Method for Obtaining Cleaned Putrefactive Anaerobe 3679 Spores.

Sporulating clostridial suspensions tend to recycle through subsequent phases such as germination, outgrowth, and further multiplication. The result is a mixture of vegetative cells and of spores at various developmental stages. Recently, activated-culture techniques have been used to achieve almost complete sporulation in semisynchronized populations of several clostridia (Collier, 1). 10, in H. 0. Halvorson [ed.], Spores, American Institute of Biological Sciences, Washington, D.C., 1957; Zoha and Sadoff, J. Bacteriol. 76:203, 1958). Recycling was prevented either by lowering the temperature or by aerating the culture medium after sporulation. Vegetative cells and debris have been removed from spore suspensions by various means (see Greez, Anellis, and Schneider, J. Bacteriol. 84: 552, 1962), including centrifugation, partitioning in two-phase polymer systems, lytic enzymes, ultrasonic treatment, autolysis, and by combinations of these methods. This communication describes a useful procedure for growing and cleaning suspensions of ungerminated Putrefactive Anaerobe (PA) 3679 spores. Preparation of spore crops. PA 3679h, a highly sporogenic variant strain isolated by Lund, Janssen, and Anderson (J. Bacteriol. 74:577, 1957), was used throughout. Incubation was at 35 C under nitrogen in an anaerobic incubator. The culture medium consisted of 6% Trypticase and 0.1% glucose (pH 7.0). Agitation of the medium during incubation was unneces-