Sandwich Technique Auxotroph

Re-test

Fig. 3.13. The use of the penicillin selection method for the isolation of auxotrophic mutants of C. glutamicum (Abe, 1972).

medium only the prototrophs will germinate and subsequent treatment of the spore suspension with a suitable compound would kill the germinated prototrophic spores but leave the ungerminated auxotrophic spores unharmed. The auxotrophic spores may then be isolated by washing, to remove the inhibitor, and cultured on supplemented medium. Ganju and Iyengar (1968) developed a technique of this type using sodium pen-tachlorophenate against the spores of Penicillium chrysogenum, Streptomyces aureofaciens, S. olivaceus and Bacillus subtilis.

The mechanical separation of auxotrophic and prototrophic spores of filamentous organisms has been achieved by the 'filtration enrichment method' (Catcheside, 1954). Liquid minimal medium is inoculated with mutated spores and shaken for a few hours, during which time the prototrophs will germinate but the auxotrophs will not. The suspension may then be filtered through a suitable medium, such as sintered glass, which will tend to retain the germinated spores resulting in a concentration of auxotrophic spores in the filtrate.

The visual identification of auxotrophs is based on the alternating exposure of suspected colonies to supplemented and minimal media. Colonies which grow on supplemented media, but not on minimal, are auxotrophic. The alternating exposure of colonies to supplemented and minimal medium has been achieved by replica plating (Lederberg and Lederberg, 1952). The technique consists of allowing the survivors of a mutation treatment to develop colonies on petri dishes of supplemented medium and then transferring a portion of each colony to minimal medium. The transfer process may be 'mechanized' by using some form of replicator. For bacteria the replicator is a sterile velvet pad attached to a circular support and replication is achieved by inverting the petri dish on to the pad, thus leaving an imprint of the colonies on the pad which may be used to inoculate new plates by pressing the plates on to the pad. It may be possible to replicate fungal and streptomycete cultures using a velvet pad, but, if unsatisfactory results are obtained, a steel pin replicator may be more appropriate.

Visual identification of auxotrophs has also been achieved by the so-called 'sandwich technique'. The survivors of a mutation treatment are seeded in a layer of minimal agar in a petri dish and covered with a layer of sterile minimal agar. The plate is incubated for 1 or 2 days and the colonies developed are marked on the base of the plate, after which a layer of supplemented agar is poured over the surface. The colonies which then appear after a further incubation period are auxotrophic, as they were unable to grow on the minimal medium.

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