Permeabilization of Beauveria bassiana blastospores for in situ enzymatic assays

Vigorous agitation of an aqueous suspension of blastospores (BS) of Beauveria bassiana mixed with nine volumes of a 1:4 (v/v) mixture of toluene: ethanol (95%) for 10 min permits blastospore permeabilization. Agitation results in greater membrane permeabilization than heating blastospores in the presence of toluene:ethanol or the detergents Triton X-100, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide and Brij-35. The beta-galactosidase activity in permeabilized blastospores was determined with these methods. The effectiveness of permeabilization in detecting enzyme activity was assessed by comparison to whole BS lysates prepared by mechanical disruption and pressurized disruption of BS biomass. The toluene:ethanol method was applied to study the incorporation of H-3-thymidine triphosphate into blastospore DNA. Whole BS permeabilization allows the examination of enzyme activity and DNA synthesis at a cellular level in this important mycoinsecticide.