5. Disrupting of Fungus and Actinomycetes Cells with Cryo Clean Blaster(CCB)(Papers presented at the 36th Annual Meeting, April, 1990, Tokyo)

Abstract

Clean Cryo Blaster(CCB) is a system to disrupt cells by colliding small particles of frozen cell suspension against a metal target panel blowing with pressurised nitrogen gas flow of sonic speed. Cell suspension is rapidly frozen by spraying through nozzle into the atmosphere of ultralow temperature. CCB has hardly been applicable for most of fungi and actinomycetes which form large aggregates of filamentous cells in liquid culture, because aggregates plugged up the orifice of the spray nozzle. In order to disperse cells a modification was deviced which uses high pressure(60 kg/cm^2)for spraying. With the modified system the aggregates were dispersed into small and fairly well isolated filamentous cells, and the cells were disrupted into small fragments by the following CCB treatment. Long strands of DNA of the length over 23kbp were recovered from Penicillium chrysogenum, Streptomyces griseus and Streptomyces coelicolor. Sixteen S and 23S rRNAs were recovered from streptomysetes which appeared to be intact from electrophoregrams. The yield of RNAs from S. coelicolor was a little higher than that obtained by conventional lysis method. It was suggested that ultralow temperature throughout the disrupting process would be effective for preventing RNA from hydrolysis by indigenous or contaminating RNase.