SEPTEMBER, 1948]
JOHN
B.
POLDING
163
the relative virulence of the three principal ones is fairly accurately portrayed in Table XXIX. If
then the immunizing value of a strain is indeed proportional to its virulence, and, if a single strain
only is to be used, plainly either McEwen's No. 45(20), or Cotton's No. 19, should be selected as being
of the highest virulence consistant with safety. If however, the use of a combination of strains or
even two injections is contemplated, then the choice becomes more difficult and trials would have to
decide, whether to give a strain of low virulence followed by one of greater or whether some other
combination was preferable. There is an enormous field of research in which to decide (i) the opti-
mum age, or sexual phase, at which to inject cattle with—(a) a single dose, or (b) 2 doses ; (ii) this
optimum size of any one dose ; (iii) whether one, or more, strains should be used and if so which ;
(iv) a means, acceptable to all, of standardizing the degree of virulence of a vaccine strain and
(v) whether vaccination should be repeated in later life and, if so, when and how often. Again, there
is much work to be done, in assessing the correct vehicle for the vaccine. It has been shown for
instance that certain microbes survive longer in some fluids than in others and it is well known that
some bacteria die rapidly in a simple saline suspension. These points are of great importance in India,
where periods of transport extend into days and variations in climate are extreme. Consequently,
as the remains of the vaccine, hitherto returned from field during the present scheme, have been
invariably found to be dead, a series of experiments was devised to study the survival of Brucella
in fluids. The enumeration of the bacteria surviving in suspensions entails the use of serial dilutions
and plate-counts—a method which is subject to many small personal differences in technique. Except
in the preliminary trials (p. 163) where changes of methods were tried, the standard technique adopted
in the work to be described was as follows. With a clean sterile 1.0 c.c. pipette, 1.0 c.c. of the bacterial
suspension was transferred to exactly 9.0 c.c. of diluent in tube 1 of a dilation series and mixed by
blowing through the pipette, whilst moving its tip up and down the column of fluid thrice. With
a fresh similar pipette 1.0 c.c. of the mixture in tube 1 was transferred to 9.0 c.c. of diluent in tube 2
and this process of transferring and mixing of fluids and discarding of pipettes was continued until
the requisite number of dilutions were prepared. Although different vehicles were used in the pre-
paration of various suspensions, unless otherwise stated, the diluting fluid used throughout was
0.85 per cent NaCl in distilled water. The pH of this fluid was not adjusted, but it was generally
slightly acid—(pH 6.0 to 7.0)—a reaction which subsequent experiments showed to be favourable
to Brucella. Huddleson [1943] stipulates that, in making plate-counts of Brucella, the diluting fluid
should contain 0.1 per cent of tryptose, as this makes the counts more regular. Such an addition
was not used in the present work, as, on the whole, the count results were satisfactory and the use
of tryptose entailed the introduction of another unknown.
Plates were prepared from the dilutions by pipetting with a fresh 1.0 c.c. pipette, 1.0 c.c. from the
selected dilution tubes in ascending order of bacterial density, without changing the pipette. Then
the melted media—about 12 c.c.—was poured at a temperature of 45° to 48°C. and the plate's contents
mixed by the usual rotating and reciprocating movements. Plates were in 4 in. in diameter.
It is perhaps unnecessary to give full details of the earlier series of experiments (Tables XXXIX
to XLII) but their findings may be summarized as follows. (1) The death-rate of Brucella in a
saline vehicle is probably proportional to the temperature of storage (Table XXXIX). (2) The
death-rate of Brucella in a saline vehicle, stored at 26°C. is regular and relatively slow (Table XL).
(3) Conversely, the death rate of Brucella in a saline vehicle, stored at 42°C. is relatively rapid
for the first day or two and thereafter less rapid, while in this environment the death-rate is little
affected by the addition of small concentrations of gelatine or tryptose (Table XLI). (4) Brucella
stored at 42°C. in a saline vehicle probably increase in viable numbers for upwards of 12 hours,
but before the twenty fourth hour is reached a very rapid death-rate supervenes and the viable
count decreases speedily (Table XLII). These preliminaries suggested that the period of storage
during which the effect of different environments could best be studied would coincide with the phase
of rapid death, say from 18 to upwards of 48 hours of storage. Moreover, as limited facilities
prevented the use of more than three variables in each test, a standard storage temperature—consi-
dered to be an average adverse plains temperature—was adopted in most tests, viz., 42°C. (103°—5Fº).
G