title: Optimizing Bacillus subtilis spore isolation and quantifying spore harvest purity.
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.
authors: Earth and Space Science Department, University of Washington, Seattle, WA 98195, United States. zharrold@uw.edu
authors: Zoë R
authors: ZR
authors: Harrold
authors: Mikaela R
authors: MR
authors: Hertel
authors: Drew
authors: D
authors: Gorman-Lewis
mesh_terms: D001331:Automation / Q000379:methods; D001412:Bacillus subtilis / Q000302:isolation & purification*; D058491:Bacterial Load / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008853:Microscopy / Q000379:methods*; D013171:Spores, Bacterial / Q000302:isolation & purification*