Anaerobic Culture
It is preferable to use freshly reconstituted and sterile medium which is inoculated as soon as it has cooled to approximately 35°C. Tubes which are not used on the day of preparation should be placed in a boiling water bath or steamer for about 15 minutes to remove dissolved oxygen. They should be allowed to cool without agitation and then inoculated.
Inoculation should be made near the bottom of the tube in the meat particles.
Clostridia may be divided into two main groups by their action on the medium.
(i) Saccharolytic Organisms
There is rapid production of acid and gas but no digestion of the meat. Cultures may have a slightly sour smell, with reddened protein.
(ii) Proteolytic Organisms
Proteolysis causes decomposition of the meat with the formation of foul-smelling sulphur compounds and blackening. However, some saccharolytic strains also produce Hydrogen sulphide which will cause blackening but to a lesser degree.
Aerobic Culture
The tube of medium is incubated with the cap loose and no seal is required. Aerobes grow at the top whilst more anaerobic species grow deeper in the medium.
Incubation
Aerobic organisms
Incubate for up to 7 days at 35°C with loosened caps. Examine daily for turbidity, gas or changes in the meat particles.
Anaerobic organisms
Use freshly reduced medium and incubate for up to 21 days at 35°C. Examine daily for changes in the medium. Make films and subculture at intervals.
Maintenance of stock cultures
Hold at room temperature after the initial incubation at 35°C. Subculture every 4-6 months.
Storage conditions and Shelf life
St