Use of a single infusion bottle to culture and prepare potentially hazardous microorganisms for biochemical analysis. Culturing on solid media

Abstract

A procedure is described to culture microorganisms under aerobic and anaerobic conditions in infusion bottles internally coated with a solid agar medium, in view of headspace sampling of volatile components or extraction of cellular constituents.

This method is far less laborious than conventional petri-dish procedures and reduces the risk of contamination.

Biochemical analysis of microorganisms usually involves time-consuming and laborious procedures including sterilization of media, inoculation, culturing, killing, harvesting, washing, freeze-drying, extraction and, finally, sampling for separation and characterization of components (Booth, 1971; Constantinescu, 1974). To simplify the procedure, a ‘single bottle technique’ was introduced for liquid media (Weijman, 1978), using regular infusion bottles as applied in blood banks and hospitals. The present paper describes a similar technique for use with agar-containing solid media, as an alternative for or an addition to the use of petri dishes, Roux bottles or roll-tubes (Hirsch, 1948).

Procedure: Infusion bottles made of borosilicate glass were used with the following specifications: 500 ml capacity, 21 cm in height and 7.7 cm outside diameter. Bottles containing 50–150 ml medium with 3% agar (w/v) were autoclaved for 20 min at 120°C and cooled to ambient temperature. During cooling the bottles were rotated in a horizontal position at about 50 rpm to allow smooth and gradual coating of the inner surfaces. Subsequently, the flasks were inoculated and incubated (Fig. 1). The bottles can be closed with cotton wool plugs