An improved method for collecting and staining microorganisms for enumeration by fluorescence light microscopy

A rapid, robust method for the enumeration of total and viable microorganisms is described. A method using specific stains for viable and total cells and fluorescence light microscopy on membrane filters had been previously developed, but was suboptimal in that some non-specific staining of the filters occurred and the filters were not flat enough for automatic image analysis methods to be employed, because not all cells in a field of view were in focus simultaneously. A new membrane filter has recently become available: the Anopore membrane was described by the manufacturers as having a number of properties which would overcome these limitations. These include inorganic construction (giving resistance to solvents), high porosity (giving high flow rates), low surface adsorption (giving low background staining) and inherent hydrophilicity (simplifying wetting with aqueous solutions). Anopore membrane filters were found to produce very high contrast images of bacteria stained with ethidium bromide. Even with a relatively low power (magnification = 40) dry objective, these images could be easily thresholded for image analysis using only grey-level information. The methods developed here are considered to be a suitable basis for a fully automated procedure for the enumeration of total microbial populations.