It has been repeatedly postulated that the high heat resistance of bacterial spores is due to stabilization of biopolymers in the spore interior by a solid deposit of protective cement consisting of coordination complexes of ligands with divalent metal ions. This report presents data on metal-binding characteristics of some of the ligands related to spores as determined by means of potentiometric equilibrium measurements under conditions of temperature and ionic strength (t = 25.0 degrees C; mu = 1.0 KNO(3)) identical with those reported earlier by the authors in order to facilitate correlation by using comparable data. The spore ligands investigated in this study included 2,6-pyridinedicarboxylic acid (DPA), alpha,epsilon-diaminopimelic acid, D-glutamic acid, and D-alanine in a ratio of 1:1 with metal ions which are known to play a role in heat resistance of spores. Stability constants of the chelates of these spore ligands with metal ions such as Ca(II), Mg(II), Cu(II), Ni(II), Zn(II), Co(II), and Mn(II) have been determined. In general the metal chelates of DPA exhibited the greatest stability. On the basis of a consideration of the stability data together with the known configurations of the ligand and the coordination requirements of the metal ions, possible structures indicating the coordinate binding of the spore ligands with the metal ions are presented. All the metal chelates except those of Ca(II) were found to undergo hydrolysis and separation of solid phase in the pH range 7-8.5. The relatively greater hydrolytic stability of Ca(II) chelates and the high affinity of DPA for metal ions appear to be of biological significance insofar as these two spore components are more widely associated with the heat resistance of bacterial spores.

No abstract

No abstract

Cans of ground cooked beef, inoculated with 106 or 108 spores per can of Clostridium botulinum 33A, were irradiated with 60Co gamma rays at a series of 14 temperatures ranging from −196 to 95C. The higher inoculum level required higher sterilizing doses. The D values, computed on the basis of recoverable C. botulinum, were independent of the inoculum level, and showed that spore resistance progressively decreased with increasing temperature. A statistical analysis of these data disclosed that the change in D values from −196 to 65C followed equally well a quadratic, exponential, or linear best-fit plot; above 65C radiation death was much more rapid. An equation was derived from the linear plot to predict D values for any desired temperature between −196 and 65C. Calculations of Ea and Q10 values, based on the linear curve, indicated a very small thermodynamic effect on radiation kill. An Arrhenius analysis of the temperature effect suggested that there was no simple physicochemical mechanism occurring in the inoculated beef pack which might explain the change in spore kill as a function of temperature. Theoretical commercial radiation processes for beef, based on the 12D concept and strain 33A spores, are presented for several easily controlled irradiation temperatures.

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 pig/ml) and trypsin (100 ,ug/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 heatsensitive toxin, and partition behavior in a twophase system composed of polyethylene glycol and 3 M potassium phosphate buffer (l)H 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.