332
W. B. SCHAEFER
When the titre has reached 1 : 160, the animal is exsanguinated, the serum
is collected, preserved with merthiolate 1 : 10,000, and stored at 4°C.
A method for obtaining type-specific antisera in mice by injecting intraperitoneally the bacteria suspended in Freund’s adjuvant and collecting
the intraperitoneal exudate has been described by Thoen and Karlson
(1970).
C. Serotyping procedure
The antisera may be stored in the refrigerator as 1 : 10 stock dilutions,
with single working dilutions available for each antiserum. Each serotype
is represented by two type-specific antisera in order to determine the crossreactivity of two strains (see Table 11). The working dilutions are pipetted
into 12 x 75 mm tubes in volumes of 0.5 ml. A saline control tube is also
included. T o all tubes an equal volume of the bacterial suspension is added.
The tubes are briefly shaken, then deposited in an incubator at 37°C. After
3-5 h, the agglutination results are read without shaking the tubes, and
read again the following day.
D. Interpretation of results
A strain is classified as a particular serotype if the bacterial suspension
is agglutinated by both antisera of this type and is not agglutinated by the
antisera of other types. The type-specific agglutination usually becomes
visible within the first 3 h of incubation. With some strains it becomes
visible only after 6 h, or over-night. Sometimes agglutination remains incomplete. It is then necessary to carry out the agglutinin absorption test.
If agglutinated by a single serum only, the specificity of this agglutination
should be verified by an absorption test. Certain strains possess such small
amounts of the type-specific antigens that they are agglutinated only by the
stronger of the two antisera of the homologous type.
E. Agglutinin absorption test
A 0.5 ml portion of the 1 : 10 diluted antiserum is mixed in a centrifuge
tube with an equal volume of a very dense suspension of the bacteria to be
tested. This suspension is made by suspending the bacterial sediment,
obtained by centrifugation, in a double volume of PPBS. The mixture is
kept at 4°C for an hour, centrifuged at high speed, and the supernatant
which now corresponds to a dilution of 1 : 20 is transferred to an agglutination tube, and two-fold dilutions made with equal volumes of PPBS to
1 : 640. A parallel series of dilutions is made beyond the titre of the unabsorbed serum. A 0.5 ml aliquot of a stable homologous bacterial suspension of the homologous type is added to all tubes. The tubes are incubated
at 37°C and the agglutination results read the following day. Strains which
W. B. SCHAEFER
When the titre has reached 1 : 160, the animal is exsanguinated, the serum
is collected, preserved with merthiolate 1 : 10,000, and stored at 4°C.
A method for obtaining type-specific antisera in mice by injecting intraperitoneally the bacteria suspended in Freund’s adjuvant and collecting
the intraperitoneal exudate has been described by Thoen and Karlson
(1970).
C. Serotyping procedure
The antisera may be stored in the refrigerator as 1 : 10 stock dilutions,
with single working dilutions available for each antiserum. Each serotype
is represented by two type-specific antisera in order to determine the crossreactivity of two strains (see Table 11). The working dilutions are pipetted
into 12 x 75 mm tubes in volumes of 0.5 ml. A saline control tube is also
included. T o all tubes an equal volume of the bacterial suspension is added.
The tubes are briefly shaken, then deposited in an incubator at 37°C. After
3-5 h, the agglutination results are read without shaking the tubes, and
read again the following day.
D. Interpretation of results
A strain is classified as a particular serotype if the bacterial suspension
is agglutinated by both antisera of this type and is not agglutinated by the
antisera of other types. The type-specific agglutination usually becomes
visible within the first 3 h of incubation. With some strains it becomes
visible only after 6 h, or over-night. Sometimes agglutination remains incomplete. It is then necessary to carry out the agglutinin absorption test.
If agglutinated by a single serum only, the specificity of this agglutination
should be verified by an absorption test. Certain strains possess such small
amounts of the type-specific antigens that they are agglutinated only by the
stronger of the two antisera of the homologous type.
E. Agglutinin absorption test
A 0.5 ml portion of the 1 : 10 diluted antiserum is mixed in a centrifuge
tube with an equal volume of a very dense suspension of the bacteria to be
tested. This suspension is made by suspending the bacterial sediment,
obtained by centrifugation, in a double volume of PPBS. The mixture is
kept at 4°C for an hour, centrifuged at high speed, and the supernatant
which now corresponds to a dilution of 1 : 20 is transferred to an agglutination tube, and two-fold dilutions made with equal volumes of PPBS to
1 : 640. A parallel series of dilutions is made beyond the titre of the unabsorbed serum. A 0.5 ml aliquot of a stable homologous bacterial suspension of the homologous type is added to all tubes. The tubes are incubated
at 37°C and the agglutination results read the following day. Strains which