Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity |
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Authors: | Harrold Zoë R Hertel Mikaela R Gorman-Lewis Drew |
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Institution: | Earth and Space Science Department, University of Washington, Box # 351310, Seattle, WA 98195, 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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Keywords: | Bacillus subtilis Endospore Isolation Purification Separation |
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