FRACTIONATION OF DIFFERENT LIFE-CYCLE STAGES OF MICROSPORIDIA NOSEMA-GRYLLI FROM CRICKETS GRYLLUS-BIMACULATUS BY CENTRIFUGATION IN PERCOLL DENSITY GRADIENT FOR BIOCHEMICAL-RESEARCH

A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli, parasitizing the fat body of c ricket Gryllus bimaculatus, by centrifugation in Percoll density gradi ent is elaborated. The whole procedure can be summarized as: 1) infect ed fat body preparation, 2) homogenization in buffer and filtration th rough cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different l ife cycle stages, 4) second centrifuging of the chosen layer in Percol l density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour a nd may become a routine procedure for biochemical studies on microspor idia.