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SUMMARY: Nutritional mutants of Neurospora crassa may be isolated by incuba- tion of microconidia in a liquid medium deficient in specific growth factors followed by filtration and then growth on supplemented media of the conidia remaining in the filtrate. A method for the isolation of nutritional mutants of Neurospora crassa by filtration and subsequent selective enrichment of the medium has been developed as an adaptation of the one devised by Fries (1947) for Ophiostom multiannulatum. It involves the use of a strain which produces microconidia abundantly, and preferably a strain that will grow as a colony or respond well by growing as a colonial paramorph on a suitable medium (Tatum, Barratt & Cutter, 1949). The significance of the use of microconidial strains is that only in a microconidium will a mutant nucleus have a chance of growing ultimately into a colony with the mutant character. A macroconidium con- taining a mutant nucleus would germinate and grow on minimal medium by virtue of the presence of one or more non-mutant nuclei, and so would be discarded in the course of the operations designed to eliminate as many as possible of the conidia which contain non-mutant nuclei, Stocks of N. crmsa such as Y 8743, which is colonial and peach-microconidial (Barratt & Garnjobst, 1949), or the peach fluffy double mutant, which is not colonial, are suitable. They should be grown on a medium which induces the production of a large number of conidia. Some strains produce microconidia almost exclusively if the inoculum is buried under a layer of agar. The mycelium grows through this and produces a dense crop of microconidia on the surface. The microconidia are harvested by washing them off the surface of the culture with sterile distilled water, The suspension is then filtered by means of a sterile cotton-wool filter to remove fragments of mycelium, yielding a dense opalescent suspension of microconidia. The filter is made by blowing a hole in the bottom of a Pyrex or other hard glass test-tube, and fusing to this hole a short length of Pyrex tube of about 7 mm. diameter to form a drainage stem. A small piece of absorbent cotton-wool is inserted into the base of the filter and just within the drainage stem. The open end of the filter is plugged with non-absorbent cotton-wool and a wrapping of this is also made round the stem and inserted into an intact test-tube. The whole assembly is then autoclaved. The filtered suspension is irradiated with ultraviolet light by either of two methods: (1) the suspension is transferred to a sterile Petri dish and rocked

Isolation of Nutritional Mutants of Neurospora crassa by Filtration Enrichment