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Hamouda et al., Sporicidal Activity of BCTP Nanoemulsion Page 3 of 12 Surfactant lipid preparations. BCTP is a water-in-oil nanoemulsion, in which the oil phase is made from soybean oil, tri-n-butyl phosphate, and Triton X-100. Stock solutions contained 80% lipid components and 20% water. Three different preparations of BCTP, 2, 8, and 16 months old, were tested for their stability. BCTP 401 was prepared by mixing equal volumes of BCTP with P10, the latter being a liposome-like compound. P10 is made of glycerol monosterate, refined soya sterols, Tween 60, soybean oil, a cationic ion halogen-containing cetylpyridinium chloride, and peppermint oil. The average size of these nanoemulsions is in the range of 400-800 nm, as determined by laser light scatter (LS230; Coulter, Hialeah, FL). These surfactant lipid preparations were stable after boiling for 1 h or exposure to 1 Nnitric acid or 1 N sodium hydroxide for 2 h. This treatment resulted in a <20% reduction in the emulsion mean particle size [16]. These solutions were stored at room temperature and were diluted before each experiment to the working dilution. All dilutions herein are in reference to the stock solution. Spore preparation. For induction of spore formation, B. cereus (ATCC 14579), B. circulans (ATCC 4513), B. megaterium (ATCC 14581), and B. subtilis (ATCC 11774) were grown for 1 week at 37°C on nutrient agar with 0.1% yeast extract and 5 mg/L MnSO,. The plates were scraped, and the bacteria and spores were suspended in sterile 50% ethanol and incubated at 22°C for 2 h with agitation to lyse the remaining vegetative bacteria. The suspension was centrifuged at 2500 g for 20 min, and the pellet was washed twice in cold distilled water. The spore pellet was resuspended in trypticase soy broth (TSB) and used immediately for experiments. B. anthracis spores, Ames and Vollum 1B strains, were supplied by Bruce Ivins (US Aimy Medical Research Institute of Infectious Diseases [USAMRID}, Fort Detrick, Frederick, MD) and were prepared as described elsewhere [5]. Four other strains of B. anthracis were provided by Martin Hugh-Jones (Louisiana State University, Baton Rouge). These strains (from South Africa; Mozambique; Bison, Canada; and Del Rio, TX) represent isolates with high allelic dissimilarity. In vitro sporicidal assays. For assessment of sporicidal activity on solid medium, trypticase soy agar (TSA) was autoclaved and cooled to 55°C. BCTP was added to the TSA at a1 : 100 final dilution and continuously stirred while the plates were poured. The spore preparations were serially diluted (10- fold), and 10-#L aliquots were plated in duplicate (highest inoculum, 10° spores/plate). Plates were incubated for 48 h aerobically at 37°C and evaluated for growth. For assessment of sporicidal activity in liquid medium, spores were resuspended in TSB. Next, 1 mL of spore suspension containing 2 x 10° spores (final concentration, 10° spores/mL) was mixed with 1 mL of BCTP or BCTP 401 (at 2x final concentration in distilled water) in a test tube. The tubes were incubated in a tube rotator at 37°C for 4 h. Treatment of B. anthracis was done at 37°C, which promotes spore germination, and at 22°C, which does not promote spore germination [5]. After treatment, the suspensions were diluted 10-fold in distilled water. Duplicate aliquots from each dilution were then streaked on TSA and incubated overnight at 37°C; then colonies were counted. Sporicidal activity expressed as percentage of killing was calculated as follows: {[cfu(initial) - cfu(posttreatment)]/[cfu (initial)]} x 100. The experiments were repeated at least 3 times, and the mean and SE of the percentage of killing http://www journals.uchicago.edu/JID/ ournal/issues/v180n6/990281/990281 text html 2/18/2005