◆ SpookStack

HE In UNCLASSIFIED DATE Le-11-2008 BY 60324 uc bawyrs/mis @ ‘ ALL JN FORMATION CONTAINED Heat Stability of SPS 02.266 The object of this study is to compare the heat stability (at 70°C in a water bath) of SPS 02.266 spores (in water for injection, WFI) with the stability of spores from the following B. anthracis strains: Ames (from Dugway, 1997), 488 (from England), 462 (Ames strain passed through a guinea pig, from Porton Down, England). The spores from the four preparations will be diluted to about 5 X 10° CFU/ml in WFI. A "0 time" sample will be diluted in WFI to approximately 5 X 10? CFU/ml and plated onto Tryptic Soy Agar plates (TSA). The spore preparations will then be incubated for 1, 6, and 12 hours. After dilutions are made in WFI, one-tenth-ml aliquots will be plated out for 1:1,000 and 1:10,000 dilutions of the heated spore preparations. All plates will be incubated overnight at 37°C. Counts will be made and the heat stability will be determined and expressed as the length of time at 70°C required for a 50% drop in viability. Preparations to be examined: Approximate Initial Concentration 1 = Dugway Ames spores 3.5 X 10% CFU/ml 2 = SPS 02.266 spores 1X 101 CFU/ml 3 = 488 spores 9 X 108 CFU/ml 4 = 462 Ames spores 4.6 X 10’ CFU/ml Dilution Schemes in tubes of WFI (to get to 5 X 10° CFU/ml): . 1 - (Dugway spores) - 0.1 ml into 9.9 ml, mix, then 0.1 ml into 6.9 ml 2 - (SPS 02.266 spores) - 0.1 ml into 9.9 ml, mix, then 0.5 ml into 9.5 ml 3 - (488 spores) - 0.1. ml into 9.9 ml, mix, then 5.0 ml into 4.0 ml 4 - (462 spores) - 1.0 ml into 8.2 ml At each time point (0 hours,1 hour, 6 hours, 12 hours), dilute as follows: 0.1 ml into 9.9 ml, mix; from this dilution, put 1.0 ml into 9.0 mi, mix; from this dilution put 1.0 ml into 9.0 ml and mix. For each of the last two dilutions, dispense 0.1 ml onto 5 TSA plates. Spread the plates to dryness with a glass spreader, then incubate overnight. Count the colonies on the plates.