Colourless Agar for Enhancing Colour Contrast between Microbial Colonies and Agar

Abstract

Lack of colour contrast hampers automated identification of colonies on agar, given that many microbial colonies are of the same colour as the background colour of most agar: beige colour. On the other hand, a colourless agar could increase the colour contrast between the agar background and microbial colonies. But, the challenge in preparing a colourless agar comes from the formation of coloured compounds when sugars and ammonium compounds are sterilized together in an autoclave. Hence, by separating glucose and ammonium compounds into different solutions for autoclave sterilization, a method was developed for preparing colourless agar that remained colourless even with 1 g/L of yeast extract supplementation. Specifically, three separate solutions were used in reconstituting the colourless agar at ~48c after individual sterilization. Solution A comprised glucose, MgSO4 and agar powder; Solution B contained NH4Cl, K2HPO4, KH2PO4, and NaCl; and Solution C was yeast extract solution for providing vitamins and trace elements needed by microorganisms unable to grow in minimal medium. Reconstituted colourless agar could be poured into agar plates using standard techniques and had a viscosity similar to many commercial agar. Composition of the colourless agar medium was [g/L]: D-Glucose, 2.0; NH4Cl, 0.5; K2HPO4, 0.5; KH2PO4, 0.1; NaCl, 0.5; MgSO4.7H2O, 1.0; Yeast extract, 1.0; Agar, 15.0. On observation against varied background images, the formulated colourless agar lacked the yellow tinge present in LB Lennox agar and had greater optical transparency. Good growth of common bacteria such as Escherichia coli DH5α (ATCC 53868), Bacillus subtilis NRS-762 (ATCC 8473) and Pseudomonas protegens Pf-5 (ATCC BAA-477) was observed in both liquid and solid versions of the formulated colourless agar medium. Specifically, colonies of E. coli DH5α, B. subtilis NRS762 and P. protegens Pf-5 exhibited similar morphology and characteristics compared to growth on LB Lennox agar. Use of the formulated agar medium in cultivating microbial flora present in deionized water recovered many types of colonies, which suggested that the medium was able to support a variety of microbial species. Collectively, by separately sterilizing sugars and ammonium compounds, a method was developed for preparing colourless agar which helped improve colour contrast for colony identification and counting. The formulated colourless agar could find use in various microbial ecology studies seeking to profile microbes from different environmental niches.