A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil

Microbial cell concentration is a particularly important bioindicator of soil health and a yardstick for determining biological quotients which are likely to gain in ecological significance if they are calculated in relation to the viable, rather than total, microbial density. A dual-staining technique with fluorescent dyes was used for the spectrofluorimetric quantitative determination of the concentration of viable microbial cells present in three different soil types. This is a novel and substantially modified application of the dual-staining procedure implemented in the LIVE/DEAD BacLight viability kit which has never been successfully applied to the quantification of naturally occurring soil microbial communities. Indigenous microbial cell concentrations were quantified using an internal standard, i.e. spiking environmental samples with suspensions containing different concentrations of live E. coli cells, and external calibration, by comparing fluorescence emission by indigenous bacteria and known concentrations of E. coli in nutrient saline. Two types of environmental samples were tested: bacterial preparations obtained by density gradient centrifugation and soil suspensions. In both cases, prior dilution of the sample was necessary to minimise fluorescence quenching by soil particulate matter. Spectrofluorimetric measurements of indigenous cell concentration in bacterial preparations were in close agreement with those found using epifluorescence microscopy. Limits of detection of 5x10(6) for the soil bacterial preparations and 8x10(7) for the soil suspensions were estimated. Deviations observed when soil suspensions are dealt with are likely due to the selection of a unique bacterial strain for standardisation and calibration. Thorough testing of a variety of reference bacteria and fungi is suggested to determine a more accurate average fluorescence enhancement per microbial cell or mass unit.