As a well characterized prototrophic derivative of B. subtilis 168
strain, B. subtilis PS533 spores (kindly donated by Dr. P. Setlow,
University of Connecticut) were used in all inactivation experiments.
Prior to their use, spores were maintained in Microbank
(Pro-Lab Diagnostics, Canada) at 80 C. Initial pure culture was
grown overnight in lactose broth (LB) medium (Oxoid, Hampshire,
UK) at 37 C, 150 rpm. Sporulation was made according to the
method described by Nicholson and Setlow (1990) in 1 L flasks
filled with 200 ml liquid 2 SG medium at 37 C, 225 rpm within
5e7 days. Harvested spores were centrifuged and washed multiple
times until the resulting suspension had >98% purity. Presence of
germinated spores or cell debris was determined with phase
contrast microscopy. Spores were suspended in sterile MiliQ water
(1010e1011 spores/ml) and stored in the refrigerator without
exposure to light for a limited time