Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity |
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Affiliation: | 1. U.S. Environmental Protection Agency, National Homeland Security Research Center, 26 W. Martin Luther King Drive, MS NG16, Cincinnati, OH 45268, United States;2. Pegasus Technical Services, Inc., 46 East Hollister St., Cincinnati, OH 45219, United States;3. U. S. Geological Survey, Coastal and Marine Science Center, 600 4th Street South, St. Petersburg, FL 33701, United States;4. U.S. Environmental Protection Agency, National Homeland Security Research Center, 1200 Pennsylvania Avenue, N.W., MS 8801R, Washington D.C. 20460, United States |
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Abstract: | Investigating the biochemistry, resilience and environmental interactions of bacterial endospores often requires a pure endospore biomass free of vegetative cells. Numerous endospore isolation methods, however, neglect to quantify the purity of the final endospore biomass. To ensure low vegetative cell contamination we developed a quality control technique that enables rapid quantification of endospore harvest purity. This method quantifies spore purity using bright-field and fluorescence microscopy imaging in conjunction with automated cell counting software. We applied this method to Bacillus subtilis endospore harvests isolated using a two-phase separation method that utilizes mild chemicals. The average spore purity of twenty-two harvests was 88 ± 11% (error is 1σ) with a median value of 93%. A spearman coefficient of 0.97 correlating automated and manual bacterial counts confirms the accuracy of software generated data. |
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