Fluorometric estimation of surface associated microbial abundance

Surface associated microbes have historically been difficult to accurately and effectively enumerate. In the current study, we propose a rapid and simple method for estimating abundance of surface associated microbial cells by fluorescence of SYBRGreen stained bacteria and in vivo chlorophyll a fluorescence of benthic diatoms in 24 and 48-well microtiter plates. The effectiveness of this high-throughput technique is demonstrated by assessing sensitivity of a clinical strain of Vibrio cholerae, a benthic bacterial isolate and the benthic microalgae Cylindrotheca closterium to three antibiotics — tylosin, lincomycin and ciproflaxacin. We report on the significant linear relationships between spectral chl a fluorescence and cell abundance and between microalgal growth rates derived from cell counts and fluorescence. Additionally, we provide a simplified and improved method for preparation of a silica gel matrix (SGM), which is an ideal plating media for fluorescence applications. These findings indicate that spectrofluorometry is an inexpensive tool for rapidly estimating abundance of surface associated microbiota and can be employed for assessing antibiotic sensitivity.

► Enumerating surface microbes is challenging among microbiological disciplines.
► Autofluorescence of surface matrices was evaluated in micro-titer plates.
► Fluorescent assessment of microbial density validated across concentration gradient.
► Fluorescence effective for assessing antibiotic sensitivity in micro-titer plates.