The Effect of Water Activity, Solutes and Temperature on the Viability and Heat Resistance of Freeze-dried Bacterial Spores

Abstract

SUMMARYFreeze-dried spores of Bacillus megaterium, B. stearotkmphilus, Clostridium bifermentaw and C . botulinum type E suffered little or no loss in viability after storage a t 25" at water activity (a,) values between 0-2 and 0.8. When stored over P,O, (0.00 a,) the spores of all four species showed a marked loss in viability. The above results were similar for spores whether stored in air or in vacuum. With spores stored over distilled water (1.00 a,) the Bacillus spores underwent a large loss of viability in vacuum, but not in air; for spores of the clostridia the reverse was true. The addition of DL-glyceraldehyde, diacetyl or ribose (0.05 M) to the spore suspensions before drying caused increased death during storage at 0.50 aw and to a lesser extent a t 0.20 a,. Death was greater at 30' than at 10". The addition of sucrose, glutamate or semi-carbazide did not decrease the viability. When the dried spores were resuspended in dilute phosphate buffer after storage for 2-6 years their resistance to heating was greatest after storage at a, values of 04, 0.6 and 0.8.

The Heat Resistance of Bacterial Spores at Various Water Activities

“…2). We found previously (Marshall et al 1963) that freeze-drying and storage of the dried spores damaged a proportion of the spores so that they became stainable on resuspension and heat labile, but not necessarily non-viable. The present results suggest that the damaged spores are also more susceptible t o destruction by dry heat.…”

“…6). The corresponding water contents of spores a t 0.2-0-4 a, are of the order of 5-10y0 of the dry weight (Marshall et al 1963). There is, of course, no certainty that the a, value or water contents which give greatest heat resistance will be the same for all species.…”

“…The spores of Bacillus megaterium (Knaysi, Strain cl), B. stearothermophilus ATCC 7953, and Clostridium botulinum type E ATCC 9564 were grown, harvested and enumerated as described by Marshall, Murrell & Scott (1963). Similar methods were used for the other three organisms except that C. botulinum type B ATCC 7949 was grown in peptic digest medium (Dubovsky & Meyer, 1922) Equilibration and control of a,.…”

(Symposium on Bacterial Spores : Paper IX). Biophysical Analysis of the Spore

“…Direct experimental evidence of the permeability of spores to water is based on two lines of evidence: (i) most of the water of a spore pellet equilibrates in concentration with external heavy water (Murrell & Scott, 1958;Black & Gerhardt, 1962). An early summary of our experiments on water permeability has been reported (Murrell 6 Scott, 1958) and discussed elsewhere (Murrell, 1961); (ii) there appears to be no restrictions to the movement of water vapour in or out of the spore (Marshall et al, 1963;Murrell & Scott, 1966;Murrell, 1967;Neihof et al, 1967). The results of the first method will be affected by exchange occurring between the protons of the spore and deuterons or tritions of the labelled water.…”

“…It is possible that very heat resistant species may bind more water or water more strongly a t certain a, than do the less resistant species. The averaged data for 6 species differing considerably in heat resistance has been tabulated already (Marshall et al, 1963). These results together with data for disrupted spores and some spore fractions are reported in this paper.…”