I. TYPING METHODS FOR CLOSTRIDIUM
19
utilised in typing. For use in bacteriocin titration, the indicator strain is
grown in cooked meat medium for 18 h after which it is subcultured by
adding 1 ml to 10 ml of freshly boiled and cooled Brain Heart Infusion
Broth. After 3 h of growth at 37"C, this culture is diluted 1 : 100 in Brain
Heart Infusion Broth and swabbed on to the surface of a blood agar plate
(Difco Brain Heart Infusion Base plus 10% human blood). The bacteriocin
to be titrated is serially diluted in broth and either loopfuls or lop1
volumes of these dilutions are spotted on to the seeded plate. After 18 h of
anaerobic incubation at 37°C the reciprocal of the highest dilution showing any inhibition of bacterial growth is expressed as the bacteriocin titre.
An end-point dilution of 1/64 would be expressed as 64 units of activity.
VII. TECHNIQUE OF BACTERIOCIN TYPING
The methodology involves testing the susceptibility of unknown strains
to ten bacteriocins of C. perfringens. The bacteriocin-producing strains and
their origin are recorded elsewhere (Mahony, 1974). Before typing, the
activity of these bacteriocins is adjusted to 320 units wherever possible.
Although most titres obtainable by ammonium sulphate precipitation
exceed 320 units, some strains consistently produce lower titres. Such
bacteriocins are used at the highest concentration possible. Blood agar
plates are swabbed with the test strains of C. perfringens grown as described
for the indicator strain. Drops (10 p1) of bacteriocin are then systematically
placed on these plates with a Pipetman P20 pipette (Mandel Scientific Co.).
Multiple inoculating devices delivering an adequate volume might alternatively be used. After the inocula have dried, the plates are incubated
anaerobically for 18 h and examined for zones of growth inhibition.
VIII. INTERPRETATION OF BACTERIOCIN TYPING
A. Reading of plates
The patterns of growth inhibition produced by the bacteriocins are
recorded. Very weak zones of growth inhibition are recorded as negative;
however, the majority of reactions are unequivocal. Figure 1 demonstrates
two different typing patterns.
B. Patterns of susceptibility
In testing 274 strains of C. perfringens from many sources (laboratory
strains, faecal isolates, food poisoning strains and soil strains), typable
strains yielded 49 different typing patterns (Mahony, 1974). Since that time,
16 new typing patterns have been observed and the complete list appears
in Table 11.
19
utilised in typing. For use in bacteriocin titration, the indicator strain is
grown in cooked meat medium for 18 h after which it is subcultured by
adding 1 ml to 10 ml of freshly boiled and cooled Brain Heart Infusion
Broth. After 3 h of growth at 37"C, this culture is diluted 1 : 100 in Brain
Heart Infusion Broth and swabbed on to the surface of a blood agar plate
(Difco Brain Heart Infusion Base plus 10% human blood). The bacteriocin
to be titrated is serially diluted in broth and either loopfuls or lop1
volumes of these dilutions are spotted on to the seeded plate. After 18 h of
anaerobic incubation at 37°C the reciprocal of the highest dilution showing any inhibition of bacterial growth is expressed as the bacteriocin titre.
An end-point dilution of 1/64 would be expressed as 64 units of activity.
VII. TECHNIQUE OF BACTERIOCIN TYPING
The methodology involves testing the susceptibility of unknown strains
to ten bacteriocins of C. perfringens. The bacteriocin-producing strains and
their origin are recorded elsewhere (Mahony, 1974). Before typing, the
activity of these bacteriocins is adjusted to 320 units wherever possible.
Although most titres obtainable by ammonium sulphate precipitation
exceed 320 units, some strains consistently produce lower titres. Such
bacteriocins are used at the highest concentration possible. Blood agar
plates are swabbed with the test strains of C. perfringens grown as described
for the indicator strain. Drops (10 p1) of bacteriocin are then systematically
placed on these plates with a Pipetman P20 pipette (Mandel Scientific Co.).
Multiple inoculating devices delivering an adequate volume might alternatively be used. After the inocula have dried, the plates are incubated
anaerobically for 18 h and examined for zones of growth inhibition.
VIII. INTERPRETATION OF BACTERIOCIN TYPING
A. Reading of plates
The patterns of growth inhibition produced by the bacteriocins are
recorded. Very weak zones of growth inhibition are recorded as negative;
however, the majority of reactions are unequivocal. Figure 1 demonstrates
two different typing patterns.
B. Patterns of susceptibility
In testing 274 strains of C. perfringens from many sources (laboratory
strains, faecal isolates, food poisoning strains and soil strains), typable
strains yielded 49 different typing patterns (Mahony, 1974). Since that time,
16 new typing patterns have been observed and the complete list appears
in Table 11.