Colorless Agar for Enhancing Color Contrast between Colonies and Solid Medium

2017-09-14T02:22:29Z (GMT) by Wenfa Ng
<p><strong>Background and purpose</strong>: Solid medium is primarily used in viable cell counting and profiling of microbial diversity in various environments. Nevertheless, lack of color contrast between colonies and agar background (usually beige for commercial complex media) hampers colony identification by automated image analysis, which increasingly is replacing the tedious and time-consuming manual cell counting approach. Theoretically, a colorless agar layered on colored paper of suitable hue offers a simple method for tuning color contrast between colony and agar background. But, reaction between carbohydrate moieties (e.g., glucose, yeast extract, and agar) and ammonium compounds (e.g., ammonium chloride) during autoclave sterilization forms colored compounds, and thus, hampers colorless agar preparation.</p> <p><strong>Main conclusion(s):</strong> By sterilizing glucose and ammonium compounds separately, this work demonstrated the feasibility of preparing colorless agar using heat sterilization and standard agar preparation technique. Yeast extract (separately sterilized) could be added (up to 1 g/L, without introducing significant coloration) for supporting environmental isolates unable to synthesize essential vitamins. Reconstituted agar had similar viscosity to common agar; thus, allowing the reproducible preparation of homogeneous agar medium in standard Petri dishes. Growth performance assays in both liquid and solid medium format revealed good growth of common bacteria (<em>Escherichia coli</em> DH5α, <em>Bacillus subtilis</em> ATCC 8473, <em>Pseudomonas protegens</em> Pf-5 and <em>Pseudomonas aeruginosa</em> PRD-10). Diverse subset of microbes isolated on colorless agar from DI water inoculum further revealed the agar’s capability at supporting myriad microbes at relatively high plating density. Similar viable cell counts on LB Lennox and colorless agar for <em>P. protegens</em> Pf-5 suggested that growth inhibitory compounds were not produced during agar preparation. Collectively, segregating glucose, yeast extract, and agar powder from ammonium compounds during autoclave sterilization was shown to be critical for preparing colorless agar with high optical transparency and better color rendition. And when layered on colored paper of desired hue, the colorless agar afforded a simple method for tuning color contrast between colonies and agar, important for improving colony identification by automated image analysis.</p>