292
M. A. GERENCSER
characteristics, strain variability, and methods for doing biochemical tests
can be found in Holmberg and Hallender (1973), Georg (1974), “Bergey’s
Manual of Determinative 3acteriology” (1974), Slack and Gerencser (1975),
Smith (1975), and Holmberg and Nord (1975). It should be pointed out
that biochemical reactions of Actinomyces are highly dependent upon the
medium and method. This should always be considered when comparing
results obtained with an unknown strain with published results.
111. PRODUCTION O F FLUORESCENT ANTIBODY REAGENTS
A. Production of antiserum
Over the years a variety of methods for preparing immunising antigens
and several different injection schedules have been used in making Actinomyces antiserum. The methods currently being used in our laboratory will
be given in some detail along with information about other methods. T h e
cultures which have been used at West Virginia University for antiserum
production are listed in Table 111.
1. Preparation of immunising antigens
Cultures to be used as immunisation antigens are grown in Trypticase
Soy Broth (BBL) or Brain-Heart Infusion Broth (BBL or Difco). The
medium is prepared in 500 ml amounts in 1 litre screw-capped flasks and
autoclaved. T h e caps are tightened as soon as the flasks are removed from
the autoclave. Each flask is inoculated with 5.0-10.0 ml of a 2-3 day old
culture growing in the same medium. A stream of oxygen-free gas containing 95% Nz-5% COz is run into the flask for approximately 1 min
after which the cap is again tightened. The inoculated flasks are incubated
at 35°C for 3-5 days with periodic shaking by hand. When good growth is
obtained, the cells are harvested by centrifugation, washed three times in
phosphate buffered saline, p H 7.2 (FTA Buffer, BBL) and resuspended in
the same buffer to a concentration equal to that of a No. 8 MacFarland
standard. When necessary, the suspensions are homogenised on a Vortex
type mixer or by sonication for 15-30 s. This may be needed for any
organism but is especially useful with A . israelii strains. The suspension is
distributed in 4.0-5.0 ml amounts in screw-capped vials and frozen at
-70°C. An aliquot is removed, allowed to thaw and warmed to room
temperature for each injection.
Cultures killed by the addition of formalin to the medium to a final
concentration of 0.5% and then washed and resuspended in formalinised
saline (0.5% formalin in 0+3570 NaCl) are kqually useful as immunisation
antigens.
M. A. GERENCSER
characteristics, strain variability, and methods for doing biochemical tests
can be found in Holmberg and Hallender (1973), Georg (1974), “Bergey’s
Manual of Determinative 3acteriology” (1974), Slack and Gerencser (1975),
Smith (1975), and Holmberg and Nord (1975). It should be pointed out
that biochemical reactions of Actinomyces are highly dependent upon the
medium and method. This should always be considered when comparing
results obtained with an unknown strain with published results.
111. PRODUCTION O F FLUORESCENT ANTIBODY REAGENTS
A. Production of antiserum
Over the years a variety of methods for preparing immunising antigens
and several different injection schedules have been used in making Actinomyces antiserum. The methods currently being used in our laboratory will
be given in some detail along with information about other methods. T h e
cultures which have been used at West Virginia University for antiserum
production are listed in Table 111.
1. Preparation of immunising antigens
Cultures to be used as immunisation antigens are grown in Trypticase
Soy Broth (BBL) or Brain-Heart Infusion Broth (BBL or Difco). The
medium is prepared in 500 ml amounts in 1 litre screw-capped flasks and
autoclaved. T h e caps are tightened as soon as the flasks are removed from
the autoclave. Each flask is inoculated with 5.0-10.0 ml of a 2-3 day old
culture growing in the same medium. A stream of oxygen-free gas containing 95% Nz-5% COz is run into the flask for approximately 1 min
after which the cap is again tightened. The inoculated flasks are incubated
at 35°C for 3-5 days with periodic shaking by hand. When good growth is
obtained, the cells are harvested by centrifugation, washed three times in
phosphate buffered saline, p H 7.2 (FTA Buffer, BBL) and resuspended in
the same buffer to a concentration equal to that of a No. 8 MacFarland
standard. When necessary, the suspensions are homogenised on a Vortex
type mixer or by sonication for 15-30 s. This may be needed for any
organism but is especially useful with A . israelii strains. The suspension is
distributed in 4.0-5.0 ml amounts in screw-capped vials and frozen at
-70°C. An aliquot is removed, allowed to thaw and warmed to room
temperature for each injection.
Cultures killed by the addition of formalin to the medium to a final
concentration of 0.5% and then washed and resuspended in formalinised
saline (0.5% formalin in 0+3570 NaCl) are kqually useful as immunisation
antigens.