X. METHODS IN CAMPYLOBACTER
441
(b) Bacitracin 25 i.u./ml, Polymyxin B 10 i.u./ml, Novobiocin 5 mg/litre,
and Actidione 50 mg/litre (Dekeyser et al., 1972). (c) Vancomycin 10 mg/
litre, Polymyxin B 2.5 i.u./ml, and Trimethoprim 5 mg/litre (Skirrow,
1977).
The antibiotics are added to liquid or solid media. Cefalotin discs
(30pg) may also be placed on solid media. Colonies growing within the
inhibition zone suggest C. fetus (Ullmann, 1976).
(3) The Brilliant-green medium of Florent (1959), is used for mixed
cultures also : fluid thioglycollate medium is supplemented with 10%
defibrinated sheep blood plus 11-4 ml of 0.22% aqueous Brilliant-green
solution/litre (Bisping, 1974).
D. Liquid and solid media
Useful for isolation are liquid media designed for isolation of fastidious
bacteria, such as Tarozzi’s liver broth, liver broth (CM78 Oxoid, London).
Brewer’s thioglycollate medium (BBL, Difco, Oxoid), brain heart infusion
(BBL, Difco, Oxoid), tryptone soya broth (Oxoid) with addition of 0.05 g
p-amino-benzoic acid per litre of medium (Ruckdeschel, 1973). The growth
of C. fetus will be accelerated by addition of defibrinated cooked blood from
sheep or ox to the following solid media: Columbia agar (BBL, Oxoid),
brain heart infusion agar (BBL, Difco, Oxoid), D.S.T. agar (Oxoid,)
Brucellu agar (Albimi). Some investigators supplement these media with
5 or 10% defibrinated fresh blood. My own experience shows that some
strains do not grow on fresh blood media but grow spontaneously on
chocolate blood agar. Nutrient agar, Endo-agar, media for Salmonella and
Shigelkz isolation, C.L.E.D. medium or other simple nutrient substrates
are useless.
E. Incubation of cultures
The inoculated plates and tubes are incubated at 37°C in an anaerobic
incubator in which 70% of the volume of air has been replaced by a
mixture of 10% carbon dioxide and 90% nitrogen. Solid media can also be
put into the GasPak system using the carbon dioxide generator; incubation
following the Fortner method is very useful for isolation. Thereby one half
of the solid medium is inoculated with an oxygen consuming bacterial
strain (for example Serratia mavcescens): on the other is spread the sample
required for anaerobes. The Petri dishes are closed with paraffin or an
airtight plastic tape (Leukoflex, Beiersdorf, Hamburg), and incubated
upside down. The aerobic bacteria grow, consuming the oxygen and giving
rise to a COz-enriched atmosphere for anaerobes. Sometimes the growth
fails to appear in subcultures on solid media especially if the GasPak system
441
(b) Bacitracin 25 i.u./ml, Polymyxin B 10 i.u./ml, Novobiocin 5 mg/litre,
and Actidione 50 mg/litre (Dekeyser et al., 1972). (c) Vancomycin 10 mg/
litre, Polymyxin B 2.5 i.u./ml, and Trimethoprim 5 mg/litre (Skirrow,
1977).
The antibiotics are added to liquid or solid media. Cefalotin discs
(30pg) may also be placed on solid media. Colonies growing within the
inhibition zone suggest C. fetus (Ullmann, 1976).
(3) The Brilliant-green medium of Florent (1959), is used for mixed
cultures also : fluid thioglycollate medium is supplemented with 10%
defibrinated sheep blood plus 11-4 ml of 0.22% aqueous Brilliant-green
solution/litre (Bisping, 1974).
D. Liquid and solid media
Useful for isolation are liquid media designed for isolation of fastidious
bacteria, such as Tarozzi’s liver broth, liver broth (CM78 Oxoid, London).
Brewer’s thioglycollate medium (BBL, Difco, Oxoid), brain heart infusion
(BBL, Difco, Oxoid), tryptone soya broth (Oxoid) with addition of 0.05 g
p-amino-benzoic acid per litre of medium (Ruckdeschel, 1973). The growth
of C. fetus will be accelerated by addition of defibrinated cooked blood from
sheep or ox to the following solid media: Columbia agar (BBL, Oxoid),
brain heart infusion agar (BBL, Difco, Oxoid), D.S.T. agar (Oxoid,)
Brucellu agar (Albimi). Some investigators supplement these media with
5 or 10% defibrinated fresh blood. My own experience shows that some
strains do not grow on fresh blood media but grow spontaneously on
chocolate blood agar. Nutrient agar, Endo-agar, media for Salmonella and
Shigelkz isolation, C.L.E.D. medium or other simple nutrient substrates
are useless.
E. Incubation of cultures
The inoculated plates and tubes are incubated at 37°C in an anaerobic
incubator in which 70% of the volume of air has been replaced by a
mixture of 10% carbon dioxide and 90% nitrogen. Solid media can also be
put into the GasPak system using the carbon dioxide generator; incubation
following the Fortner method is very useful for isolation. Thereby one half
of the solid medium is inoculated with an oxygen consuming bacterial
strain (for example Serratia mavcescens): on the other is spread the sample
required for anaerobes. The Petri dishes are closed with paraffin or an
airtight plastic tape (Leukoflex, Beiersdorf, Hamburg), and incubated
upside down. The aerobic bacteria grow, consuming the oxygen and giving
rise to a COz-enriched atmosphere for anaerobes. Sometimes the growth
fails to appear in subcultures on solid media especially if the GasPak system