Method for removal of vegetative cells from Bacterial spore preparations.

One of the more difficult and important problems encountered in analytical work on bacterial spores is the removal of the nonsporulating and unlysed vegetative cells found in all cultures which have attained maximum sporulation. A number of methods reported in the literature which have given satisfactory removal of vegetative cells of a number of species gave no appreciable separation of the spores and cells of Bacillus stearothermophilus NCA numbers 1518 and 7900, or Bacillus cereus. Reported methods which were here unsuccessful included attempted destruction of the vegetative cells by alteration of the osmotic environment, autolysis in distilled water (Church et al., J. Bacteriol., 68, 393-399, 1954), slow freezing and thawing (Stewart and Halvorson, J. }acteriol., 65, 160-166, 1953), and separation by differential centrifugation in plain water or in high viscosity, aqueous solutions of sucrose. Satisfactory purification of spore material was accomplished by the procedure given here. An advantage of the method lies in the use of commonly available laboratory equipment. The B. stearothermophilus strains were grown in the tryptose-basamine-dextrose broth of Long and Williams (Bacteriol. Proc., 1957, 15, 1957). B. cereus was grown in a glucose-yeast extractglutamic acid broth. After maximum obtainable sporulation had occurred, the spores and remaining vegetative cells were removed from the growth media by centrifugation at 2000 rpm for 1 hr in the cold. The supernatant was carefully removed with sterile pipettes and discarded. The residues were then dispersed by shaking vigorously with glass beads in cold sterile distilled water. The spore and cell materials were again packed by centrifu-