11. SEROTYPING OF LISTERIA MONOCYTOGENES AND RELATED SPECIES 41
motility should be checked by hanging drop preparations. Cultures
showing filaments indicating the presence of rough forms, should not be
used for the production of H-antigens. For unknown reasons H-antigens
from broth cultures are not always easily agglutinable by homologous
immune sera. Thus it may be advisable to use H-antigens grown on the
surface of semi-solid agar medium, for example Gard agar supplemented
by 1% glucose and phosphate buffer. Its surface must be kept moist.
On such media H-antigens of Listeriu develop well and motility is pronounced.
The growth is harvested in 0.3% formolised saline and is adjusted to
pH 7-2 after killing the organisms by allowing the suspensions to stand
for 3 4 days at room temperature.
The test for sterility is carried out as follows: 1 ml of suspension is
plated on sheep-blood-agar and incubated for 48 h.
C. Production of antisera
Before immunisation is started the animal ought to be tested for the
possible presence of Listeriu agglutinins, which are frequent, but usually
found in low titres only. Whether they are Listeriu-specific is not known.
Rabbits with serum titres against Listeriu of 1 : 80 and above should not be
used for the production of Listeriu antisera.
For the production of antisera rabbits are immunised intravenously
with increasing quantities of antigen at 3-4 day intervals. The initial
immunising dose consists of 0.5 ml of an antigen suspension corresponding
to a density of McFarland 3 (0.5 x 109 organisms/ml). This dose is increased by 0.5 ml every time until 2.5 ml are reached. The injections are
usually tolerated well. After five injections the animals are allowed to rest for
a week. If sufficiently high titres (about 1 : 1600 for 0-antisera and 1 : 6400
for H-antisera) are found the animals are exsanguinated.
Sera are divided into small aliquots and are preserved in the deep-freeze
without preservative. For use the sera may also be kept in the refrigerator
at +4"C. Then it is advisable to preserve 0-sera by addition of 0.5%
phenol and H-sera by 10% glycerine or merthiolate (final concentration
OH-antisera may easily be produced by injection of formalised broth
cultures or agar suspensions as described. For routine work OH-antisera
may be used in the study of 0-antigens if the flagellar antigens of the whole
cells to be tested are destroyed by previous boiling. Injections of living
organisms do not seem to offer any advantage.
Pure 0-antisera are obtained by injection of suspensions boiled for at
least 1 h.
1 : 10,000).
motility should be checked by hanging drop preparations. Cultures
showing filaments indicating the presence of rough forms, should not be
used for the production of H-antigens. For unknown reasons H-antigens
from broth cultures are not always easily agglutinable by homologous
immune sera. Thus it may be advisable to use H-antigens grown on the
surface of semi-solid agar medium, for example Gard agar supplemented
by 1% glucose and phosphate buffer. Its surface must be kept moist.
On such media H-antigens of Listeriu develop well and motility is pronounced.
The growth is harvested in 0.3% formolised saline and is adjusted to
pH 7-2 after killing the organisms by allowing the suspensions to stand
for 3 4 days at room temperature.
The test for sterility is carried out as follows: 1 ml of suspension is
plated on sheep-blood-agar and incubated for 48 h.
C. Production of antisera
Before immunisation is started the animal ought to be tested for the
possible presence of Listeriu agglutinins, which are frequent, but usually
found in low titres only. Whether they are Listeriu-specific is not known.
Rabbits with serum titres against Listeriu of 1 : 80 and above should not be
used for the production of Listeriu antisera.
For the production of antisera rabbits are immunised intravenously
with increasing quantities of antigen at 3-4 day intervals. The initial
immunising dose consists of 0.5 ml of an antigen suspension corresponding
to a density of McFarland 3 (0.5 x 109 organisms/ml). This dose is increased by 0.5 ml every time until 2.5 ml are reached. The injections are
usually tolerated well. After five injections the animals are allowed to rest for
a week. If sufficiently high titres (about 1 : 1600 for 0-antisera and 1 : 6400
for H-antisera) are found the animals are exsanguinated.
Sera are divided into small aliquots and are preserved in the deep-freeze
without preservative. For use the sera may also be kept in the refrigerator
at +4"C. Then it is advisable to preserve 0-sera by addition of 0.5%
phenol and H-sera by 10% glycerine or merthiolate (final concentration
OH-antisera may easily be produced by injection of formalised broth
cultures or agar suspensions as described. For routine work OH-antisera
may be used in the study of 0-antigens if the flagellar antigens of the whole
cells to be tested are destroyed by previous boiling. Injections of living
organisms do not seem to offer any advantage.
Pure 0-antisera are obtained by injection of suspensions boiled for at
least 1 h.
1 : 10,000).