I. TYPING METHODS FOR CLOSTRIDIUM
7
active at p H values 3 and 9, while the L type displayed a narrower activity
range. T h e bacteriocin of Clarke et al. (1975) was stable from pH 2 to 12 and
we have observed two bacteriocins which are similarly stable (Mahony, unpublished data). Tubylewicz (1966b) found the bacteriocins to be nondialysable; activity was able to pass through Seitz E.K. filters. Hirano and
Imamura (1972a, b, c) reported that some activity of the S type was lost on
dialysis, while Uchiyama (1966b) found that the substance was not filterable
through Seitz and Chamberland L3 bacterial filters. Also the bacteriocin
was not sensitive to chloroform. Mahony and Butler (1971) found that their
bacteriocin 28 was not affected by commercially available antitoxin.
The first attempt to purify the bacteriocins of C. perfringens and to determine their chemical composition was carried out by Tubylewicz (1966b).
The procedure was modified in 1968. It involved precipitation of bacterial
supernatant fluids with ammonium sulphate (60-70% of saturation) followed by ether-ethanol extraction. Active perfringocin was obtained from
the supernatant fluids of four bacteriocinogenic cultures following this
method. All the preparations of active perfringocins studied by Tubylewicz
(1968) were described as protein-saccharide complexes.
Of the two types of bacteriocin studied by Hirano and Imamura (1972c),
the L type appeared to be protein in nature-possibly a lytic enzyme involved in bacteriophage replication. The S type seemed to be a more stable
toxic substance with a lower molecular weight. We (Mahony, unpublished
data) have shown by column chromatography using Sephadex G150 that
bacteriocin 28 of C. perfringens has a molecular weight of about 100,000.
The biological activity of the bacteriocin becomes very unstable as purification progresses. The perfringocin described by Clarke et al. (1975)
seemed to be a pure protein with a molecular weight of about 76,000 as
shown by gel electrophoresis.
Tubylewicz (1970) studied the antigenic properties of four bacteriocins
using several serological methods. Immune sera were obtained by immunisation of rabbits with partially purified bacteriocins. The bacteriocin activity
was neutralised by such immune sera. The results of double diffusion
tests in gels indicated that bacteriocins a, b, c and dare related antigenically,
although not identical. Immunoelectrophoresis of bacteriocins a and b
caused them to move towards the negative pole. Two components of these
bacteriocins had different mobilities. Complement fixation tests were performed, but were not specific in that cross-reactions occurred.
5 . Mode of action
Studies on the mode of action of the bacteriocins of C. perfringens have
only recently been reported. Mahony and Butler (1971) followed the growth
of a broth culture of a sensitive strain of C. perfringens after the addition of
7
active at p H values 3 and 9, while the L type displayed a narrower activity
range. T h e bacteriocin of Clarke et al. (1975) was stable from pH 2 to 12 and
we have observed two bacteriocins which are similarly stable (Mahony, unpublished data). Tubylewicz (1966b) found the bacteriocins to be nondialysable; activity was able to pass through Seitz E.K. filters. Hirano and
Imamura (1972a, b, c) reported that some activity of the S type was lost on
dialysis, while Uchiyama (1966b) found that the substance was not filterable
through Seitz and Chamberland L3 bacterial filters. Also the bacteriocin
was not sensitive to chloroform. Mahony and Butler (1971) found that their
bacteriocin 28 was not affected by commercially available antitoxin.
The first attempt to purify the bacteriocins of C. perfringens and to determine their chemical composition was carried out by Tubylewicz (1966b).
The procedure was modified in 1968. It involved precipitation of bacterial
supernatant fluids with ammonium sulphate (60-70% of saturation) followed by ether-ethanol extraction. Active perfringocin was obtained from
the supernatant fluids of four bacteriocinogenic cultures following this
method. All the preparations of active perfringocins studied by Tubylewicz
(1968) were described as protein-saccharide complexes.
Of the two types of bacteriocin studied by Hirano and Imamura (1972c),
the L type appeared to be protein in nature-possibly a lytic enzyme involved in bacteriophage replication. The S type seemed to be a more stable
toxic substance with a lower molecular weight. We (Mahony, unpublished
data) have shown by column chromatography using Sephadex G150 that
bacteriocin 28 of C. perfringens has a molecular weight of about 100,000.
The biological activity of the bacteriocin becomes very unstable as purification progresses. The perfringocin described by Clarke et al. (1975)
seemed to be a pure protein with a molecular weight of about 76,000 as
shown by gel electrophoresis.
Tubylewicz (1970) studied the antigenic properties of four bacteriocins
using several serological methods. Immune sera were obtained by immunisation of rabbits with partially purified bacteriocins. The bacteriocin activity
was neutralised by such immune sera. The results of double diffusion
tests in gels indicated that bacteriocins a, b, c and dare related antigenically,
although not identical. Immunoelectrophoresis of bacteriocins a and b
caused them to move towards the negative pole. Two components of these
bacteriocins had different mobilities. Complement fixation tests were performed, but were not specific in that cross-reactions occurred.
5 . Mode of action
Studies on the mode of action of the bacteriocins of C. perfringens have
only recently been reported. Mahony and Butler (1971) followed the growth
of a broth culture of a sensitive strain of C. perfringens after the addition of