PROCEDURE FOR CLEANING OF CLOSTRIDIUM BOTULINUM SPORES

Abstract

Liberation of clean spores from vegetative sporangia of Clostridium botulinum strains was accomplished by the use of lytic enzymes and sonic oscillation. Suspensions of crude spores in phosphate buffer (pH 7) were digested with lysozyme (200 [mu]g/ml) and trypsin (100 [mu]g/ml). Rapid lysis of sporangia was induced by ultrasonic oscillation of the reacting mixture at 10 kc for 5 min. at 0, 0.5, 1, 2, 4 and 6 hr. of incubation at 45 C. Intermittent washing of the reacting spore suspension with a solution of lysozyme and trypsin hastened purification of the spore crop. The cleaning procedure was completed by repeated washing of the spores with distilled water. The spores produced by this procedure were clean as judged by their microscopic appearance, retractility to staining, loss of heat-sensitive toxin and partition behavior in a two-phase system composed of polyethylene glycol and 3 M potassium phosphate buffer (pH 7.1). The cleaning procedure appeared not to affect the viability, resistance to heat and gamma radiation or the toxic nature of C. botulinum spores.