Albert Besson.

Practical bacteriology, microbiology and serum therapy (medical and veterinary) A text book for laboratory use online

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find any indol in cultures of this organism : and Remy has shown that when the
colon bacillus is grown with the typhoid bacillus the former may lose its capacity
to produce indol.

Recent work demonstrates that the negative results obtained by the earlier
observers were due to the imperfections of their technique. " The property of
producing indol is far less variable than is generally believed," and the indol reaction
furnishes one of the best tests there is for identifying the organism, provided that
the following precautions be observed, viz. :

1. That peptone water and not ordinary broth be used as the culture medium.

2. That the culture be examined between the third and the eighth day but never

3. That the test be performed exactly as described at p. 374.

4. Cultures on synthetic media. The colon bacillus as a rule grows
luxuriantly in the different liquid media of Nsegeli, Maasse, Frankel, Remy
and Sugg '(p. 375).

5. Growth on vaccinated media. If the colon bacillus be sown on a tube
of agar or gelatin on which the typhoid bacillus has already been grown and
scraped off as described above, some amount of growth generally takes place
which though distinct is less abundant than on tubes of new media.

6. Growth on coloured media. The colon bacillus decolourizes both


Nceggerath's medium and fuchsin-agar. The typhoid bacillus gives similar

7. Growth on arseniated broth. A typical colon bacillus grows in broth
containing as much as 2 grams of arsenious acid per litre (Thoinot and

8. Growth on artichoke. A typical colon bacillus grows luxuriantly on
artichoke, and turns the medium green (p. 375).

9. Growth on media containing caffeine. The colon bacillus does not
grow on media containing 0*5 per cent, of caffeine (p. 408).

10. Growth on malachite-green media. According to Loeffler the addition
of a small quantity of malachite-green to culture media prevents the growth
of the colon bacillus, but does not interfere with the growth of the typhoid
bacillus. As a matter of fact, the colon bacillus grows on media containing
either malachite green or crystal violet (pp. 409 and 407).

2. Variability of flagella.

The variability of the flagella is very limited, their characteristics being
little influenced by antiseptics, temperatures unfavourable to growth, etc.
(Kemy and Sugg).

Examination of the flagella should never be neglected when it is desired to
identify the colon bacillus.

3. Vitality and Virulence.

Vitality. All that has been said with regard to the vitality of the typhoid
bacillus is equally applicable to the colon bacillus.

Virulence. The virulence of the colon bacillus is subject to great variation
(vide experimental inoculation, p. 394).

4. Toxin.

Malvos has shown that porcelain-filtered broth cultures are toxic. Broth
cultures also yield a toxic precipitate when heated with sulphate of ammonia.
As a rule, the toxin is not very harmful and large doses of filtered cultures
must be inoculated to produce a fatal result in experimental animals.

The inoculation of a large dose of toxin into the ear- vein of a rabbit pro-
duces the following symptoms : At first there is muscular weakness, sub -normal
temperature, drowsiness and coma : later, convulsions set in and finally a
generalized tetanic condition which continues till the animal dies (Gilbert).
A smaller dose produces a chronic intoxication with diarrhoea, drowsiness
and wasting, the animal often dying of cachexia.

In guinea-pigs, the inoculation of large quantities of toxin into the peri-
toneal cavity is followed by a sub-normal temperature and leads to collapse
and death (Boix). The blood may contain organisms (especially the colon
bacillus) which have found their way from the intestinal canal (Achard and

Colilysin. In suitable media the colon bacillus forms an haemolytic sub-
stance (Kayser).

Colilysin is only produced in any quantity if the broth has a markedly acid
reaction (80 c.c. of decinormal oxalic acid per litre).

The hsemolysin is present after incubating for 2 days at 37 C. but continues to
increase in amount until the fourth day and remains at its maximum until the end
of the second week.

Colilysin is a powerful solvent of dog red-cells ; it has less action on horse,
ox, and rabbit cells, and very little and in some cases no action at all on the


red cells of other animals (man, guinea-pigs, birds etc.). Colilysin can be
kept for months at the ordinary temperature of the laboratory and is not
destroyed by heating to 120 C. for half an hour.

Some normal serums (those of man, the horse etc.) neutralize the hsemolytic
property of colilysin : and an anti-colilysin can be readily produced by inoculating
various animals sub-cutaneously with four-day old broth cultures of the colon

5. Vaccination and serum therapy.

Guinea-pigs and rabbits can be immunized by repeatedly inoculating them
either with small doses of living and virulent organisms or with filtered
cultures of similar strains. Albarran and Mosny produced a very high degree
of immunity in dogs and rabbits by repeatedly inoculating them with small
doses of filtered cultures and with the filtrates derived from macerating the
internal organs of animals dead of a colon bacillus infection. Rodet im-
munized horses and sheep by inoculating them repeatedly with increasing
doses of living or dead cultures.

The serum of vaccinated animals has marked immunizing properties and
also, to some extent, therapeutic properties. These properties are mani-
fested against the strains used for immunization but may be wanting against
strains from other sources.

Antityphoid serum is neither prophylactic nor curative for the colon

According to the experiments of Sanarelli and some other observers animals
vaccinated against the colon bacillus should be immune to both the colon and typhoid
bacilli, and the serum of the animals should immunize against the typhoid bacillus.
These results have however not been confirmed.

6. Agglutination.

(a) The serum of animals infected with the colon bacillus or immunized
against that organism, as well as the serum of persons suffering from infections
due to the colon bacillus, have the property of agglutinating the bacillus.
The agglutination reaction is always obtained with the strain producing the
infection, but the results are often negative if other than the infecting organism
be employed for the reaction, though the latter may be an authentic colon
bacillus. This method of diagnosis cannot therefore be relied upon. The
capacity of the colon bacillus to agglutinate is increased to a very marked
extent by sub-culturing it on artificial media (Rodet).

(b) The colon bacillus is not agglutinated by the serum of animals vaccinated
against the typhoid bacillus nor by the serum of persons suffering from enteric
fever. But for this reaction to be of any value it is important that certain
precautions be observed (vide footnote on p. 389).

All human serums whether taken from enteric fever patients or not exert
a slight agglutinating action on the colon bacillus when diluted five or ten
times. Unless this fact be borne in mind it may lead to error. All mistakes
may be avoided by adopting the following methods.

Determine carefully first of all the agglutinating power of the typhoid
serum which is to be used in the reaction : then mix a drop of the highest
dilution of the serum which will definitely agglutinate the typhoid bacillus
with a culture of the colon bacillus. Thus, for example, if the highest dilution
in which a given typhoid serum will agglutinate the typhoid bacillus be
1-100 this dilution of the serum should be used in testing the suspected colon
bacillus. Under these conditions the agglutination of the colon bacillus is
never observed, and the serum reaction can be employed as an excellent
means for differentiating the two organisms provided that it be always remem-


bered that a strain of the typhoid bacillus which is not agglutinated by a
typhoid serum may very occasionally be encountered.


The methods of detecting the colon bacillus in the tissues and fluids of the
body are similar in principle to those employed for the detection of the typhoid
bacillus. These methods as well as the differentiating tests, etc. are fully
dealt with in Chap. XXIII.

It must be remembered that the colon bacillus often multiplies in the
body immediately after death, and even during the last few hours of life :
the finding of the colon bacillus in the tissues or fluids under these conditions
is therefore of no diagnostic value whatever.

The bacillus of Green Diarrhoea.

According to Lesage and Thiercelin the bacillus of green diarrhoea is merely a
chromogenic variety of the colon bacillus. The organism is found in practically
pure culture in the stools of children suffering from the disease.

Experimental inoculation. The organism is only slightly pathogenic for laboratory
animals. Rabbits, when inoculated intra-venously or fed with cultures of the bacillus,
suffer from an attack of green diarrhoea from which they recover in a few days.

Microscopical appearance. Morphologically the bacillus is a short rod-shaped
organism with rounded ends in every way similar to the colon bacillus.

Cultures. The bacillus of green diarrhoea is a facultative aerobe. It grows on
all the ordinary media and gives rise to a disagreeable odour. The green colouring
matter is only produced in aerobic culture.

A pure culture is very easily obtained by plating a trace of the stool of an infected
child on gelatin.

Broth. At first the medium is uniformly cloudy but later a greenish sediment
is deposited.

Gelatin is not liquefied. In stab culture, the bacillus gives rise to a scanty whitish
growth in the substance of this medium and on the surface to a smalf greenish
lenticular pellicle. On sloped gelatin, the growth is poor, greenish in colour and has
a tendency to spread away from the line of sowing : after a few days the gelatin is
tinted uniformly green. Isolated colonies form small greenish granular points.

On agar. The growth is poor, greenish in colour and spreading. The agar acquires
a green tint.

On potato. The growth is luxuriant, covers the whole surface of the medium
and is of a dirty green mucous appearance.

M ilk is rapidly coagulated.

Carbohydrate media are strongly fermented.




Section I. The isolation of the typhoid and colon bacilli, p. 402.

1. Original methods, p. 402. 2. Eisner's method and its modifications, p. 403.
3. Precipitation methods, p. 406. 4. Method based upon the motility of the typhoid
bacillus, p. 406. 5. Chantemesse's carbolic media, p. 407. 6. Conradi-Drigalski's
method, p. 407. 7. Endo's medium, p. 408. 8. Caffeine media, p. 408. 9. Malachite
green media, p. 409. 10. China green medium, p. 410. 11. Bile media, p. 410. 12.
Brilliant green medium, p. 411. 13. Neutral red media, p. 411. 14. Methods based
upon agglutination, p. 412. 15. MacConkey's media, p. 412.

Section II. The identification of the typhoid and colon bacilli, p. 412.

THE isolation of the typhoid bacillus from water, etc. in which it is mixed
with other species of organisms, and especially when the colon bacillus is
also present, presents certain difficulties which may be summed up under
four headings.

1. On gelatin media, at the ordinary temperature of the atmosphere,
colonies of the typhoid bacillus develop slowly (requiring about 48 hours)
while saprophytic organisms which liquefy the medium grow more quickly
and so put an end to the investigation.

2. The colon bacillus very often retards the growth of the typhoid bacillus
when the two organisms are sown together on artificial culture media, with
the result that the presence of the latter may pass unnoticed. There is, in
fact, a true antagonism between the colon bacillus and the typhoid bacillus
(Grimbert). A similar antagonism also exists between certain other micro-
organic species and the typhoid bacillus when sown together on artificial
media (Besson).

3. Remy, though he does not admit that the typhoid bacillus is crowded
out by the colon bacillus, nevertheless lays stress on the difficulty of isolating
the former when the latter organism is also present. He shows that by
growing the two organisms together their properties may be profoundly
modified : thus the typhoid bacillus occasionally loses its property of being
agglutinated by a specific serum, and the colon bacillus may under like con-
ditions lose its indol-producing and fermentation properties.

4. The ordinary method of gelatin-plating only permits of a very small
quantity of a suspected water being sown and it is therefore possible that if
the typhoid bacillus be present only in small numbers as compared with other
organisms, it may escape notice.



It is not a matter for surprise therefore to find that much experimental
work has been done with a view to perfecting a method or methods of detecting
with certainty the presence of the typhoid bacillus in material in which it
may be suspected to occur.


1. Original methods.

Under this heading will be briefly considered various methods which though
in use until recently do not give dependable results, being practically useless
for detecting the typhoid bacillus when the latter is mixed with the colon
bacillus. These methods are now almost entirely discarded.

(a) Rodet's method. Rodet showed that the typhoid and colon bacilli would grow
at 45 C. while most other organisms failed to do so, and on this fact based the following
method of analysis. To a flask containing sterilized broth he added 20-100 c.c. of the
suspected water and incubated at 45 C. for 20-24 hours. If on taking the flask out of
the incubator the broth was cloudy a strong presumption was raised that the typhoid
or colon bacillus or both were present in the water. Microscopical examination of the
culture and, if need be, isolation on gelatin plates removed all doubt.

(6) Method of Chantemesse and Widal. Chantemesse and Widal found that both
typhoid and colon bacilli would grow in artificial media containing 2'5 grams of carbolic
acid per litre, and utilized the fact in order to detect these organisms in water.

To tubes containing 20 c.c. of liquefied gelatin add 1 c.c. of a 5 per cent, solution of
carbolic acid and a few drops of the water to be examined and pour plates. Unfortunately
a certain number of organisms develop in the plates which, as they grow, liquefy the
medium and consequently soon put an end to the experiment. A large number of plates
must be sown with each of the suspected samples because only a very small amount of
water can be used for each plate.

(c) Vincent's method. Vincent devised a method, which for a long time was in general
use, based upon a combination of the two preceding observations. He used broth con-
taining O'l per cent, of carbolic acid as the culture medium and incubated the cultures

To each of half-a-dozen tubes containing 10 c.c. of broth add, immediately before use,
5 drops of a 5 per cent, solution of carbolic acid. Sow with O'5-l c.c. of the suspected
water, cover with india-rubber caps to prevent evaporation of the carbolic acid, and
incubate at 41 '5 or 42 C. If the medium in any of the tubes becomes cloudy after
incubating for 12 or 20 hours, transfer a little of the culture to a fresh tube of carbolic-
broth and incubate it similarly at 41 '5 C. As a rule, when the suspected water contains
the colon bacillus the first sub-culture yields a pure growth of the latter organism. It
must, however, be borne in mind that some saprophytes (Bacillus subtilis, Bacillus mesen-
tericus, B. luteus, the white streptococcus of water, Proteus vulgaris, etc.) will also grow
under these conditions. These latter organisms cannot be excluded by further sub-
cultivation in carbolic -broth because once they become accustomed to carbolic media
they grow in them just as well as the colon bacillus. A watered silk appearance in the
tubes is a fairly reliable indication of the presence of the colon or typhoid bacillus, but
the investigation must always be carried further by microscopical examination and
isolation on gelatin. It is well to remember that in carbolic-broth the colon and typhoid
bacilli often occur as very short rods (cocco-bacilli) arranged in pairs and devoid of

(d) Method of P6re". This is merely Vincent's method modified in such a way as to
allow large quantities of the suspected water to be examined.

Prepare a concentrated broth (meat, 1000 grams,, water 1000 grams, and peptone
50 grams), distribute in quantities of 50 c.c. in a series of flasks, and autoclave.

To each flask add 3 c.c. of a 5 per cent, solution of carbolic acid and 100 c.c. of the
suspected water. Sow five or six flasks and incubate them at 41 C. As soon as the
medium becomes cloudy (15-20 hours) sow a series of broth tubes each containing O'l per
cent, carbolic acid with a trace of the growth from any of the flasks that may be cloudy.
Incubate at 41 C. and continue the experiment as in Vincent's method.

(e) Method of Pouchet and Bonjean. This also is a modification of Vincent's method.
To each of a series of flasks containing 100 c.c. of sterile broth add 150 c.c. of the water
to be examined and 5 c.c. of a 5 per cent, solution of carbolic acid. Incubate at 42 C.


If the medium becomes cloudy in any of the flasks sow sub-cultures for three generations
in O'l per cent, carbolic acid broth and incubate at 42 C. Finally, sow a tube of ordinary
broth from the last carbolic broth culture, incubate at 36 C. for 8 days and then inoculate
a guinea-pig with 0*3 c.c. of culture per 100 grams of animal. If the animal die sow
cultures with fragments of the internal organs and heart blood.

The five methods just described are available for the isolation of the typhoid bacillus
provided that the colon bacillus is not also present but if, as is most often the case, the
two organisms are present together the isolation of the former is impossible by these

2. Eisner's method and its modifications.
A. Eisner's method.

The method is available according to Eisner for the isolation of the typhoid
bacillus from sources such as water or stools in which the colon bacillus is
also present.

The technique is based upon the fact that the typhoid and the colon bacilli
grow, to the exclusion of most other organisms, on a potato-jelly containing
iodide of potassium. Disappointing results are however frequently obtained ;
sometimes the plates are rapidly liquefied and the experiment brought to an
end ; at other times the typhoid bacillus cannot be found even though it
has been purposely introduced into a sample of water as a control. Several
attempts have been made to improve the method, and these will be considered

Technique. A. Isolation from water. 1. Prepare and sterilize : (i) a number
of tubes each containing 10 c.c. of potato gelatin (p. 41).
(ii) The following solution :

Distilled water, 50 grams.

Potassium iodide, - 10

2. Immediately before use, melt the potato-gelatin tubes and add 1 c.c. (20 drops)
of the iodide solution.

The gelatin will then contain 1 per cent, of iodide.

3. Sow ten to fifteen tubes each with 0*5 or 1 c.c. of the suspected water and

4. According to Eisner, the colon bacillus appears on these plates as early as the
second day (at 22 C.) as circular, opaque, slightly brown colonies while the typhoid
bacillus does not develop until the plates have been incubated for 4 days and then
as smaller, transparent, barely visible colonies. Other organisms fail to grow.

As a matter of fact, various organisms other than the typhoid and colon bacilli,
and some of which liquefy the gelatin, do grow on the medium ; and then again the
colonies of the typhoid bacillus are not so easily differentiated as Eisner makes out.
It must be distinctly realized that Eisner's medium possesses no specific property
which ensures the development of the typhoid and colon bacilli to the exclusion of
other organisms. Its only advantage is that it allows the typhoid bacillus an equal
opportunity with the colon bacillus to grow. It is necessary, therefore, to examine
carefully every colony on the plates which does not liquefy the medium and which
does not form pigment. This is easily done by transferring them each to a separate
tube of broth and then incubating at 37 C. After incubating for 24 hours the
morphology of the organisms is determined by examining the cultures microscopically
and only those tubes which show short, gram-negative bacilli with rounded ends
need be reserved for the further tests to be described later.

If any of the broth cultures prove to be impure they must be plated out again on
Eisner's jelly. Sow a loopful of the broth in a fresh tube of the jelly, a drop of this
on a second tube, and three drops of the second into a third tube (p. 77).

B. Isolation from stools. The technique to be adopted in this case is similar to
that just described. Dilute a loopful of the stool in a tube of sterile water and use
a drop of the dilution to sow a tube of Eisner's gelatin : mix thoroughly and transfer
a drop to a second tube and from the second tube two or three drops to a third tube.
Pour plates and incubate. All the non-liquefying colonies which develop must be
picked off for further investigation in the manner described above.


B. Grimbert's method.

Grimbert attributes the failure of Eisner's method partly to the want
of uniformity of the medium due to variations in the chemical composition
of potatoes, and partly to the fact that Eisner did not test the reaction of
his medium. According to Grimbert the addition of iodide of potassium is
not essential : ordinary gelatin can be used if the reaction be such that
10 c.c. are neutralized by 5 c.c. of lime water, though it is better to have a
medium of constant chemical composition. Grimbert's medium is used in
the same way as Eisner's, but the colonies are more slow in developing and
the earliest do not appear before the third day. The method, as a matter of
fact, has hardly any advantage over Eisner's original method.
Technique. To 1,000 c.c. of water add :

Maltose, - 1 gram.

Soluble starch, 2 grams.

Asparagin, - 2

Neutral phosphate of potassium. 2 ,,

Potassium sulphate, - 2

Magnesium sulphate, 2 ,,

Ammonium bimalate, - 2 ,.

Magnesium carbonate, - 1 gram.

Dissolve 15 per cent, of gelatin in the mixture, clear with white of egg, heat to
115, filter, and test the reaction thus : dilute 10 c.c. of the gelatin with 50 c.c. of
warm distilled water, add a few drops of an alcoholic solution of phenol-phthalein,
then run in lime water until a permanent rose pink colour is obtained. If more
than 3 c.c. of lime water are required to neutralize the gelatin reduce the acidity by
the addition of a small quantity of normal soda solution until 10 c.c. of the gelatin
are neutralized with 5 c.c. of lime water.

Immediately before use 1 per cent, of iodide or bromide of potassium may be

C. Remy's method.

Kemy suggests the use of a medium which is more nutritive and less acid
than Grimbert's. By means of his " differential gelatin " he has been able
to isolate the typhoid bacillus from stools in all the cases of enteric fever
which he has investigated.

This " differential gelatin " has no greater selective property than Eisner's
medium and the majority of micro-organisms grow in it. Still, liquefying
species are to some extent checked and the inhibiting influence of the colon
bacillus on the typhoid bacillus is not apparent on this medium.

Technique. Preparation of the " differential gelatin." To a litre of water in a
flask add :

Asparagin, - 6 grams.

Oxalic acid, - - 0'5 gram.

Lactic acid, - - 0'15

Citric acid, - - .... Q*15

Di- sodium phosphate, - - 5 grams.

Potassium sulphate, - - - - 1*25 ,,

Sodium chloride, - - - - - - - - 2

Witte's peptone, - .... 30

Heat to 110 C. for 15 minutes, and on taking the flask out of the autoclave pour
the boiling liquid into another flask containing 120-150 grams of best quality gelatin.
Shake the flask until the gelatin is dissolved, add soda solution until the mixture is

Online LibraryAlbert BessonPractical bacteriology, microbiology and serum therapy (medical and veterinary) A text book for laboratory use → online text (page 50 of 110)