Bacterial strains, media, and growth conditions

Clostridium tetani strains were obtained through the American type culture collection (ATCC) and maintained as glycerol stocks at -70°C. Strain C2, obtained by CBER in 1962 from a commercial vaccine manufacturer, was passaged once in 1965 and stored lyophilized in heat-sealed glass vials. Bacterial culture stocks were propagated in thioglycollate media containing resazurine (Sigma Aldrich, USA) in anaerobic boxes (BioMerieux, Craponne, France) at 35°C. For tetanus toxin expression, 24 hour cultures were inoculated into Massachusetts medium as originally described in Latham et al. [17] containing NZ-Case TT (Kerry Bio-Science, Beloit WI) and harvested after 6 days. For swarming behavior and motility, 24 hr cultures were plated onto 1.5% Trypticase soy agar plates (TSAII, BD Biosciences, Franklin Lakes, NJ) containing 5% defibrillated sheep blood (Hemostat Laboratories, Dixon CA) and incubated at 30°C for 48 hrs before colonies were imaged.