THE SEPARATION AND ISOLATION OF PARTICULAR BIOCHEMICAL MUTANTS OF NEUROSPORA BY DIFFERENTIAL GERMINATION OF CONIDIA, FOLLOWED BY FILTRATION AND SELECTIVE PLATING

Abstract

The wild type strains used in the study were macroconidial, haying largely mono- and dinucleate conidia. Conidia from cultures which had grown on minimal medium for 5 days at 25[degree]C, were suspended in sterile water in Petri plates and irradiated with a 15-watt G-E UV germicidal lamp for 30 to 90 min., with agitation during treatment. The conidial suspension was then inoculated into phosphate-buffered minimal medium and incubated with aeration. As the wild-type mycelia became perceptible they were filtered through cheesecloth and the remaining non-germinating conidia were plated on minimal medium with growth supplements and sorbose to induce colonization. After incubation isolations were made from individual colonies to test-tubes and identity tests were run. Fifteen different kinds of mutant strains were isolated, with arginineless mutants being recovered most efficiently and nicotinicless mutants with the greatest difficulty. When platings were made on complete agar instead of on minimal + supplements, a number of mutants did not grow on any of the test media, indicating a complex or unusual growth pattern. Use of the filtration method with X-rays gave results comparable to those with UV radiation. In order to test the efficiency of the filtration and selective plating method for the recovery of mutants, synthetic mixtures were made using wild-type and one of several known mutant strains. By hemo-cytometer counts the proportion of mutant conidia was adjusted to approx. 1% of the total no. of conidia incubated. At the start of incubation and every 24 hrs. thereafter aliquots of incubation medium were plated to determine changes in proportions of viable wild-type and mutant conidia. These tests showed that mutant conidia as well as wild-type conidia were reduced in numbers but at a slower rate, and the proportion of mutant conidia was greatly increased. The advantages and limitations of the filtration and selective plating method are discussed and possible causes for loss of mutant spores in the synthetic mixtures are set forth.