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immunisation of animals against typhoid bacilli have been
applied by Wright and Semple to man in the following
way. Typhoid bacilli are obtained of such virulence that
a quarter of a twenty -four hours' old agar culture when
administered hypodermically will kill a guinea-pig of from
350 to 400 grammes. Such a culture is emulsified in
bouillon and killed by being raised to 60 C. and kept at
that for five minutes. . The vaccination is accomplished by
the hypodermic injection of from one-twentieth to one-fourth
of such a dead culture. The effects of such an injection
are some local tenderness, and it may be swelling, and a
general feeling of restlessness for some hours with occa-
sionally a slight rise of temperature. The possibility of
this vaccination preventing the development of typhoid


fever after exposure to natural infection must rest on
experience, but as most of those who have been subjected
to it are medical officers in the British services, who are
likely to be exposed to such infection abroad, this ex-
perience may soon be gained. It has been found in cases
where the method has been practised that in the course of
a fortnight a positive Widal's reaction was obtained, and
such an occurrence is probably evidence of the acquisition
of a certain degree of immunity.

Anti-typhoid Serum. Bokenham grew virulent typhoid bacilli for
three weeks on bouillon containing ten per cent of alkali albumin, and
filtering the cultures through porcelain obtained a filtrate which,
though non-toxic to guinea-pigs, probably had immunising properties.
Starting with this and afterwards using it alternately with killed
cultures, he immunised a horse and found that the serum had neutral-
ising power for typhoid bacilli when the latter mixed with it were
injected into guinea-pigs. When injection of the serum was followed
by injection of bacilli, the pathogenic action of the latter was to a
certain extent prevented, and there was also evidence of the serum
possessing curative properties.

Methods of Examination. The methods of microscopic
examination, and of isolation of typhoid bacilli from the
spleen post mortem, have already been described. They may
be isolated from the Peyer's patches, lymphatic glands, etc.
by a similar method.

During life, typhoid bacilli may be obtained in culture
in the following ways :

(a) from the Spleen. This is the most certain method
of obtaining the typhoid bacillus during the continuance
of a case. The skin over the spleen is purified and, a sterile
hypodermic syringe being plunged into the organ, there is
withdrawn from the splenic pulp a droplet of fluid, from
which plates are made. In a large proportion of cases of
typhoid the bacillus may be thus obtained, failure only
occurring when the needle does not happen to touch a
bacillus. Numerous observations have shown that pro-
vided the needle be not too large, the procedure is quite
safe. Its use, however, is scarcely called for.


(b) From the Urine. Typhoid bacilli are present in the
urine in about twenty-five per cent of cases, especially late
in the disease, probably chiefly when there are groups in
the kidney substance. For methods of examining sus-
pected urine, see p. 78.

(c) From the Stools. During the first ten days of a case
of typhoid fever, the bacilli can be isolated from the stools
by the ordinary plate methods preferably in phenolated
gelatine. After that period, though the continued in-
fectiveness of the disease indicates that they are still
present, their isolation is practically hopeless. We have
seen that after ulceration is fairly established by the slough-
ing of the necrosed tissue, the numbers present in the
patches are much diminished and therefore there are fewer
cast off into the intestinal lumen, and that in addition
there is a correspondingly great increase of the B. coli,
which thus causes any typhoid bacilli in a plate to be quite
outgrown. From the fact that the ulcers in a case of
typhoid may be very few in number, it is evident that there
may be at no time very many typhoid bacilli in the
intestine. We may add that the microscopic examination
of the stools is useless as a means of diagnosing the
presence of the typhoid bacillus.

Isolation from Water Supplies. A great deal of work
has been done on this subject. It is evident that if it
is difficult to isolate the bacilli from the stools it must
a fortiori be much more difficult to do so when the latter
are enormously diluted by water. Some have held that
the typhoid bacillus has never been isolated from suspected
water, and have adduced this as an argument against its
etiological relationship to the disease. The considerations
just advanced, however, militate against such a view. The
B. typhosus has been isolated from water during epidemics.
This was done by Klein in the outbreaks in recent years
at Worthing and Rotherham. The B. coli is, as might be
expected, the organism most commonly isolated in such
circumstances. In the case of both bacteria, the whole
series of culture reactions must be gone through before


any particular organism isolated is identified as the one or
the other ; probably there are saprophytes existing in nature
which only differ from them in one or two reactions. In
the examination of water, the addition of .2 per cent
carbolic acid to the medium inhibits to a certain extent
the growth of other bacteria, while the B. typhosus and the
B. coli are unaffected. In examining waters, the ordinary
plate methods are generally used. Klein, however, filters
a large quantity through a Berkefeld filter and, brushing
off the bacteria retained on the porcelain, makes cultures.
A much greater concentration of the bacteria is thus


THERE is no better example of the valuable contributions
of bacteriology to scientific medicine than that afforded in
the case of diphtheria. Not only has research supplied,
as in the case of tubercle, a means of distinguishing true
diphtheria from conditions which resemble it, but the study
of the toxines of the bacillus has explained the manner by
which the pathological changes and characteristic symptoms
of the disease are brought about, and has led to the dis-
covery of the most efficient means of treatment, namely,
the anti-diphtheritic serum.

Historical. As in the case of many other diseases, various organisms
which have no causal relation to the disease were formerly described in the
false membrane. The first account of the bacillus now known to be the
cause of diphtheria was given by Klebs in 1883, who described its charac-
ters in the false membrane, but made no cultivations. It was first culti-
vated by Loftier from a number of cases of diphtheria, his observations
being published in 1884, and to him we owe the first account of its
characters in cultures and of some of its pathogenic effects on animals.
The organism is for these reasons known as the Klebs-Loflfler bacillus,
or simply as Loffler's bacillus. By experimental inoculation with the
cultures obtained, Loftier was able to produce false membrane on damaged
mucous surfaces, but he hesitated to conclude definitely that this organ-
ism was the cause of the disease, for he did not find it in all the cases
of diphtheria examined, he was not able to produce paralytic pheno-
mena in animals by its injection, and, further, he obtained the same
organism from the throat of a healthy child. This organism became
the subject of much inquiry, but its relationship to the disease may be



said to have been definitely established by the brilliant researches of
Roux and Yersin, who made an extensive study of its characters and
life history, and showed that the most important features of the disease
could be produced by means of the separated toxines of the organism.
Their experiments were published in 1888-90. A considerable amount
of further light has been thrown on the subject by the work of Sidney
Martin, who has found that there can be separated from the organs in
cases of diphtheria substances which act as nerve poisons, and also
produce other phenomena met with in diphtheria.

General Facts. Without giving a description of the
pathological changes in diphtheria, it will be well to men-
tion the outstanding features which ought to he considered
in connection with its bacteriology. In addition to the
formation of false membrane, which may prove fatal by
mechanical effects, the chief clinical phenomena are the
symptoms of general poisoning, great muscular weakness,
tendency to syncope, and albuminuria ; also the striking
paralyses which occur later in the disease, and which may
affect the muscles of the pharynx, larynx, and eye, or less
frequently the lower limbs (being sometimes of paraplegic
type), all these being grouped together under the term
" post-diphtheritic paralyses." It may be stated here that
all these conditions have been experimentally reproduced
by the action of the bacillus of diphtheria, or by its toxines.
Other bacteria are, however, concerned in producing various
secondary inflammatory complications in the region of the
throat, such as ulceration, gangrenous change, and suppura-
tion, which may be accompanied by symptoms of general
septic poisoning.

The detection of the bacillus of Loffler in the false
membrane or secretions of the mouth is to be regarded as
the only certain means of diagnosis of diphtheria. With
the exception of the tubercle bacillus, there is probably no
organism which has been the subject of so much routine
examination, and the opinion of all who are competent to
judge may be said to be unanimous on this subject.

Bacillus Diphtheria Microscopical Characters. If a
film preparation be made from a piece of diphtheria mem-
brane (in the manner described below) and stained with


methylene-blue, the bacilli are found to have the following
characters. They are slender rods, straight or slightly
curved, and usually about 3 /x in length, their thickness
being a little greater than that of the tubercle bacillus.


<* *


~J ,g^*.

FlG. 89. Film preparation from diphtheria membrane ; showing
numerous diphtheria bacilli. One or two degenerated forms are seen
near the centre of the field. (Cultures made from the same piece of
membrane showed the organism to be present in practically pure

Stained with methylene-blue. x 1000.

The size, however, varies somewhat in different cases, and
for this reason varieties have been distinguished as small
and large, and even of intermediate size. It is sufficient
to mention here that in some cases most are about 3 //.
in length, whilst in others they may measure fully 5 yu.
Corresponding differences in size are found in cultures.


They stain deeply with the blue, sometimes being uniformly
coloured, but often showing, in their substance, little
granules more darkly stained, so that a dotted or beaded
appearance is presented. Sometimes the ends are swollen
and more darkly stained than the rest; often, however, they
are rather tapered off (Fig. 89). In some cases the terminal
swelling is very marked, so as to amount to clubbing, and
with some specimens of methylene-blue these swellings and
granules stain of a violet tint. Distinct clubbing, however,
is less frequent than in cultures. There is a want of
uniformity in the appearance of the bacilli when compared
side by side. They usually lie irregularly scattered or in
clusters, the individual bacilli being disposed in all direc-
tions. Some may be contained within leucocytes. They
do not form chains, but occasionally forms longer than
those mentioned may be found, and these specially occur
in the spaces between the fibrin as seen in sections.

Distribution of the Bacilli. The diphtheria bacilli may
be found in the membrane wherever it is formed, and may
also occur in the secretions of the pharynx and larynx in
the disease. It may be mentioned that distinctions formerly
drawn between true diphtheria and non-diphtheritic con-
ditions from the appearance and site of the membrane,
have no scientific value, the only true criterion being the
presence of the diphtheria bacilli. The occurrence of a
membranous formation produced by streptococci has
already been mentioned (p. 178).

In diphtheria the membrane has a somewhat different
structure according as it is formed on a surface covered
with stratified squarnous epithelium as in the pharynx, or on
a surface covered by ciliated epithelium as in the trachea.
In the former situation necrosis of the epithelium occurs
either uniformly or in patches, and along with this there
is marked inflammatory reaction in the connective tissue
beneath, attended by abundant fibrinous exudation. The
necrosed epithelium becomes raised up by the fibrin, and
its interstices are also filled by it. The fibrinous exudation
also occurs around the vessels in the tissue beneath, and in


this way the membrane is firmly adherent. In the trachea,
on the other hand, the epithelial cells rapidly become shed,
and the membrane is found to consist almost exclusively of
fibrin with leucocytes, the former arranged in a reticulated
or somewhat laminated manner, and varying in density in

FIG. 90. Section through a diphtheritic membrane in trachea, show-
ing diphtheria bacilli (stained darkly) in clumps, and also scattered
amongst the fibrin. Some streptococci are also shown, towards the
surface on the left side.

Stained by Gram's method and Bismarck-brown. x 1000.

different parts. The membrane lies upon the basement
membrane, and is less firmly adherent than in the case of
the pharynx.

The position of the diphtheria bacilli varies somewhat in
different cases, but they are most frequently found lying in
oval or irregular clumps in the spaces between the fibrin,


towards the superficial, that is, usually, the oldest part of
the false membrane (Fig. 90). There they may be in a
practically pure condition, though streptococci and occa-
sionally some other organisms may be present along with
them. They may occur also deeper, but are rarely found
in the fibrin around the blood vessels. On the surface
of the membrane they may be also seen lying in large
numbers, but are there usually accompanied by numerous
other organisms of various kinds. Occasionally a few
bacilli have been detected in the lymphatic glands. As
Loffler first described, they may be found after death in
pneumonic patches in the lung, this being a secondary
extension by the air passages. They have also been occa-
sionally found by various observers in the spleen, liver, and
other organs after death. This occurrence is probably to
be explained by an entrance into the blood stream shortly
before death, similar to what occurs in the case of other
organisms, e.g., the bacillus coli communis. With these
exceptions, however, it may be stated that the bacillus of
diphtheria occurs only locally in the false membrane and
in the fluids of the mouth, and does not invade even the
subjacent tissues to any extent.

Association ivith other Organisms. The diphtheria
organism is sometimes present alone in the membrane,
but more frequently associated with some of the pyogenic
organisms, the streptococcus pyogenes being the com-
monest. The staphylococci, and occasionally the pneumo-
coccus or the bacillus coli, may be present in some cases.
Streptococci are often found lying side by side with the
diphtheria bacilli in the membrane, and also penetrating
more deeply into the tissues. In some cases of tracheal
diphtheria, we have found streptococci alone, at a lower
level in the trachea than the diphtheria bacilli, where the
membrane was thinner and softer, the appearance in these
cases being as if the streptococci acted as exciters of in-
flammation and prepared the way for the bacilli. It is
still a matter of dispute as to whether the association of
the diphtheria bacillus with the pyogenic organisms is a


favourable sign or the contrary, though on experimental
grounds the latter is the more probable. We know, however,
that some of the complications of diphtheria may be due to
their action. The extensive swelling of the tissues of the
neck, sometimes attended by suppuration in the glands,
and also various haemorrhagic conditions, have been found
to be associated with their presence, in fact, in some cases
the diphtheritic lesion enables them to get a foothold in the
tissues, where they exert their usual action and may lead
to extensive suppurative change, to septic poisoning or to
septicaemia. In cases where a gangrenous process is super-
added, a great variety of organisms may be present, some
of them being anaerobic.

Against such complications anti-diphtheritic serum pro-
duces no favourable effect, as its action is specific and only
neutralises the toxines of the diphtheria bacillus. In view
of this fact, in some cases the anti-streptococcic serum has
been used along with it, and it is apparent that in such con-
ditions the bacteriological examination of the parts affected
may afford valuable indications as to treatment.

Cultivation. The diphtheria bacillus grows best in
cultures at the temperature of the body ; growth still takes
place at 22 C, but ceases at 20 C. The best media are
the following: Loffler's original medium (p. 52), solidified
blood serum, alkaline blood serum (Lorrain Smith), blood
agar, and the ordinary agar media. If inoculations be made
on the surface of blood serum with a piece of diphtheria
membrane, colonies of the bacillus appear within twenty-
four hours, and often before any other growths are visible.
The colonies are small circular discs of opaque whitish
colour, their centre being thicker and of darker greyish
appearance when viewed by transmitted light than the
periphery. On the second or third day they may reach
4 mm. in size, but when numerous they remain smaller.
On the agar media the colonies have much the same
appearance (Fig. 91) but grow less quickly, and sometimes
they may be comparatively minute, so as rather to resemble
those of the streptococcus pyogenes. In stroke cultures

3 6


the growth forms a

FIG. 91. Cultures of the
diphtheria bacillus on an agar
plate; twenty-six hours'growth.
(a) Two successive strokes ; (b)
isolated colonies from the same

of toxine. Ordinary
bouillon becomes
acid during the first
two or three days,
and several days later
again acquires an
alkaline reaction. If,
however, the bouillon
is glucose-free (p. 87)
the acid reaction does
not occur.

In these media
the bacilli show the
same characters as in
the membrane, but
the irregularity in
staining is more
marked (Figs. 92,

continuous layer of the same dull
whitish colour, the margins
of which often show single
colonies partly or completely
separated. On gelatine at 22
C. a puncture culture shows a
line of dots along the needle
track, whilst at the surface a
small disc forms, rather thicker
in the middle. In none of the
media does any liquefaction
occur. In bouillon the organism
produces a turbidity which soon
settles to the bottom and forms
a powdery layer on the wall of
the vessel. By starting the
growth on the surface and keep-
ing the flasks at rest a distinct
scum forms, and this is speci-
ally suitable for the development

FIG. 92. Diphtheria bacilli from a twenty-
four hours' culture on agar.

Stained with methylene-blue. x 1000.


93). They are at first
fairly uniform in size
and shape, but if a
culture is examined
from day to day it will
be found that their
appearance gradually
becomes irregular.
Many become swollen
at their ends into club-
shaped masses which
are stained deeply,
and the protoplasm
becomes broken up
into globules with
unstained parts be-
tween (Fig. 94). Some
become thicker
throughout, and seg-

FIG. 93. Diphtheria bacilli of larger size
than in previous figure, showing also irregular
staining of protoplasm. From a three days'
agar culture.

Stained with weak carbol-fuchsin. x 1000.

mented so
pear like


FIG. 94. Involution forms of the diph-
theria bacillus ; from an agar culture of seven
days' growth.

Stained with carbol-thionin-blue. x 1000.

to ap-
large cocci,
and others show
globules at their ends,
the rest of the rod
appearing as a faintly-
stained line. These
are to be regarded as
involution forms, and
they occur more
quickly and abundant-
ly on the media less
suitable for their
growth, e.g., more
quickly on glycerine
agar than on serum.
The bacilli are non-
motile, and do not
form spores.


Staining. They take up the basic aniline dyes, e.g.,
methylene-blue in watery solution, with great readiness,
and stain deeply, the granules often giving the meta-
chromatic reaction as described. They also retain the
colour in Gram's method.

Neisser has recently introduced the following stain as an aid to the
diagnosis of the diphtheria bacillus. Two solutions are used as
follows : (a) I grin, methylene-blue (Griibler) is dissolved in 20 c.c.
of 96 per cent alcohol, and to the solution are added 950 c.c. of distilled
water and 50 c.c. of glacial acetic acid ; (/) 2 grms. Bismarck-brown
(vesuvin) dissolved in a litre of distilled water. Films are stained in
(a) for 1-3 seconds or a little longer, washed in water, stained for 3-5
seconds in (6), dried, and mounted. The protoplasm of the diphtheria
bacillus is stained a faint brown colour, the granules a blue colour.
Neisser considers that this reaction is characteristic of the organism,
provided that cultures on Loffler's serum are used and examined 9-24
hours after incubation at 34-35 C. The same satisfactory results are
not obtained in the case of films prepared from membrane, etc.

Powers of Eesistance, etc. In cultures the bacilli
possess long duration of life. Even when kept at 37 C.
for one or two months they may be shown by subcultures
to be still alive ; at the room temperature they survive still
longer. In the moist condition, whether in cultures -or in
membrane, they have a low power of resistance, being
killed at 60 C. in a few minutes. On the other hand, in
the dry condition they have great powers of endurance.
In membrane which is perfectly dry, for example, they can
resist a temperature of 98 C. for an hour. Dried diphtheria
membrane, kept in the absence of light and at the room
temperature, has been proved to contain diphtheria bacilli
still living and virulent at the end of several months. The
presence of light, moisture, or a higher temperature, causes
them to die out more rapidly. Corresponding results have
been obtained with bacilli obtained from cultures and kept
on dried threads. These facts, especially with regard to
drying, are of great importance, as they show that the con-
tagium of diphtheria may be preserved for a long time in
the dried membrane. It follows, of course, that cultures
can be obtained from membrane even after it has been
dried, a fact of some practical importance.


Effects of Inoculation. In considering the effects pro-
duced in animals by experimental inoculations of pure
cultures, we have to keep in view the local changes
which occur in diphtheria, and also the symptoms of general

Loffler in his original paper stated that in the case of
rabbits, guinea-pigs, pigeons, and fowls the bacilli taken
from pure cultures produced no change on healthy mucous
membranes, but when the latter were injured by scarification
or otherwise the production of false membrane resulted. A
similar result was obtained when the trachea was inoculated
after tracheotomy had been performed. In this case the
surrounding tissues became the seat of a blood stained
oedema, and the lymphatic glands were enlarged, the general
picture resembling pretty closely that of laryngeal diphtheria.
These results have been amply confirmed by other ob-
servers. The membrane produced by such experiments is
usually less firm than in human diphtheria, and the bacilli
are not generally found in such large numbers in the mem-
brane. Rabbits inoculated after tracheotomy often die, and
Roux and Yersin were the first to observe that in some
cases paralysis may appear before death.

Subcutaneous injection in guinea-pigs, of diphtheria bacilli
in a suitable dose, produces death within thirty-six hours.
On section at the site of inoculation there is seen a small

Online LibraryRobert MuirManual of bacteriology → online text (page 30 of 47)