• Clostridium perfringens;
  • spore germination;
  • wet heat inactivation;
  • spores;
  • dipicolinic acid



To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens.

Methods and Results

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl. perfringens with various germinants. When incubated in water at 90–100°C for 10–30 min, more than 90% of spores were inactivated but 50–80% retained their Ca2+-dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640–1680 cm−1 (amide I) and 1230–1340 cm−1 (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of Tlag, ΔTrelease and ΔTlys, during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer Tlag and ΔTrelease values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased Tlag, ΔTrelease and ΔTlys values, as did wet heat-treated sleC spores germinating with dodecylamine.


(i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat.

Significance and Impact of the Study

This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.