Part III: Culture
inoculation and incubation procedures

Inoculation procedures
Condensed moisture is frequently observed at the bottom of culture medium slants. This should be removed before inoculation is attempted.

A common fault in inoculation is the use of too small an inoculum. Either loops (wire or disposable) or pipettes can be used for primary cultivation, although plastic Pasteur pipettes are recommended. Each slope should be inoculated with 0.2-0.4ml (2-4 drops or 2-4 loopfuls) of the centrifuged sediment, distributed over the surface. Fluid media can accommodate up to 1ml used for each specimen.

Two slopes of LJ medium should be inoculated per specimen. In areas where M. bovis may be a problem, an additional slope containing pyruvate should be added.

Incubation of cultures
All cultures should be incubated at 35E-37EC until growth is observed or discarded as negative after eight weeks.

Inoculated media should preferably be incubated in a slanted position for at least 24 hours to ensure even distribution of inoculum. Thereafter, if incubator space is needed, bottles could be placed upright. Tops should be tightened to minimise evaporation and drying of media.

The various Middlebrook agars require an atmosphere of 10% CO2and 90% air to ensure growth. CO2 is not essential to initiate growth on egg-based medium but does stimulate earlier and more luxuriant growth. A separate CO2incubator is not necessary. Inlet and outlet petcocks can be attached to an airtight metal or plastic box, built to fit on a shelf of the incubator. This box, which contains the incubating cultures, should be flushed daily with a compressed mixture of 10% CO2and 90% air. Alternatively, agar plates can be placed in impermeable Mylar plastic bags and these charged three times a week with CO2.


Dr Martie van der Walt

Dr Roxanna Rustomjee
E-mail: roxanna.rustomjee@

Prof Valerie Mizrahi
E-mail: mizrahiv@

Prof. Paul van Helden


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