Image-based Quantification of Surface-attached Saccharomyces cerevisiae Coverage on Silicone Following Enzyme and Hydrogen Peroxide Treatments

Abstract

The aim of this study was to develop a low-cost transmitted-light microscopy and Fiji/ImageJ workflow for quantifying visible methylene-blue-stained Saccharomyces cerevisiae-associated surface coverage on silicone and to compare water, hydrogen peroxide, bromelain, amylase, and combined enzyme treatments. Three sequential method-development trials were conducted; all quantitative treatment inference was based exclusively on the final flat-silicone trial (Trial 3), with five independent silicone samples per group. After 48 h of yeast exposure and treatment with distilled water, 1% hydrogen peroxide, bromelain (10 mg/mL), amylase (10 mg/mL), or combined bromelain/amylase (10 mg/mL total; 5 mg/mL of each preparation), samples were stained with methylene blue and imaged. Three fields were obtained per silicone sample and averaged to a sample-level percent-area value. Because variability differed substantially among groups, Welch's one-way analysis of variance was used, followed by two-sided Welch comparisons with the water control and Holm adjustment for multiplicity. Mean stained coverage was 1.9934% for water, 0.7474% for 1% hydrogen peroxide, 1.8520% for bromelain, 0.9847% for amylase, and 1.0146% for combined bromelain/amylase. Welch's ANOVA indicated an overall group effect, F(4, 9.46) = 5.803, p = .0124. Relative to water, hydrogen peroxide (Holm-adjusted p = .011) and amylase (p = .022) showed statistically lower stained coverage; bromelain (p = .831) and combined bromelain/amylase (p = .053) did not. The Triangle auto-threshold sensitivity analysis was descriptive and showed threshold-dependent absolute values with a broadly similar ordering of group means. The assay measures stained surface coverage and does not establish killing, sterilisation, or complete yeast removal.