PREPARATION OF A CLEAN SAMPLE OF BACTERIAL SPORES

Abstract

For the preparation of clean bacterial spore samples, cultural conditions were examined under which the least possibility of contamination of vegetative cells and of non-refractile spores could be attained. The cultivation of bacilli on the nutrient agar of 5mm in thickness for 3 days was found to be favorable for this purpose.
For the purification of spores, an improved method of SACKS and ALDERTON was adopted. After analyzing the composition of the upper layer of their Y-system, a single layer system of 25% Carbowax 4000 solution in 0.5M phosphate buffer (pH 7.1) was found to be favorable solvent for the separation of spores and vegetative cells. When impure sample was mixed vigorously with this solvent and centrifuged at 600×g for 30min, spores remained homogeneously dispersed in the supernatant, while vegetative cells sedimented at the bottom. Recommended procedure for the purification of bacterial spores is described.