18
D. E. MAHONY
(Prescott and Altenbern, 1967b) showed that one phage had a sheathed tail
while that of a second was simple in structure. Each was about 100 nm
long, the head diameter being about 65 'nm. Prescott and Altenbern could
not demonstrate plaque formation with their induced cultures, but Roseman and Richardson (1969) successfully showed plaques of phages from
11 of 26 toxin producing strains of C . tetani.
3. Other bacteriophages
This review has been restricted to the bacteriophage and bacteriocins of
the pathogenic species of Clostridium. A number of other papers deal with
non-pathogenic species. One might draw attention, in particular, to the
work of Hongo and Murata in Japan and their extensive studies on the
bacteriophages of Clostridium saccharoperbutylacetonicum (Hongo et al.,
1969). For a review on the classification of bacteriophages of Clostridium,
see Ackerman (1 974).
VI. PRODUCTION OF BACTERIOCIN FOR
TYPING OF C . PERFRINGENS
A. Preparation of bacteriocin
The bacteriocin-producing strain of C . perfringens is grown for 18 h in
cooked meat medium (Difco). At this time, the bacteria are subcultured by
adding a 10% inoculum to Brain Heart Infusion Broth (Difco) which has
been previously boiled for 10 min and cooled to 37°C. After 2.5 h incubation at 37"C, 30 ml are transferred to 500 ml of freshly boiled and cooled
Brain Heart Infusion Broth. After 5 h of incubation, the culture is centrifuged at 6000 x g for 10 min to sediment the bacteria. The supernatant is
removed and the bacteriocin is precipitated by the addition of powdered
ammonium sulphate to 40% saturation (28 g/100 ml) at 4°C. The fluid is
constantly stirred by a magnetic stirrer during this procedure. Good yields
of bacteriocin are attainable in 1-2 h, although we usually allow precipitation to continue overnight. The precipitate is collected by centrifugation at
6000 x g for 10 min and resuspended in 5 ml of sterile Brain Heart Infusion
Broth. Buffers might be substituted for'broth, although we have found
broth preferable for preserving bacteriocin activity. The bacteriocin should
be stored at 4°C or preferably at - 70°C for long-term storage. The method
of obtaining higher yields of some bacteriocins by induction with Mitomycin C is described elsewhere (Mahony, 1977).
B. Titration of bacteriocin
We have consistently used strain no. 2 in our collection as an indicator
of bacteriocin activity because it is very sensitive to all of the bacteriocins
D. E. MAHONY
(Prescott and Altenbern, 1967b) showed that one phage had a sheathed tail
while that of a second was simple in structure. Each was about 100 nm
long, the head diameter being about 65 'nm. Prescott and Altenbern could
not demonstrate plaque formation with their induced cultures, but Roseman and Richardson (1969) successfully showed plaques of phages from
11 of 26 toxin producing strains of C . tetani.
3. Other bacteriophages
This review has been restricted to the bacteriophage and bacteriocins of
the pathogenic species of Clostridium. A number of other papers deal with
non-pathogenic species. One might draw attention, in particular, to the
work of Hongo and Murata in Japan and their extensive studies on the
bacteriophages of Clostridium saccharoperbutylacetonicum (Hongo et al.,
1969). For a review on the classification of bacteriophages of Clostridium,
see Ackerman (1 974).
VI. PRODUCTION OF BACTERIOCIN FOR
TYPING OF C . PERFRINGENS
A. Preparation of bacteriocin
The bacteriocin-producing strain of C . perfringens is grown for 18 h in
cooked meat medium (Difco). At this time, the bacteria are subcultured by
adding a 10% inoculum to Brain Heart Infusion Broth (Difco) which has
been previously boiled for 10 min and cooled to 37°C. After 2.5 h incubation at 37"C, 30 ml are transferred to 500 ml of freshly boiled and cooled
Brain Heart Infusion Broth. After 5 h of incubation, the culture is centrifuged at 6000 x g for 10 min to sediment the bacteria. The supernatant is
removed and the bacteriocin is precipitated by the addition of powdered
ammonium sulphate to 40% saturation (28 g/100 ml) at 4°C. The fluid is
constantly stirred by a magnetic stirrer during this procedure. Good yields
of bacteriocin are attainable in 1-2 h, although we usually allow precipitation to continue overnight. The precipitate is collected by centrifugation at
6000 x g for 10 min and resuspended in 5 ml of sterile Brain Heart Infusion
Broth. Buffers might be substituted for'broth, although we have found
broth preferable for preserving bacteriocin activity. The bacteriocin should
be stored at 4°C or preferably at - 70°C for long-term storage. The method
of obtaining higher yields of some bacteriocins by induction with Mitomycin C is described elsewhere (Mahony, 1977).
B. Titration of bacteriocin
We have consistently used strain no. 2 in our collection as an indicator
of bacteriocin activity because it is very sensitive to all of the bacteriocins