SEPTEMBER, 1948]
JOHN
B.
POLDING
151
In all, 21 optimum proportion tests were made and, on the whole, the results confirmed that,
no matter whether a weak, medium, or strongly positive serum was used, if tests are made with
dilutions approaching the serum's end titre, the optimum primary density of antigen is slightly less
than 1 on the Brown's scale. When, however, serum is diluted to considerably less than its end-
titre, the density of antigen that gives optimum reaction is greater, i.e., near about a final dilution
of 2 on the Brown's scale.
Now, if it is agreed that an agglutination system should be most sensitive towards the end-
point of a titration, these results show that antigen should be primarily diluted to about tube 1 on
the Brown's scale. Its dilution when admixed with serum then becomes one half of tube 1 and it
has been found that, when antigen is prepared to about this density, it agglutinates with Stable-
forth's reconstructed serum in a dilution of about ++1/1280. Stableforth [1936] himself prepares
antigen at a density of 4 on the Brown's scale and his end-point with reconstructed serum is +1/480.
A third variable in antigen preparation is salt concentration. In the test being described, 12
per cent NaCl and 0.5 per cent phenol is used. A 12 per cent salt concentration is said to abolish
zoning; it also ensures adequate self-mixing of the antigen and serum and does not appear to interfere
with the test.
Antigens are prepared as follows : The selected strain is tested for S-R change and smooth
strains only are used. After this test, the strain is sown on liver agar and incubated for 48 to 72
hours at 37°C. The growth is then washed off in carbol saline (12 per cent NaCl, 0.5 per cent phenol)
and the bacterial density reduced to between 2 and 3 on the Brown's scale. This suspension is again
tested for S-R change. A sample of the bulk is then diluted 19 parts to 1 and 18 parts to 2 and so
on, in 12 per cent saline and each dilution is tested against reconstructed serum in the primary
dilutions of 1/320, 1/400, 1/480, 1/560 and 1/640. The density of antigen that agglutinates nearest
to +1/1280 is selected and the bulk antigen diluted accordingly.
Serum
The collection, preservation, and transportation of serum for the agglutination test have already
been described [Polding 1943]. In selecting the serum dilutions for the standard test the series
1/10, 1/20 etc. has been chosen, rather than the other popular range of 1/25, 1/50 etc. The initial
primary dilution (1/5) of the former is easier to prepare than that of the latter (1/12½). Further, in
the former range there are four closely spaced dilutions (1/10, 1/20, 1/40, 1/80) in the critical
portion of the range, whilst in the latter there are but 3 (1/25, 1/50, 1/100). For these reasons
the former range is more practical.*
The quick agglutination test
Antigen for the quick agglutination test as recommended in India is prepared exactly in the
manner of Huddleson [1939]. The actual technique and interpretation of the test is, however, a
simplification of his method [Polding 1943].
In a country like India, the value of the quick test, as a supplement to the tube test, is unqualified.
For, whilst its results are somewhat subordinate to the personal judgement of the operator and are
therefore only a rough guide to the incidence of Brucella infection, the virtues of the test are manifold,
e.g., (i) the abolition of the need for sending field specimens to the laboratory with all the attendant
difficulties, and delays, (ii) the obtaining of a diagnosis on the day of the visit to the field outbreak,
and (iii) the cheapness and compactness of the apparatus required. On the other hand, on account
of its lesser accuracy, the quick agglutination test is only recommended for use in rough village survey
work, for making preliminary tests before taking specimens for the isolation of Brucella, and for
simple diagnosis, when all that is required is to establish the existence of Brucellosis in a herd. More-
over, this method can also provide valuable information in tests on fluids other than blood serum.
For example, it is advantageous to quick-test the synovial fluid of bursal enlargements and the milk
of aborting dams and only to attempt isolation of Brucella from these sources if their reaction is
positive. Further, if time is available, it is advisable, in survey work, to quick-test the blood and
milk of aborting anmals and the blood serum and synovial fluid of animals with bursal enlargements