NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
could any development of colonies of Comma-Bacilli be detected, and the gene-
ral result of the series of experiments was thus to show that the Bacilli, even
when introduced in very large numbers, do not find conditions in ordinary sam-
ples of milk favourable to their multiplication or even to their continued survival
for any considerable time.
So much having been ascertained, it next appeared to be desirable to follow
the course of events subsequent to inoculation somewhat more closely, and a
series of cultivations derived from one and the same sample of milk, but set at
successive periods shortly after inoculation had been effected, was accordingly
carried out. The results of these are embodied in Table VII :—
TABLE VII.—Results of successive Cultivations of a Specimen of Unboiled
Milk inoculated with Comma-Bacilli.
Medical Officers of the Army of India.
29
Inoculation was performed at 8 A.M., and successive cultivations set at in-
tervals of 2, 4, 6, 10 and 24 hours thereafter. The first four of these showed
evidences of excessive multiplication of common lacteal schizomycetes occur-
ring within the first 10 hours of the experiment, and they also showed that
Comma-Bacilli survived in the fluid for a like period. So far as the evidence
No. of specimen
in Table VI.
Date and
time at
which
inoculated.
No. of
Plate-
culti-
vation.
Date and time at
which set.
RESULTS.
V.—Milk in a
sterilised
tube inocu-
lated with
0.03 C.C.
sterilised salt
solution full
of the Com-
ma-Bacilli of
a tube-cul-
tivation of 48
hours' stand-
ing.
August
3rd, 8 A.M.
1 August 3rd, 10
A.M. Plate;
inoculated by a
looped needle
dipped in the
milk.
August 4th. Numerous colonies of lacteal schizomy-
cetes, and a few of characteristic Comma-Bacilli ;
smell non-choleraic.
2 August 3rd, 12
noon. Plate set
as abo
August 4th. A greatly increased number of colonies
or common lacteal schizomycetes, and a somewhat
larger number of colonies of Comma-Bacilli ; smell
non-choleraic.
Reaction at
first neutral ;
at noon faint-
ly acid.
...
3 August 3rd, 2
P.M. Plate set
as above.
August 4th. Excessive increase in the number of
colonies of common lacteal schizomycetes, and
great diminution in the number of colonies of
Comma-Bacilli ; smell non-choleraic.
August 5th.
Strongly
acid ; fully
coagulated
and separa-
ted.
...
4 August 3rd, 6
P.M. Plate;
inoculated as
above.
August 4th. Absolutely crowded with colonies of
common lacteal schizomycetes ; only a single
colony of Comma-Bacilli detected ; smell non-
choleraic.
5 August 4th, 10
A.M. Plate ;
inoculated by
a common
needle.
August 5th. Numerous colonies of common lacteal
schizomycetes ; not a trace of Comma-Bacilli ;
smell non-choleraic.
Scientific Memoirs by
goes, it would appear to indicate, not mere survival, but also multiplication of the
Comma-Bacilli as occurring for some time subsequent to their introduction into
the milk, for a larger number of colonies of the organism were present in the
cultivation set at four hours after inoculation than in that set only two hours after
it. The period of multiplication would seem, however, to be a brief one, for the
third cultivation of the series, set at six hours subsequent to inoculation, showed
a diminution in the number of colonies, while that set 10 hours after inoculation
contained only a single recognisable one. The last cultivation of the series gave
the normal result for cultivations established after the lapse of 24 hours from
the period of inoculation, in containing large numbers of colonies of common
lacteal schizomycetes and a complete absence of Comma-Bacilli. The cessa-
tion of multiplication and onset of disappearance of the Bacilli would appear to
have been coincident with the development of acidity in the medium—permanent,
although primarily only faint, acidity having replaced neutrality within four hours
subsequent to the time of inoculation, and acid fermentation having thereafter
progressed rapidly so as to cause the reaction 24 hours after inoculation to be
strongly acid, and the milk to be fully coagulated and separated.
Milk in its ordinary condition, therefore, appears to be anything but a
favourable medium for Comma-Bacilli, the rapidly developed acid fermentation
normally setting in in it connected with the presence and multiplication of other
schizomycetes tending to arrest their multiplication and presently to destroy
their vitality. That it is the presence of the common lacteal schizomycetes
and the fermentive changes developed in the fluid in connection with it which is
the actually determinant of the repression of multiplication and vitality of the
Comma-Bacilli is clearly shown by the data recorded in the two next tables,
which deal with the results following the introduction of Comma-Bacilli into
boiled and sterilised samples of milk
TABLE VIII.—Results of Inoculations of Specimens of Boiled Milk with Comma-
Bacilli.
No. o f
speci-
men.
Nos. of corre-
s p o n d i n g i
specimens in
Tables III &
I V .
Nature and
treatment of
specimen.
Date of
inocu-
lation.
Nature of
cultivations.
Date
at
which
set.
RESULTS.
III. IV.
I XVI XV Milk boiled in
a sterilised
tube, July
20t h.
July
22nd.
Inoculated with
0.03 of sterilised
salt solution full
of Comma-Ba-
cilli of 48 hours'
g r o w t h .
... July 23rd. Reaction faintly acid,
completely coagulated into a large,
white, powdery deposit and clear
colourless fluid.
July 24th. Reaction almost neutral.
30
Medical Officers of the Army of India. 31
No. of
speci-
men.
Nos. of corre-
sponding
specimens in
Tables III &
IV.
Nature and
treatment of
specimen.
Date
of
inocu-
lation.
Nature of
cultivations.
Date
at
which
set.
RESULTS.
III. IV.
1. Plate; inoculat-
ed with a com-
mon needle
dipped in ste-
rilised salt
solution in-
oculated by
one looped
needle dip-
ped in the
milk.
July,
23rd.
July 24th. Full of large colonies
of Comma-Bacilli ; scattered and un-
healthy looking rods of Bacillus
subtilis also present, seemingly due
to the presence of a thin, diffuse,
surface growth ; smell strongly cho-
leraic ; practically a cultivation of
Comma-Bacilli.
I I XVIII ... Milk boiled in
a sterilised
tube, July
29th.
July
31st.
Inoculated as
a b o v e .
. . . August 1st. Reaction neutral ; smell
putrid; coagulated.
August 6th. Reaction faintly acid
at first, faintly alkaline on drying.
1. Plate; inocu-
lated as above.
Aug.
1st.
August 2nd. Covered with discrete
and confluent colonies of Comma-
Bacilli and a thin, diffuse growth
of Bacillus subtilis.
2. Plate ; inocu-
lated as above.
Aug.
6th.
August 7th. Apparently a pure cul-
tivation of Bacillus subtilis.
III XIX XVII Milk boiled in
a sterilised
tube, August
3rd.
Aug.
9th.
. . .
. . . At the time of inoculation the milk
was coagulated and partly separat-
ed ; coagulum of the loose, pul-
verulent character nominally asso-
ciated with Bacillus subtilis; full of
Bacillus subtilis; smell strong, pu-
trid ; reaction faintly acid, almost
neutral on drying.
1. Plate ; inocu-
lated as above.
Aug.
14th.
August 15th. Covered by a continu-
ous, thick stratum of Bacillus subti-
lis and containing a limited number
of colonies of large Comma-Bacilli ;
smell non-choleraic.
IV XX XVIII Milk boiled in
a sterilised
tube, August
15th.
Aug.
17th.
. . .
. . . At the time of inoculation the milk
showed coagulation characteristic
of the continued presence of Bacil-
lus subtilis, being full of fine, loose,
powdery flakes, with a little free,
supernatant fluid ; reaction very
faintly acid.
August 18th. Separation of coagu-
lum and fluid considerably advanc-
ed ; reaction neutral. August 21st.
Reaction neutral. August 22nd.
Reaction very faintly acid. Sep-
tember 29th. Reaction neutral.
1. Plate ; inocu-
lated as above.
Aug.
18th.
August 19th. An abundant growth
of colonies of Comma-Bacilli, and
a partial and very thin, diffuse
growth of Bacillus subtilis.
32
Scientific Memoirs by
No. of
speci-
men.
Nos. of corre-
sponding
specimens in
Tables III &
IV.
Nature and
treatment of
specimen.
Date
of
inocu-
lation.
Nature of
cultivations.
Date
at
which
set.
RESULTS.
III. IV.
XX XVIII
... ... 2. Plate; inocu-
lated as pre-
viously.
Aug,
19th.
August 20th. A very limited num-
ber of colonies of Comma-Bacilli,
and one or two very thin, localised
films of Bacillus subtilis.
3. ,, ,,
Aug.
20th.
August 21st. A single large colony
of Bacillus subtilis ; no Comma-
Bacilli present.
4. ,, ,,
Aug.
21st.
August 22nd. Surface almost cover-
ed by a thick stratum of sporing
Bacillus subtilis; one or two large
colonies of small, feebly-staining
Comma-Bacilli.
5. ,,
,,
Aug.
22nd
August 23rd. A solitary colony of
large Comma-Bacilli ; no Bacillus
subtilis.
6. ,,
,,
Aug.
23rd.
August 24th. One or two colonies
of short, thick Comma-Bacilli, and
a localised very thin diffuse film
of Bacillus subtilis.
7. ,, ,,
Aug.
24th.
August 26th. An abundant growth
of sporing Bacillus subtilis and
small, feebly-staining Comma-
Bacilli.
8 ,,
,,
Aug.
31st.
September 1st. Smell very faint,
non-choleraic ; crowded with a
diffuse growth and ill-defined colo-
nies of small, feebly-staining, vacuo-
late Comma-Bacilli, and a thin
diffuse growth of Bacillus subtilis.
9. Plate ; inocu-
lated by one
common
needle dipped
in the milk.
Sept.
19th.
September 20th. An abundant,
mixed growth of Bacillus subtilis
and Comma-Bacilli.
In the case of the boiled samples of milk referred to in the above table, it
will be seen that inoculation with Comma-Bacilli was carried out at some time
subsequent to the boiling—the interval elapsing between the two operations hav
-
ing amounted to 48 hours in three of the experiments and to 144 hours in the
remaining one. The interval was allowed to occur in order to permit of the
growth and multiplication of Bacillus subtilis, which normally takes place in boil
-
ed samples of milk, going on, without any possible interference on the part of
the intrusive Bacilli, for such a time as would certainly secure its presence in
very large amount within the medium at the period of inoculation. By following
34
Scientific Memoirs by
number, and in some cases apparent total absence of Comma-Bacilli with a pro
- p o r t i o n a t e i n c r e a s e i n t h e q u a n t i t y o fB
a c i l l u s s u b t i l i s .
F o r e x a m p l e , i n t h e
case of Sample II of the Table, the first cultivation, which was set twenty-four
hours after inoculation, yielded an excessive crop of Comma-Bacilli, and only a
feeble growth of Bacillus subtilis, whilst the second cultivation, which was set five
days later, showed a very abundant and seemingly pure growth of the latter
species only. Again, in the case of Sample III, where only a single cultivation
was carried out, and that five days subsequent to inoculation, the growth of
Bacillus subtilis was excessive, and Comma-Bacilli, although not entirely absent,
were present in relatively very small numbers only. This result does not, how
- ever, appear to be constant in regard to the time at which it occurs, for in Sam
- ple IV a cultivation set a fortnight after the date of inoculation showed exces
- s i v e g r o w t h o f C o m m a - B a c i l l i , a n d o n l y a l i m i t e d d e v e l o p m e n t o fBacillus sub-
t i l i s .
I n t h e c a s e o f t h i s s a m p l e a n e x t e n d e d s e r i e s o f c u l t i v a t i o n s w a s m a d e .
The results of these so far agreed with those in the previous cases that they
indicated an initial period of excessive growth of Comma-Bacilli, succeeded by
a period during which a great diminution in their numbers occurred, with simul
- t a n e o u s l y i n c r e a s e d a c t i v i t y i n t h e g r o w t h o fB
a c i l l u s s u b t i l i s ,
b u t , a f t e r t h i s ,
neither persistent depression of growth of the former organisation, nor persistent
excess of the latter manifested themselves. On the contrary, the successive cul
- t i v a t i o n s , w h i c h w e r e c a r r i e d o u t s u b s e q u e n t l y , a p p e a r e d t o i n d i c a t e a n a l t e r n a t i o n
of periods of relatively excessive and depressed activity in the case of both
species alike. The results in this instance clearly showed that there is not
necessarily any rapid suppression of Comma-Bacilli in association with Bacillus
subtilis in samples of boiled milk, for the fluid continued to yield abundance of
both species, up at all events to the 19th September, when the last cultivation
of the series was carried out, or up to a period of almost five weeks subsequent
to inoculation.
The general results of this series of experiments are to show that boiled
milk affords a much more favourable medium for the growth of Comma-Bacilli-
than the unboiled fluid does, providing conditions certainly favouring excessive
multiplication of the species for some time and not definitely precluding its
continued presence in large numbers and in a living state for a very considerable
period.
The last set of experiments were devoted to the investigation of the pheno
- m e n a f o l l o w i n g i n t r o d u c t i o n o f C o m m a - B a c i l l i i n t o s p e c i m e n s o f m i l k w h i c h h a d
not merely been boiled but actually sterilised, and the results are embodied in
Table IX.
Medical Officers of the Army of India.
35
PLATE IX.—Results of Inoculations of Specimens of Sterilised Milk
with Comma-Bacilli.
No. of
speci-
men.
Nos. of corre-
sponding spe-
cimens in
Tables I & III.
Nature and
treatment of
specimens.
Date
of
in
ocu-
lation .
Nature of
cultivations.
Date
of
inocu-
lation.
REMARKS.
I.
III.
I
XIII XV Sample of milk
in a tube, steri-
lised between
7th and 10th
June. Reac-
tion faintly
acid, becom-
ing almost
neutral on
drying;
odourless.
July
11th.
Inoculated with
0.059 of steri-
lised salt solu-
tion containing
Comma-Bacilli
of 24 hours'
growth.
... July 12th. Quite fluid ; reaction
faintly alkaline; containing numer-
ous Comma-Bacilli, many of them
of large size and vacuolate.
1. Plate ; inocu-
lated by one
looped needle
dipped in the
milk.
July
12th.
July 13th. Smell strongly choleraic,
crowded with colonies of Comma-
Bacilli, the majority of which were
short and thick.
2. Plate; inocu-
lated as above.
July
15th
July 16th. Absolutely crowded with
colonies of Comma-Bacilli.
3. Plate; inocu-
lated as above.
July
24th.
July 25th. Absolutely sterile.
II
"
" As above . July
15th.
Inoculated as
above, save that
the Comma-
Bacilli were de-
rived from a
cultivation of
48 hours' stand-
ing.
... July 25th. Coagulated; coagula soft,
powdery.
1. Plate ; inocu-
lated by one
common needle,
dipped in the
milk.
July
17th.
July 18th. Smell choleraic; crowded
with colonies of Comma-Bacilli ; the
latter generally very short.
2. Plate ; inocu-
lated with 0.03
of the milk.
July
25th.
July 26th. Crowded with colonies of
Comma-Bacilli.
3. Plate ; inocu-
lated by one
looped needle,
dipped in the
milk.
July
29th.
July 30th. Crowded with colonies of
Comma-Bacilli.
III
"
" As above . Aug.
9th.
Inoculated as
above; the
Comma-Bacilli
from a tube of
48 hours' growth.
... August 14th reduced to a soft gela-
tinous clot, but with hardly any
separation of fluid ; smell very faint,
mouldy.
F2
36
Scientific Memoirs by
No. of
speci-
men.
Nos. of corre-
sponding spe-
cimens in
Tables I & III.
Nature and
treatment of
specimens.
Date
of
inocu-
lation.
Nature of
cultivations.
Date
at
which
set.
REMARKS.
I
III
XIII XV
...
... I. Plate ; inocu-
lated by a
looped needle
dipped in ste-
rilised salt so-
lution which
had been pre-
viously inocu-
lated by a
looped needle,
dipped in the
milk.
Aug.
15th.
August 16th. An enormous and pure
growth of Comma-Bacilli.
2. Plate ; treated
as above.
Aug.
24th
August 26th. No Comma-Bacilli ; an
abundant growth of intrusive Bacil-
lus subtilis.
3. Plate ; treat-
ted as above.
Aug.
30th
August 31st. Exactly like the pre-
vious plate.
The milk in all the experiments consisted of sterilised specimens of the
sample numbered XV in Table III, and which were experimentally shown to be
completely sterilised on the 10th of June, cultivations from that time onwards
failing to show the presence of any living elements in it. Up to the present
time, September 19th, the specimens of it which yet remain show absolutely no
change save a diminution in bulk due to gradual evaporative loss. They retain
their fluidity completely, save for the presence of a thin dry tough skin which
has gradually formed on the surface as evaporation has progressed. The re
-
action is in some instances faintly acid at first, becoming neutral on drying, and
in others absolutely neutral throughout, and were it not for the presence of the
superficial film above described, and the slightly brownish tinge which they
acquired as the result of the prolonged exposure to an elevated temperature
during the process of sterilisation, they present all the essential characters of
specimens of fresh milk.
The inoculation of those specimens which formed the subjects of special ex
-periment was carried out in the same fashion as in the cases where fresh or
merely boiled specimens of milk were dealt with. The introduction of Comma-
Bacilli was ultimately followed by coagulative changes in the fluid. These were
unconnected with any conspicuous alterations in its re-action, and the resultant
coagula rather resembled in character those associated with the fermentive
changes accompanying the persistent presence of Bacillus subtilis than those
occurring in the case of the ordinary spontaneous acid fermentation and co
-
agulation of common specimens of milk. In one case, where the fluid was
directly examined in regard to its content of Comma-Bacilli at a period 24 hours
subsequent to inoculation, the cover-glass preparations showed the presence of
Medical Officers of the Army of India.
numerous specimens of them. Cultivations also afforded unequivocal evidence
of the occurrence of their excessive multiplication in the fluid subsequent to
their introduction. This, moreover, was not limited to periods shortly following
inoculation, as in the case of merely boiled specimens, but appeared to persist
continuously for a very considerable time. For example, in the case of sample
II, cultivations set at 48 hours, ten days, and a fortnight subsequent to inoculation
all alike produced excessive growths of Comma-Bacilli. The effect of freedom
from a struggle for existence is very clearly indicated by this phenomenon, and
even more emphatically when we compare the results in this instance with those
recorded in the case of sample III. In the first instance the medium through
-out remained free of any schizomycetes save the inoculated Comma-Bacilli,
in the second an intrusive organism in the shape of Bacillus subtilis accident
-ally entered the medium at some period subsequent to inoculation. In the first
instance Comma-Bacilli remained in the medium in excessive numbers up to a
fortnight subsequent to inoculation; in the second they were present in great
numbers six days subsequent to inoculation, and when the medium still remained
uncontaminated by intrusive Bacillus subtilis, but at some period between that
time and the date at which the next cultivation was set-15 days after inocula
-tion—a complete suppression of Comma-Bacilli had taken place, coincidently
with abundant growth of intrusive Bacillus subtilis.
It now only remains briefly to consider the results of the entire series of
experiments. These appear clearly to warrant the following conclusions:-
1. The milk which is in ordinary use in Calcutta normally contains a large number
of schizomycete organisms, and in many instances an excessive number of them.
2. The number of distinct species which are normally present is, however, very
limited.
3. All of these, save Bacillus subtilis, are destroyed by boiling the fluid for a very
short time.
4. A certain, but apparently usually only a small number of spores of Bacillus subtilis
is, however, normally present, and these serve to give rise to an excessive development
of that species in the boiled milk.
5. Acid fermentation and coagulation normally occur very rapidly in ordinary speci
-mens of milk.
6. These phenomena are associated with processes of rapid growth and multiplication
of the schizomycete organisms which are destroyed by simple boiling of the fluid, and,
therefore, in many cases fail to manifest themselves in boiled specimens.
7. In certain cases, however, boiling, while demonstrably destroying the common
lacteal schizomycetes, with the exception of Bacillus subtilis, fails to prevent acid ferment
-ation and coagulation.
8. This is related to the particular stratum of any sample of milk from which the
specimens have been obtained—specimens derived from the upper strata having a much
greater tendency to undergo acid fermentation and coagulation than those derived from
the lower strata.
37
Medical Officers of the Army of India.
39
is not at all likely to be favourable to the development or continued existence of
those forms which require an alkaline or neutral environment. The very con
-tamination of the fluid comes to render it an unfavourable medium for organisms
which, were it pure, would find conditions extremely favourable to their growth
and multiplication in it. But, if this be so, unless it were clearly proven that the
normal schizomycete population of the milk contains species which are capable,
directly or indirectly, of giving rise to disease when they come into relation to
the human organism, it can only be regarded as a protective arrangement tend
-ing to render the medium unfavourable to the development of other and possibly
noxious organisms. It is no doubt unpleasant to realise that the milk supply in
India is so highly impure as it unequivocally is, but it does not follow that the
contamination is an unmixed evil. For example, if Comma-Bacilli really be the
specific cause of cholera, there can be no doubt that the common impurities in milk
must tend to render the latter an unfavourable vehicle for the communication of the
cause of the disease, and as it is one to which there can be no doubt that Comma-
Bacilli are very likely to gain access in any locality in which the disease prevails,
those who see in them the essential cause of the disease must regard the normal
contamination, or, in other words, the normal schizomycete population of the
milk, as playing a beneficent part. The evidence, in so far as it goes, seems
to indicate that the ordinary milk supply in this country is an unfavourable me
-dium for schizomycete organisms requiring alkalinity or neutrality in their en
-vironment in order to their successful propagation and continued existence
apart from the presence of spores, and, this being so, all those who ascribe the
development of any disease to the influence of pathogenic schizomycetes may
fairly be called on to demonstrate that these are capable of surviving the con
-ditions to which they must normally be exposed on gaining access to milk, ere
blaming the latter as a means of communicating the disease. In any case it
must be a comfort to realise that if the population of India be condemned to
the use of impure milk, the very contamination is likely to act more or less in
protective fashion, and is normally due to the presence of organisms which, with
the exception of the harmless Bacillus subtilis, may be completely destroyed by
simple boiling.