Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores

Assessing true numbers of viable anthrax spores is complex. Optimal heat activation conditions vary with species, media and germinants. Published time/temperature combinations for Bacillus anthracis spores range from 60 °C for ≤90 min to boiling for 1 min. Results presented here indicate that temperatures are best kept to ≤70 °C and holding times need not exceed 15–30 min. Under conditions of 60 °C for 90 min, 62–23 °C for 15 min and 70 °C for 15 or 30 min, although the ratio of heated:unheated counts ranged from <1 to >1, post-heating counts were less than their pre-heating counterparts on between 71% and 88% of occasions. A high probability was found of viable spore counts differing significantly from counts determined microscopically, with differences of almost 1 log possible. Viable counts were lower than microscopic counts in 15 of 18 tests.