W. L. HINDMARSH
259
opinion is against this practice on account of
danger to the laboratory staff, and because the
bacteria may be killed without any appreciable
effect on agglutinability. In this connection
Henry and Traum have stated that tricresol
0.2 per cent and carbolic 0.5 per cent have
scarcely any effect on the results of the test,
but that formalin is not desirable.
As previously mentioned the density of the
suspension is very
important.
Various
methods have been adopted in the fixation of
a standard of density. Opacity standards,
enumeration of bacilli,
centrifugalization,
nephelometers and opacity meters have all
been used. Probably the best method is by
centrifugalization, using Fitch's modification
of Hopkins' vaccine tube. Whilst some writ-
ers have stated that within certain limits the
density of the suspension is without influence
on the results of the test, others, notably Fitch,
Donham, Bishop and Boyd, are of opinion
that the maximum titre of agglutination of
sera containing a moderate quantity of agglu-
tinin changes with the density of the antigen.
Stableforth has shown that with an increase
in the density of the antigen there was a
diminution in the titre of the serum.
The agglutinability of strains of Brucella
from different sources varies to a great degree.
Traum and Henry tested the agglutinability
of 22 strains of Br. abortus and found consi-
derable variation. Frei states that many strain
react with sera which give negative results
with other strains. On this account most
workers use a suspension made of a number
of strains.
In putting up the test it is usual to use a
series of serum dilutions, although in the
routine testing of large numbers of sera the
series is a small one. Some workers make
their serial dilutions in normal saline or carbol-
saline before adding the sera to the antigen.
Others, notably in America, add undiluted
serum direct to the antigen. Stableforth states
that with the latter method higher titres are
obtained. The actual volume of fluid in the
tubes is of some importance, as agglutination
is more rapid in the smaller volumes, although
the final result is probably the same.
The tubes are usually incubated at from
37° C. to 40° C.. although agglutination will
take place at lower temperatures. Frei agrees
with the finding of the Commission on Conta¬
gious Abortion of U. S. A. (1932-33) that the
test should remain in the incubator 42 hours
at least.
READING THE TEST
On reading the test it is usual to refer to
different degrees of agglutination, indicating
the reaction by the sign +.
Complete sedimentation, supernatant
clear
.
.
.
.
Almost complete
sedimentation,
supernatant almost clear
Much sedimentation,
supernatant
hazy
.
.
.
.
.
Light sedimentation,
supernatant
cloudy
.
.
.
No sedimentation . . . .
Contin-
ental
+ + + +
+ + +
+ +
+
. .
Fitch
U.S.A.
+
1
. .
INTERPRETATION OF THE TEST
With regard to Brucella infection of cattle,
we must bear in mind that:—
(a) Agglutinins may appear in the blood in
from one to three months after infection, so
that an animal may be recently infected and
give a negative reaction.
(b) Agglutinins may persist in the blood for
a long period, even if the infection be lost, so
that all animals giving a positive reaction do
not necessarily harbour the living organism.
(c) The intensity of the reaction does not
necessarily indicate the virulence or degree of
the infection.
(d) There is a relationship between continu-
ous high titre and under infection.
(e) The greatest percentage of abortions
occur in animals with a high titre (Huddle-
son).
Willems [1937] suggests that an agglutina-
tion titre should be determined, and he recom-
mends the calculation of a constant of aggluti-
nation. He takes as an index of agglutinability
of the suspension that dilution of a standard
positive serum which gives 50 per cent of
agglutination.
Calculating in this way he found that 50
per cent agglutination was given in a serum
dilution of 1 in 700 with the suspension used
in his laboratory, and with 1 in 400 with the
suspension used by Stableforth.
The indices of agglutinability therefore were
700 for the Belgian suspension and 400 for the
English suspension.
This index is a measure of the degree cf
sensitivity of the suspension.
Utilizing this suspension with an index of
agglutinability of 700, Willems regards -as a
positive reaction a 50 per cent agglutination
with a serum dilution of 1/50.
The constant of agglutination is calculated
as 50/700 or 0.07142.