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 degrees C for <or=90 min to boiling for 1 min. Results presented here indicate that temperatures are best kept to <or=70 degrees C and holding times need not exceed 15-30 min. Under conditions of 60 degrees C for 90 min, 62-23 degrees C for 15 min and 70 degrees 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.