Germination and outgrowth of Schizosaccharomyces pombe ascospores isolated by Urografin density gradient centrifugation

Abstract

A simple method for the isolation of single ascospores of the fission yeast Schizosaccharomyces pombe was examined. Single spores in the 7-day-old sporulating culture of a homothallic strain were separated from remaining vegetative cells by isopycnic centrifugation in the linear gradient from 10 to 60% of Urografin solution at 700 X g for 20 min. Protein content of isolated spores was very low as compared with that of vegetative cells. The isolated spores germinated through the following steps when cultured in a liquid medium at 25--35 degrees C; loss of refractility (darkening) under a phase-contrast microscope, spherical growth (swelling), emergence of germ tubes, elongation of germ tubes, cell plate formation, and cell separation. The absorbance at 650 nm of the spore suspension initially decreased, accompanied by darkening of spores, and then increased with spherical growth. The germination rate of isolated spores reached almost 100%.