4. Cryo-disruption of Bacterial Cells by Cryo Clean Blaster(CCB)(Lectures presented at the Meeting on the 30th Anniversary of Japanese Society for Research of Freezing and Drying(in part))

Abstract

The efficiency and the mode of bacterial cell disruption was investigated for a newly developed cell disrupting apparatus, Cryo Clean Blaster(CCB), which collides rapidly frozen cells onto a hard metal surface with pressurized nitrogen gas flow of sonic speed. The yield of high molecular weight DNA extracted by phenol reagent treatment followed by CCB cell disrupting procedure was used as index for cell disruption. In Escherichia coli cells from both logarithmic and stationary phases, the yield of purified DNA was twice as much as those from cells disrupted by lysis method which employed lysozyme and sodium dodecyl sulfate. Hyperchromicity of DNA was about 40% and the length of DNA strands was over 23kbp. The original shape of cells was almost maintained after CCB treatment as observed by phase contrast and electron microscopy and their appearance was like a"ghost"cell. It was supposed that the mode of cell disruption was such that the cell constituents were extruded without disrupting cells into small debris. In the cells of Bacillus subtilis and Lactobacillus plantarum from both logarithmic and stationary phases, DNA yields were as high as those by lysis methods. Lysozyme treatment of cells followed by CCB disrupting procedure improved DNA yield to about twice. Two-stepped disrupting device, which collides the once collided cells onto another metal surface gave the DNA yield thrice as much as that by lysis method in Arthrobacter globiformis cells from both logarithmic and stationary phases.