Lack of Influence of Suspending Media on Heat Activation of Bacillus subtilis Spores and Absence of Deactivation1

BiUSTA, F. F. (U.niversity of Illinois, UTrbana), AND Z. -JOHN ORDAL. Lack of influence of suspending media on heat activation of Bacillus subtilis spores and absence of deactivation. Appl. Microbiol. 12: 111-114. 1964.-Bacillus subtilis strain 5230 endosl)ores suslended in a variety of suspending media at a concentration of ca. 108 per ml were heated at 95 and 75 C. The effect of the heating at 75 C was measured by plate count, and was reported as the heat-activated decimal fraction of the total viable-spore lpopulation. Thermal inactivation at 95 C was influenced by the suslending medium. No effects on heat activation at 75 C were noted for suspending media containing glucose, xylose, ribose, NaCl, or sodium phosphate, nor was there any marked effect due to a change in pH from 5 to 8. The heatactivation response was retained during postheating storage at 5 C in water ul) to 215 (lays. Postheating storage in several susp)ending media for 7 (lays also indicated no deactivation. A numlber of investigators reported on the influence of miedia used to suspend bacterial spores for a heat-activatioln tIeatmnent (CurraIn and EvaIns, 1945; Brachfeld, 1955; Treadwell, ,Janni, and Salle, 1958; Hermiier, 1958; Splittstoesser and Steinkraus, 1962; Finley and Fields, 1962). The reported effects have been varied and numerous. The effects of suspending miedia on heat activation generally were miieasured by plate counts, and the differenices in heat-activation responises due to the suspending miedia were based on the differenices in plate counts after heating. Simiiilar plate counit aiialyses were used to report the loss of the heat-activatioil responise during postheating storage. This deactivation and also subsequent reactivation was reported by Curran and Evans (1947), Brachfeld (1955), and Desrosier and Heiligmuan (1956). The present investigation was designed to evaluate past reports on the effect of the suspending media on heat activation, and to evaluate the occurrence of deactivation, by emiiploying new miiethods for the measurement of heat activation, namely, the use of the heat-activated decimal fraction of the total numiiber of viable spores (Busta and Ordal, 1964). l Based on part of a dissertation submaitted by the senior author in partial fulfillment of the requirements for the Ph.). degree in Food Science, University of Illinois. 2 Predoctoral Fellow, National Institutes of Health, IT.S. Public Health Service. Present address: l)epartment of Food Science, North Carolina State College, Raleigh. M\IATERIALS AND ]METHODS The test culture, recovery media, miethod of heatinig, and especially the formulation and use of CaDPA-TGE were described previously (Busta and Ordal, 1964). Preparation of the spore suspension also was described previously (Busta and Ordal, J. Food Sci. in press). Measurement of heat activation. The amount of heat activation is reported as a deciimial fraction, i.e., the n1uimiber of heat-activated spores divided by the total nuimber of viable spores. Standard plate count mlethods were used throughout. The diluent was sterile deionized distilled water. Spore suspensions were heated at concentratioiis of ca. 108 spores per nml. Subsequent dilutions were such that ca. 100 spores were deposited into each plate. For each samiiple, 13 plates were prepared. The average counts from ten plates with fortified TGE eniumnerated the heatactivated number, and the average counts froml three plates with CaDPA-TGE eniumiierated the total viable number. Plates were incubated for 24 hr at 37 C. Suspending media. In all instances, the water was sterile, deionized, distilled water. The NaCl solutions conitained only salt and water. The phosphate buffer was made fromii the sodiumii salts and used at pH 7.0 in inost instances (Gomnori, 1955); however, phosphate buffers were adjusted to pH 5.2, 6.0, 7.2, and 8.0 for one trial. The study of heating at 95 C enmployed ribose, xylose, and glucose solutionis prepared with water anid autoclaved pIrior' to use, as were the salt solutions. The sugar solutions used in heatiing at 75 C were prepared by adding the sugar to sterile phosphate buffer with nio additional heat treatmiient prior to inoculationi.