6533b873fe1ef96bd12d4ad4

RESEARCH PRODUCT

Micromanipulation Techniques for the Isolation of Single Microorganisms

subject

description

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. The only absolute criterion of purity for a bacterial culture is that it has been derived from the progeny of a single cell. Failure to apply this criterion may lead to much effort in proving the purity of a culture. All strains upon which research is to be based should therefore be rigorously purified before starting to investigate the properties of individual organisms (Johnstone 1969). Ecologically oriented microbiologists are faced especially with the problem of how to obtain a pure culture of certain microbial strains from their densely populated natural habitats. The methods used range from simple devices up to very complex machines. The principal procedures for obtaining pure cultures of bacterial strains have not been greatly improved since Robert Koch (Koch 1881). The isolation according to conventional methods, like the separation of microorganisms by agar plates (Koch 1881) and agar shake tubes (cf. Pfennig and Truper 1981), cannot avoid the fact that individual colonies are formed by cell aggregates. This occurs particularly with filamentous bacteria, which could easily agglomerate. More sophisticated electronic enumeration and sampling systems such as Coulter counter or flow cytometry cannot prevent the formation of cell aggregates. In addition to the isolation of single cells with optical tweezers (Huber 1999), there are alternative approaches, e.g. the “Bactotip” and “Membrane” methods for the micromanipulation of individual microorganisms. With the aid of these methods single cells can be picked out of a mixed population under direct visual control. The isolated aerobic or anaerobic species can be grown in pure culture or can be subjected to single cell PCR (Frohlich andKonig 1999a, 2000; cf. Prescott et al. 2002; Frohlich et al. 2002).