Heat Sensitization of Bacterial Spores After Exposure to Ethidium Bromide, Acriflavine, or Daunomycin

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.