Aims:  To determine the mechanism of wet heat killing of spores of Bacillus cereus and Bacillus megaterium. Methods and Results:  Bacillus cereus and B. megaterium spores wet heat‐killed 82–99% gave two bands on equilibrium density gradient centrifugation. The lighter band was absent from spores that were not heat‐treated and increased in intensity upon increased heating times. These spores lacked dipicolinic acid (DPA) were not viable, germinated minimally and had much denatured protein. The spores in the denser band had viabilities as low as 2% of starting spores but retained normal DPA levels and most germinated, albeit slowly. However, these largely dead spores outgrew poorly if at all and synthesized little or no ATP following germination. Conclusions:  Wet heat treatment appears to kill spores of B. cereus and B. megaterium by denaturing one or more key proteins, as has been suggested for wet heat killing of Bacillus subtilis spores. Significance and Impact of the Study:  This work provides further information on the mechanisms of killing of spores of Bacillus species by wet heat, the most common method for spore inactivation.

Aims:  To analyse the germination and its heterogeneity of individual spores of Clostridium perfringens. Methods and Results:  Germination of individual wild‐type Cl. perfringens spores was followed by monitoring Ca‐dipicolinic acid (CaDPA) release and by differential interference contrast (DIC) microscopy. Following the addition of KCl that acts via germinant receptors (GRs), there was a long variable lag period (Tlag) with slow release of c. 25% of CaDPA, then rapid release of remaining CaDPA in c. 2 min (ΔTrelease) and a parallel decrease in DIC image intensity, and a final decrease of c. 25% in DIC image intensity during spore cortex hydrolysis. Spores lacking the essential cortex‐lytic enzyme (CLE) (sleC spores) exhibited the same features during GR‐dependent germination, but with longer average Tlag values, and no decrease in DIC image intensity because of cortex hydrolysis after full CaDPA release. The Tlag of wild‐type spores in KCl germination was increased significantly by lower germinant concentrations and suboptimal heat activation. Wild‐type and sleC spores had identical average Tlag and ΔTrelease values in dodecylamine germination that does not utilize GRs. Conclusions:  Most of these results were essentially identical to those reported for the germination of individual spores of Bacillus species. However, individual sleC Cl. perfringens spores germinated inefficiently with either KCl or exogenous CaDPA, in contrast to CLE‐deficient Bacillus spores, indicating that germination of these species’ spores is not completely identical. Significance and Impact of the Study:  This work provides information on the kinetic germination and its heterogeneity of individual spores of Cl. perfringens.

Aim:  To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results:  Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (Tlag and Trelease times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔTrelease times (Trelease−Tlag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions:  Since (i) ΔTrelease times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in Tlag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter Tlag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study:  This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.

Aims:  To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results:  Bacillus megaterium was sporulated with no added MnCl2 and up to 1 mmol l−1 MnCl2. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H2O2. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions:  Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study:  The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.

Aims: To determine how hydrated Bacillus anthracis spores are killed in a high-temperature gas environment (HTGE), and how spores of several Bacillus species including B. anthracis are killed by UV radiation, dry heat, wet heat and desiccation. Methods and Results: Hydrated B. anthracis spores were HTGE treated at c. 220°C for 50 ms, and the treated spores were tested for germination, mutagenesis, rupture and loss of dipicolinic acid. Spores of this and other Bacillus species were also examined for mutagenesis by UV, wet and dry heat and desiccation. There was no rupture of HTGE-treated B. anthracis spores killed 90-99Á9%, no mutagenesis, and release of DPA and loss of germination were much slower than spore killing. However, killing of spores of B. anthracis, Bacillus thuringiensis and Bacillus subtilis by UV radiation or dry heat, but not wet heat in water or ethanol, was accompanied by mutagenesis.Conclusions: It appears likely that HTGE treatment kills B. anthracis spores by damage to spore core proteins. In addition, various killing regimens inactivate spores of a number of Bacillus species by the same mechanisms. Significance and Impact of the Study: This work indicates how hydrated spores treated in a HTGE such as might be used to destroy biological warfare agent stocks are killed. The work also indicates that mechanisms whereby different agents kill spores are similar with spores of different Bacillus species.