Significance and Impact of the Study: Surfactants with emulsifying bioactivity are known to be produced by Bacillus subtilis. Here, a colorimetric assay for methylene blue active substances is adapted for use in bacterial plaque to describe surfactant bioactivity, and supplemental salts of calcium during culture are shown to enrich cultured plaque for said bioactivity. Where B. subtilis is utilized as a feed additive to food producing animals, the manufacture of bacterial plaque with controlled and enriched concentration of surfactant bioactivity is desirable, especially where the dose amount is limited by mass, total colony forming units, enzymatic activities or other limiting qualities.