SEPTEMBER, 1948]
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is likely to vary more than that of tryptose). After sowing, a slip of filter paper, previously
soaked in a saturated solution of lead acetate and sterilized in hot air, is inserted beside the plug
of each culture-tube so that it projects into its lumen. The slants are incubated at 37°C for
eight days. At the end of each 24 hours of this interval, the slips of filter paper are removed and
fresh inserted. The removed slips are labelled, dated and retained as a record of the degree
of blackening of their surface due to the formation of lead sulphide by the interaction of H 2 S and lead
acetate. Br. melitensis strains eliminate no H 2 S. Br. abortus strains eliminate medium quantities
of H 2 S for three or four days only. Br. suis strains eliminate maximum quantities of H 2 S for
upwards of six consecutive days.
(iii) The inhibitory action of dye media. A single batch of tryptose or liver agar pH 6.6 to
6.8 is prepared and divided into seven lots. Lot (1) is left unaltered. Lots (2), (3) and (4) are
melted and whilst warm a hot aqueous solution of basic fuchsin is added to each so that the final
concentration of dye in the three lots is 1/75,000, 1/85,000 and 1/100,000, respectively. In the
same way, a hot aqueous solution of thionin is added to lots (5), (6) and (7) so that the dye
concentration in the final preparation is 1/50,000, 1/60,000 and 1/70,000 respectively. Plates are
poured from each of the seven lots and two or three strains of each of the three type variants
are sown into each of the seven different media. Seeding is done by transferring standard large
loopsfull of a 48-hour old broth culture of the strains under examination. The plates are incubated
at 37°C for three days and their growth examined on the second and third day. The growth on the
plain agar is taken as a standard for each strain and is compared with the degree of growth on the
various batches of dye media. The object of this work is to select the concentration of dye which
best permits a normal growth of Br. melitensis strains on both thionin and fuchsin and which at
the same time, just inhibits the growth of Br. suis on fuchsin, whilst allowing its growth on
thionin and inhibits the growth of Br. abortus on thionin whilst permitting its growth on fuchsin.
Having selected the most suitable concentrations of dye, the unknown strains are seeded in the
same way into plates of the selected concentration of dye media and plain liver agar. The in-
hibitory effects of the dyes are noted and the unknown strains allocated to their various types
accordingly. In this work it is important that all media be freshly prepared and of the
correct pH. No delay, therefore, should be permitted between the trial run with known strains
and the tests on unknown strains.
(iv) Antigenic structure. Type specific antisera are prepared by injecting separate rabbits
with one of the type variants, Br. abortus and Br. melitensis. One intravenous injection only is
given the dose being approximately 4,000×10 6 organisms, in 1.0 cc. of saline. Sera with titres
in the order of 1/2,560 are the most convenient in use and therefore the rabbit's blood must be
tested daily after the third day and the animals killed and bled when the previous day's test shows
a titre in the order of 1/640-1/1,280, or thereabouts. The sera should then be cold-stored for a
week, during which time their titres will fall a little. To prepare the absorbing systems Wilson and
Miles [1932] who also describe the theory of this work—recommend that the sera so obtained be
diluted to 1/64 of their end-titres e.g. a serum with an end-titre of 1/2,560 would be diluted to 1/40
in saline. But, whilst this is a good rough guide, it is usually best to prepare three dilutions viz.,
1/32, 1/64 and 1/128 of the end-titre and perform a rough optimum proportion test. These three
dilutions of each type specific sera are then used to wash off the surface growth of the heter-
ologous Brucella strain from 48-hour-old agar cultures. In each case the previously diluted serum
is added until the density of bacteria is approximately 7 to 8 on the Brown's scale. The result-
ing bacterial suspensions are incubated for three hours, their containers being shaken three or four
times during this period. After incubation, the suspensions are centrifuged and the supernatent
sera collected. The effect of absorption has been to remove the minor heterogenous antigen
from each type specific serum and the Br. abortus serum has consequently become monospecific
D 2