44
ir. P. R. SEELIGER AND K. HOHNE
Preparation of 0-factor sera
1. Immunise rabbits with boiled antigen and test for presence of desired
2. Absorb sera, containing the desired factors:
0- or H-agglutinins
Mix one part of undiluted serum with two parts of centrifuged antigen
sediment. .
Mix thoroughly
3. Incubate for 24 h at 4°C
4. Separate serum from antigen by high speed centrifugation (30 min at
about 10,000 rev/min)
5. Save supernatant
6. Centrifuge again
7. Check by slide agglutination with absorbing antigen suspension
whether complete absorption is achieved (no agglutination is allowed 1)
8. If not, a second absorption will be necessary
9. Check specificity of factor sera by slide agglutination.
For this purpose dense antigen suspensions must be used. After treatment the 0-antigen is centrifuged and the supernatant is decanted but for
a little residuum which is used for resuspending the sediment. A drop of
the concentrated antigen suspension is mixed with a drop of 0-factor
serum on a glass slide. The slide is tilted between the fingers for 1-2 min
and the occurrence of agglutination is watched against a dark background
with a good source of light. Two or three factor sera may be examined at
the same time on one slide and compared with a saline control.
E. Conservation of factor sera
The factor sera preserved with phenol (0.5% final concentration) or
any other suitable preservative, may be kept for years. T o 0.9 ml factor
serum 0.1 ml of a 5% phenol-saline solution is added. Factor sera in
vials may be kept in a refrigerator at 4" to 6°C. Shaking the contents of
the vials should be avoided. When withdrawing sera, care should be taken
not to tip up the vial abruptly in order to avoid transferring the fine deposit
which tends to form after prolonged storage. The reading of the reaction
is not affected by an occasional flake.
The antigenic formulae as ascertained by Paterson have been fully
confirmed by Seeliger (1975). They proved useful for sero analysis of
listeriae and could be simplified for routine work (Table VI).
F. Results of serological analysis
Serological differentiation of L. monocytogenes as a routine procedure was
introduced by Seeliger (1954, 1958, 1961). Results of almost 3500 typings
ir. P. R. SEELIGER AND K. HOHNE
Preparation of 0-factor sera
1. Immunise rabbits with boiled antigen and test for presence of desired
2. Absorb sera, containing the desired factors:
0- or H-agglutinins
Mix one part of undiluted serum with two parts of centrifuged antigen
sediment. .
Mix thoroughly
3. Incubate for 24 h at 4°C
4. Separate serum from antigen by high speed centrifugation (30 min at
about 10,000 rev/min)
5. Save supernatant
6. Centrifuge again
7. Check by slide agglutination with absorbing antigen suspension
whether complete absorption is achieved (no agglutination is allowed 1)
8. If not, a second absorption will be necessary
9. Check specificity of factor sera by slide agglutination.
For this purpose dense antigen suspensions must be used. After treatment the 0-antigen is centrifuged and the supernatant is decanted but for
a little residuum which is used for resuspending the sediment. A drop of
the concentrated antigen suspension is mixed with a drop of 0-factor
serum on a glass slide. The slide is tilted between the fingers for 1-2 min
and the occurrence of agglutination is watched against a dark background
with a good source of light. Two or three factor sera may be examined at
the same time on one slide and compared with a saline control.
E. Conservation of factor sera
The factor sera preserved with phenol (0.5% final concentration) or
any other suitable preservative, may be kept for years. T o 0.9 ml factor
serum 0.1 ml of a 5% phenol-saline solution is added. Factor sera in
vials may be kept in a refrigerator at 4" to 6°C. Shaking the contents of
the vials should be avoided. When withdrawing sera, care should be taken
not to tip up the vial abruptly in order to avoid transferring the fine deposit
which tends to form after prolonged storage. The reading of the reaction
is not affected by an occasional flake.
The antigenic formulae as ascertained by Paterson have been fully
confirmed by Seeliger (1975). They proved useful for sero analysis of
listeriae and could be simplified for routine work (Table VI).
F. Results of serological analysis
Serological differentiation of L. monocytogenes as a routine procedure was
introduced by Seeliger (1954, 1958, 1961). Results of almost 3500 typings