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J Bacteriol. 1962 September; 84(3): 552-558
Copyright © 1962, The Williams & Wilkins Company. All Rights Reserved.

PROCEDURE FOR CLEANING OF CLOSTRIDIUM BOTULINUM SPORES¹

^a Quartermaster Food and Container Institute for the Armed Forces, Quartermaster Research and Engineering Command, U.S. Army, Chicago, Illinois

ABSTRACT

GRECZ, N. (Quartermaster Food and Container Institute, Chicago, Ill.), A. ANELLIS, AND M. D. SCHNEIDER. Procedure for cleaning of Clostridium botulinum spores. J. Bacteriol. 84:552–558. 1962.—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 µg/ml) and trypsin (100 µg/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, refractility 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.

¹ This paper has been assigned number 2211 in the series of papers approved for publication. The views or conclusions contained in this report are those of the author(s). They are not to be construed as necessarily reflecting the views or indorsement of the Department of Defense.