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THE PRODUCTION OF ALKALI IN LIQUID MEDIA
BY THE BACILLUS PESTIS.
T H E
f a c t t h a t p l a g u e m i c r o b e s c e a s e t o g r o w a b u n d a n t l y i n l i q u i d m e d i a
after a month or six weeks has hitherto been ascribed to the supposed
consumption of all the available nutriment. Thus, Mr. Haffkine*, in his evidence
before the Indian Plague Commission in 1898, says the broth becomes modified
in the course of five or six weeks, so that "the liquid is exhausted, its nutritious
elements having been incorporated in the body of the bacilli" ........."It will not
feed any further growth."
Dr. E. Klein† also assumes that the growth of plague bacilli stops because
the nutritive material in the broth is exhausted.
In this laboratory it has been common experience to find that not only
does active growth cease in flasks containing 1000 c.c. or even 2000 c.c. of
broth after six weeks or two months, but that the plague germs actually die out
altogether in from two to four months' time.
As Mr. Haffkine taught that an acid medium is inimical to the growth of the
plague germ, a trial was made to see if the medium had become acid. To our
surprise we found that, on the contrary, the medium in these old sterile flasks
had become highly alkaline, many of them containing the equivalent of from 2 to
2.5 per cent. of normal alkali.
Macé says on this point with regard to the plague bacillus (p. 925) "Le
microbe produit un peu d'acide dans les milieux, probablement aux dépens des
hydrocarbones."‡
That this alkali was not due to any change taking place in the broth itself
was proved by testing a flask containing sterile broth made seven months
previously. The reaction given by this broth was the amphoteric reaction to
litmus paper which is given by the freshly made media used here.
To prove whether the alkaline condition of the broth was the cause of the
cessation of the growth the following experiment was made.
* Minutes of evidences taken by the Indian Plague Commission with appendices, Vol. 1, pp. 2, 4, 28.
Calcutta; Office of the Superintendent of Government Printing, India, 1900.
† Thirty-first Annual Report of the Local Government Board, 1901-1902. Supplement containing the
Report of the Medical Officer for 1901-1902.
‡ Traité Pratique de Bactériologie par E. Macé, Cinquiéme édition, Paris: Libraire J. B. Balliére-
et Fils, 1904.
B
2
Six flasks containing 1000 c.c. of broth, which had been sown with plague four
months previously, were tested for plague growth and reaction with the following
result :
Growth on agar.
No. of c.c. of normal acid per litre required to produce neutralisation.
I. Sterile
14.7
II. „
22.2
III. „
25.2
IV. „
24.5
V. „
23.5
VI. „
24.1
That is to say, in four months' growth the plague germ had produced
alkalinity in the broth equivalent to from 1½ to 2½ per cent. of normal alkali as
tested by the following procedure :
Ten c.c. of the neutral broth used in the laboratory, which was amphoteric
to blue and red litmus papers, was diluted with 90 c.c. of distilled water in a
porcelain dish. To this was added 2 c.c. of solution of litmus made according
to the directions in the British Pharmacopœia.
The tint thus produced was taken as the measure of the neutrality with
which to compare the fluid from the old plague flasks.
Ten c.c. from each of the above-mentioned flasks was withdrawn by a
sterile pipette, and added to 90 c.c. of distilled water ; 2 c.c. of litmus solution
was added, and N
/20 acid added from a burette till the tint corresponded to that
of the standard broth. As a slight precipitate was produced when the acid was
added to the plague broth, this first dish of tested broth was kept as the standard
tint to be aimed at as being easier to match than the pure neutral broth.
It is allowed that the above method of arriving at the neutral point of the
broth or plague culture is not ideal, but at the time (1904) no more perfect
method had been found for the broth then used in the laboratory.
Phenolphthalein and methyl orange were both tried but proved less satisfac-
tory than the litmus.
These six flasks were, on 27th June 1904, resown with plague germs after
having been neutralised with hydrochloric acid.
Agar cultures made from them, on 4th July 1904, i.e., seven days later,
showed a good growth of plague in all, with the exception of No. III, which had
become contaminated.
3
When again tested on the 11th July the following were the results
—
I was strongly alkaline to litmus paper, and showed good growth of plague on agar.
II
Ditto
ditto
ditto
ditto.
IV was slightly alkaline to litmus paper, and showed good growth of plague on agar.
V
Ditto
ditto
ditto
ditto.
VI
Ditto
ditto
ditto
ditto.
These flasks were again tested on 22nd July 1904, i.e., three weeks from
the date of re-sowing, with the following results
I contained 1.2 per cent. alkali and produced good growth of plague on agar.
II
”
1 . 1 7 ” ”
”
ditto
ditto.
IV
”
1 . 1 5 „
„
„ but proved to be contaminated.
V
”
1 . 0 „
„
„ and produced good growth of plague on agar.
VI
,,
1 . 1 6 ” ,,
”
ditto
ditto.
The plague germs in the re-sown flasks had in three weeks produced as
much alkali as though growing in fresh nutrient medium.
This conclusively proves that the broth is not " exhausted " but still contains
nutriment sufficient for the nourishment of a new generation of plague germs.
Another proof that the growth of plague is stopped by the alkalinity of the
medium is afforded by the following experiment.
A series of flasks containing 100 c.c. of broth was prepared by adding 0.25,
0.5, and 0.75 per cent. of normal hydrochloric acid to the amphoteric medium as
used in the laboratory, and another set by similarly adding 0. 25, 0.5, 0.75, 1.0,
1. 5, and 2 per cent. of normal sodium hydroxide.
These flasks and also two control flasks of amphoteric broth as ordinarily
used in the laboratory were sown with 0.5 c.c. of a 3 days' old culture of plague
on 24th June 1904.
The subsequent history of these flasks is shown below.
25th June 1904.—The amphoteric broth flasks showed dots of growth on
the sides of the glass, and commencing stalactite formation.
The 0.25 acid broth showed dots but no stalactites.
The 0.50
„
”
”
a few dots only.
The 0. 75 „
„
,,
no growth at all.
The 0.25 alkaline broth showed dots but no stalactites.
The 0.50
„
”
„
a few dots only.
The 0.75
„
””)
”
a fewer number of dots.
Those of the higher series showed no growth at all.
26th rune 1904.— The amphoteric broth flasks showed good stalactite
formation.
The 0.25 acid broth showed growth as good as the above.
The
0 . 5 0 „
„
„
abundant dots and a few stalactites.
The 0.75 „
„
”
”
”
only.
B2
4
The
0 . 2 5
alkaline broth showed abundant dots and a few stalactites.
The
0 . 5 0
The
0 . 7 5 show similar growth but in , a less degree, and the growth is distinctly
less in the broths of higher alkalinities.
The
1 . 0 0
The flasks of 1.5 and 2 per cent. acid broth show no growth at all.
27th J une1904.—The amphoteric broth shows abund ant stalactite growth..
The o.25 acid broth shows equally g o o d g r o w t h .
The 0.50 „ „
”
excellent growth of stalactites.
The o.75 ”
„
,,
one stalactite and numerous dots.
The o.25 alkaline broth shows good growth of stalactites.
The o.50
„
”
”
”
”
”
,,
The o.75
” ”
”
slightly less growth.
The
1 . 0 0
„
”
,,
still less growth.
The 1.50 ”
”
,,
about a dozen dots only.
The 2.00
,,
”
” no growth at all.
Another similar experiment was made with flasks containing 100 c.c. of
broth acidulated by the addition of 0.5, 0.75, 1.0, 1.25, 1.5, and 2 per cent. of
normal acid.
A corresponding set of flasks was treated by the addition of similar quantities
of normal alkali,
On 8th July 1904 to each flask was added o.1 c.c. of plague culture in
broth, and along with control flasks all were put aside to observe the growth.
Three days later the flask with amphoteric broth and that with the o.5 per cent.
of acid showed a growth of abundant long stalactites, and it was difficult to say
which was best.
The flask with the 0.75 per cent. acid broth showed short stalactites and
many dots of growth on the glass.
This latter appearance was also seen on the sides of tht other acid flasks, but
no stalactites were visible at this stage. The flasks with alkaline broth showed
much inferior growths. Thus, the 0.75 alkaline broth showed one or two short
stalactites and dots on the glass, but nothing like the corresponding acid flask.
The higher series of alkaline broths showed dotting on the glass merely and no
stalactites.
From the above it is clear that, far from being inimical to the growth of
the plague germ, slightly acid broth is a favourable medium for its growth. It is
likewise clear that an alkaline medium is less favourable to its growth, and that a
broth containing the equivalent of 2 per cent. of normal alkali inhibits the growth
altogether. It does not, however, kill the plague germ, for if a few drops from
such a flask are sown on agar a growth of plague is obtained, and from old
5
prophylactic flasks that have stood in the incubation room for eighteen months,
and have a reaction approaching 2 per cent. of normal alkali, germs, can still be
recovered sufficiently alive to show growth on agar slopes.
A question naturally arising from the above is : can the plague bacillus evolve
alkali when grown in acid broth so as to produce a neutral and finally an alkaline
broth?
To solve this the following proceeding was adopted. The first set of the
above series of flasks was shaken up on 9th July 1904. When examined on
11 July 1904, it was found that the best stalactites were to be seen in the flask
containing broth originally acidulated to the extent of 0.75 per cent. On testing
the fluid in this flask it was, found that it now contained the equivalent of 0.15
per cent. of normal alkali, therefore the plague bacilli had made enough alkali in
the eighteen days they had been growing to neutralise all the acid introduced, and
had produced in addition 0.15 per cent. of normal alkali and this would no doubt
have gone on until the 2 per cent. limit of alkalinity was reached.
Similarly, the plague germs growing in the flask containing the 0.5 per cent.
acid broth were found to have produced 1.05 per cent. ; and the germs growing
in the 0.25 per cent. broth, 0.6 per cent. of alkali.
Two flasks containing a similar quantity of amphoteric broth and sown with
the same germ had in the same eighteen days produced the equivalent of 0.40
and 0.35 per cent. of alkali respectively.
In the series of alkaline broth flasks the following results were obtained.
Flask with 0.75 per cent. of normal alkali added, showed a deficiency in
alkali to the extent of 0.01 per cent. Flask with 1.0 per cent. of normal alkali
added, showed a deficiency in alkali to the extent of 0.35 per cent. Flask with
1.5 per cent. of normal alkali added, showed a deficiency in alkali to the extent
of 0.75 per cent. Flask with 2 per cent, of normal alkali added, showed a
defi-ciency in alkali to the extent of 0.91 per cent.
These two latter flasks though showing no stalactites but only a muddiness
after the eighteen days' incubation, yet gave a good growth of plague when a few
drops were placed on agar, proving that the germs were still alive though their
growth had been inhibited.
A second observation in confirmation of the above may be recorded. The
series of flasks noted above as sown on 8th July 1904 were tested on 29th July
1904. The growth was undoubtedly best developed in the flask containing 1 per
cent. acid broth. There was little growth in the 1.25 per cent. acid broth and
none apparently in the higher numbers of the acid series. A precipitate was,
however, evident in these highly acidulated broths as well as in others of the same
degree of acidulation which had not been sown with plague.
Cultures taken from this whole series showed a pure growth of plague in all
6
the alkaline broths, and in the less acid ones up to 1.0 per cent.; the 1.25 and 2.0
per cent. acid broths showed no growth at all on agar.
The results got by titration of this series, as well as those of the first
experiment, are shown below in percentages.
Denomination of flask.
Result of titration.
Alkali produced.
Alkali produced in
first experiment.
2 per cent. acid
.
.
2.0 per cent. acid.
0
1.50 „ „ „
.
.
1.38 „ „ „
0.12
1.25 „ „ „
. .
1.26 „ „ „
0
1.00 „ „ „
.
.
0.26 „ „ ,
0.74
0.75 „ „ „
.
.
0.48 „ „ alkali.
1 . 2 3
0.90
0. 5 0 „ „ „
.
.
0.67 „ „ „
1.17
1.05
Amphoteric
.
.
.
0.49 „ „ „
0.49
0.42
0.75 per cent. alkali .
.
1.04 „ „ „
0 . 2 9
0 . 3 5
— 0 . 0 1
1.00 „ „ „
.
.
1.13 „ „ „
0.13
—0.35
1.25 „ „ „
.
.
1.31 „ „ „
0.06
—
1.50 „ „ „
.
.
1.41 „ „ „
—0.09
—0.75
2.00 „ „ „
.
.
1.35 „ „ „
—0.65
—0.91
One flask containing 2 per cent. acid broth which had not been sown was
tested nine days after preparation and found on titration by the method described
above, to give an acidity of 1 . 89 per cent. only. Three flasks containing broth
with alkalinities of 1.0, 1.5, and 2 per cent., respectively, were tested after nine
day s and found to show a loss of 0.07, 0. 26, and 0.39 of alkali respectively.
A series of flasks containing 1000 c.c. of broth was tested to see at what
rate alkali was produced. The following shows the results for weekly periods
after sowing of the plague germ.
Number of weeks since sowing.
Percentage of normal alkali produced
since sowing.
1
0.41
2
0.55
3
1.11
4
1.38
5
1.42
6
2.10
7
The conclusion; then, appears to be that—
1. In the higher series of both acid and alkali broths, the production of
alkali by the plague germ is stopped.
2. In the higher alkaline series the plague germ lives for a long time
whereas in the highly acid broths it quickly dies out.
3.The more alkaline the medium is, the less alkali is produced by the
plague germs sown in it, and in the higher series little or none is
produced, the growth being apparently inhibited.
4. The production of alkali is regular, increasing from week to week to
the maximum, which is attained in about six weeks.
The above investigation was carried out in 1904, but owing to the labour
involved in organising and working this laboratory, and the urgency of other
work, nothing further could be done till this year. In the meantime the technique
used in making the broth had been changed, and it was found possible to use the
more accurate method of titration with phenolphthalein as recommended by the
Committee of American Bacteriologists. The broth used in the laboratory is
prepared by chemical digestion with hydrochloric acid, no artificial peptone
being added, so that no offence might be occasioned to the religious prejudices
of certain sections of the Indian community. The details were worked out by
the late Colonel Warden, I.M.S., and involved digestion under steam pressure
for six hours. The medium so prepared had a very high colour and varied much
in composition. Various alterations were made from time to time, such as reduc-
tion in the amount of acid, shortening the time of, and lessening the pressure
during, digestion. It was found, however, that heating under pressure led to the
production of other bodies than albumose and peptone, as proved by heating a
sample of the broth with caustic alkali, when a strong smell of ammonia and
methylamine bodies was evolved.
These considerations led to the trial of a new method of preparation where
length of maceration at a low temperature was substituted for heating for a short
time at a high temperature under pressure. The experiments on which these
opinions regarding composition of the broth are founded were devised and
carried out by Mr. F. Maitland Gibson, M.B., B.Sc., of this laboratory, to
whom is due the whole credit in the matter.
The conclusion arrived at was that broth prepared according to the follow
ing method was better for the purpose of growing the plague bacilli than any
used hitherto, that it was equal in nutritive value to bouillon made in the ordinary
way to which artificial peptone had been added, and that it no longer gave off
ammonia when heated with caustic alkali.
To each kilogramme of minced and fat-free goat's flesh, 80 c.c. of hydro-