446
U. ULLMANN
and incubated as described. The bacterial growth from each Petri dish is
harvested in 5 ml of 0.8% NaCl (pH 7.0), checked for purity and pooled
(Bokkenheuser, 1972). For production of 0 antigens the samples are centrifuged at 4080 x g for 20 min, then the cells are resuspended in 0.85%
saline solution and autoclaved for 2 h at 121°C. The cells are then removed
from saline solution by pelleting (Berg et al., 1971). For use in slide
agglutination tests, the antigen is diluted with saline solution to an optical
density of 0.8 at 700 nm. For application in the tube agglutination test, the
antigen suspension is adjusted to an optical density of 0.15 (Berg et al.,
1971).
Extraction of antigen for the indirect haemagglutination test after
Bokkenheuser (1972): the bacterial suspension is heated to 100°C for 2 h,
cooled to room temperature, and centrifuged at 12,000 x g for 45 min.
Two volumes of the supernatant fraction are treated with one volume of
0.25 N NaOH at 56°C for 30 min. The mixture is cooled to room temperature, adjusted to pH 7.2 with 0.25 N HCl, and centrifuged at 20,000 x g
for 45 min. This antigenic extract occasionally caused clumping of the
erythrocytes. Dialysis in Visking tubes, pore size 0.0024 pm, against
0.15 M NaCl at 4°C for 48 h removed the erythrocyte-damaging factor.
Antigenetically weak solutions are concentrated in the same type of tubes
by dialysis against a saturated solution of sucrose at room temperature.
The purified product is stored at - 12°C.
Also useful is phenol extraction after O’Neill and Todd (1961): acetonedried cells are extracted for 3 h successively with 0.25 N trichloroacetic
acid (10 ml/g dried bacteria) at 4°C. The suspension is then centrifuged,
the sediment washed, and extracted with 45% phenol at 65°C for 1 h.
The aqueous phase is separated and dialysed against distilled water, centrifuged and the supernatant freeze dried in 3 ml portions. For use the extract
is dissolved in 1 ml 0.85 saline solution.
The antigen prepared for haemagglutination can also be employed for
the complement fixation test in the Kolmer technique.
2. Alteration of 0 antigens and R forms
The somatic antigen can change its specific character. According to the
results of Mitscherlich and Heider (1968) C. fetus is able to change its 0
antigen from type 1 to type 2 but not from type 2 to type 1. This phenomenon is observed in strains that are subcultured in live broth for months
or years.
Rough strains are also found in viable strain collections. Then the growth
is granulated in liquid media. They will become smooth after one or two
subcultures in liver broth or on chocolate solid media containing 10-20%
serum from rabbit or guinea pig without complement.
U. ULLMANN
and incubated as described. The bacterial growth from each Petri dish is
harvested in 5 ml of 0.8% NaCl (pH 7.0), checked for purity and pooled
(Bokkenheuser, 1972). For production of 0 antigens the samples are centrifuged at 4080 x g for 20 min, then the cells are resuspended in 0.85%
saline solution and autoclaved for 2 h at 121°C. The cells are then removed
from saline solution by pelleting (Berg et al., 1971). For use in slide
agglutination tests, the antigen is diluted with saline solution to an optical
density of 0.8 at 700 nm. For application in the tube agglutination test, the
antigen suspension is adjusted to an optical density of 0.15 (Berg et al.,
1971).
Extraction of antigen for the indirect haemagglutination test after
Bokkenheuser (1972): the bacterial suspension is heated to 100°C for 2 h,
cooled to room temperature, and centrifuged at 12,000 x g for 45 min.
Two volumes of the supernatant fraction are treated with one volume of
0.25 N NaOH at 56°C for 30 min. The mixture is cooled to room temperature, adjusted to pH 7.2 with 0.25 N HCl, and centrifuged at 20,000 x g
for 45 min. This antigenic extract occasionally caused clumping of the
erythrocytes. Dialysis in Visking tubes, pore size 0.0024 pm, against
0.15 M NaCl at 4°C for 48 h removed the erythrocyte-damaging factor.
Antigenetically weak solutions are concentrated in the same type of tubes
by dialysis against a saturated solution of sucrose at room temperature.
The purified product is stored at - 12°C.
Also useful is phenol extraction after O’Neill and Todd (1961): acetonedried cells are extracted for 3 h successively with 0.25 N trichloroacetic
acid (10 ml/g dried bacteria) at 4°C. The suspension is then centrifuged,
the sediment washed, and extracted with 45% phenol at 65°C for 1 h.
The aqueous phase is separated and dialysed against distilled water, centrifuged and the supernatant freeze dried in 3 ml portions. For use the extract
is dissolved in 1 ml 0.85 saline solution.
The antigen prepared for haemagglutination can also be employed for
the complement fixation test in the Kolmer technique.
2. Alteration of 0 antigens and R forms
The somatic antigen can change its specific character. According to the
results of Mitscherlich and Heider (1968) C. fetus is able to change its 0
antigen from type 1 to type 2 but not from type 2 to type 1. This phenomenon is observed in strains that are subcultured in live broth for months
or years.
Rough strains are also found in viable strain collections. Then the growth
is granulated in liquid media. They will become smooth after one or two
subcultures in liver broth or on chocolate solid media containing 10-20%
serum from rabbit or guinea pig without complement.