, if deprived of the toxins, proved
harmless to guinea-pigs, and from 15 to 30 c.c. of
broth cultures were harmless to rabbits. But if a cul-
ture of non-pathogenic organisms was injected simul-
taneously with the spores, or if there was an effu-
sion of blood at the point of injection, or if there was
a previous bruising of the tissues, the animals surely
died of tetanus. Even irritating foreign bodies were
introduced along with the spores deprived of their
390 BACTERIOLOGY.
toxins, and tetanus did not develop; bat if the wounds
containing the foreign bodies became infected with
other bacteria, tetanus developed and the animals died.
From these experiments it seems that a mixed infection
is necessary to the development of tetanus when the
infection is produced by spores.
This fact i's of the greatest importance in natural
tetanus. Here the infection may be considered as
prubably invariably produced by the bacilli in their
spore state, and the conditions favoring infection are
almost always present. A wound of some kind has
occurred, penetrating at least through the skin, though
perhaps of a most trivial character, such as might be
caused by a dirty splinter of wood, and the bacilli or
their spores are thus introduced from the soil in which
they are so widely distributed. If in any given case,
the tissues being healthy, the ordinary saprophytic
germs are killed by proper disinfection at once, a
mixed infection does not take place, and tetanus will
not develop. If, however, the tissues infected be
badly bruised or lacerated, the spores may develop
and produce the disease. With regard to the persist-
ence of tetanus spores upon objects where they have
found a resting-place, Henrijean reports that by means
of a splinter of wood which had once caused tetanus
he was able after eleven years to again cause the dis-
ease by inoculating an animal with the same splinter.
The bacilli of tetanus are apparently more numerous
in certain localities than in others for example, some
parts of Long Island and New Jersey, which have
become notorious for the number of cases of tetanus
caused by small wounds but they are very generally
distributed, as the experiments on animals inoculated
THE BACILLUS OF TETANUS. 391
with garden earth have shown, and are fairly common
in New York City.
Man and almost all domestic animals are subject to
tetanus. On examination of an infected individual
very little local evidence of the disease can be discov-
ered. Generally at the point of infection, if there is
an external wound, some pus is to be seen, in which,
along with numerous other bacteria, tetanus bacilli or
their spores may be found. By successive inoculation
of this pus in susceptible animals the disease can often be
reproduced for from four to five generations; but some-
times there is a break in the chain, which proves that
in such cases the infection has been brought about less
by the bacilli than by the toxin which was transmitted
with them.
Not only traumatic tetanus, but also all the various
forms of tetanus, are now conceded to be produced by
the tetanus bacillus puerperal tetanus, tetanus neona-
torum, and idiopathic and rheumatic tetanus. In
tetanus neonatorum and puerperal tetanus the infection
is introduced through the navel and the inner surface
of the uterus. It should be borne in mind, however,
that when there is no external and visible wound there
may be an internal one. Carbone and Perrero report
a case of so-called rheumatic tetanus in which attenu-
ated forms of tetanus bacilli were found in the bronchial
secretions. These bacilli possessed the morphological
and cultural peculiarities of the tetanus bacilli, but
they did not produce toxin. Similar anaerobes have
been found in meat-juices and in the soil. The bacilli
found in the bronchial secretions, therefore, may have
been tetanus bacilli which, owing to certain conditions,
had lost their virulence, just as we know it to happen
392 BACTERIOLOGY.
in diphtheria. It may well be supposed that the mucous
membranes of the bronchi, and other similar mem-
branes, in a condition of catarrhal inflammation, may
be more susceptible to tetanus infection than they
normally are.
Tetanus Toxin. It is evident from the localization of
the tetanus bacilli at the point of inoculation and their
slight multiplication at this point that they owe their
action to the production of a powerful toxin. While
there are a few cases on record in which the bacilli