PROBLEMS ACCOMPANYING THE SEPARATION OF ZYGOSPORES OF THE ALGA CHLAMYDOMONAS-GEITLERI (CHLOROPHYTA) BY DENSITY GRADIENT CENTRIFUGATION

As the yield of zygospores from laboratory cultures of the green alga Chlamydomonas geitleri Ettl was usually very low (no more than 30-40%, only exceptionally 60%), it was necessary to search for a method of separation of zygospores from culture suspensions. We investigated the technique of density gradient centrifugation for this purpose. For the density gradients we used both the osmotically inactive (Ficol 400, Percoll) and osmotically active (sucrose) chemical compounds. Zygospores of C. geitleri proved to be resistant to high concentrations of sucrose (up to 40%, w/w) to a relatively high degree. Although in many cases it was possible to achieve very pure fractions of zygospores using density gradient centrifugation, this method frequently did not work. It was apparently caused by the fact that a distinct difference between the buoyant density of zygospores and that of vegetative and other non-zygospore cells (which is a necessary precondition of successful use of the technique in question) is not an invariable characteristics. The present knowledge of its probable dependence on growth conditions is not sufficient to date so that the ratio of buoyant densities of zygospores and of other cells could be regulated. Alternative ways are discussed: the separation of zygospores of C. geitleri through the selective digestion of non-zygospore cells by mosquito larvae (described in another paper) and the selective destruction of non-zygospore cells by ultrasound treatment, which has been reported to be successful in purifying the zygospores of C. reinhardtii.