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Applied and Environmental Microbiology, May 2009, p. 2712-2719, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02572-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic Clostridium botulinum Bacteria{triangledown}

Sandra C. Stringer,* Martin D. Webb, and Michael W. Peck

Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom

Received 11 November 2008/ Accepted 22 February 2009

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (tgerm), one mature cell (tC1), and two mature cells (tC2) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the tgerm, the outgrowth time (tC1 – tgerm), and the first doubling time (tC2 – tC1). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (tC1) and outgrowth (tC1 – tgerm) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.

* Corresponding author. Mailing address: Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. Phone: (44)1603 255000. Fax: (44)1603 255288. E-mail: sandra.stringer{at}bbsrc.ac.uk

{triangledown} Published ahead of print on 6 March 2009.

Applied and Environmental Microbiology, May 2009, p. 2712-2719, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02572-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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