Rapid counting of liquid-borne microorganisms by light scattering spectrometry

Fast and sensitive techniques are needed to determine microorganism presence in liquid samples. In this research, the feasibility of using light scattering spectrometry for enumerating the biological particles in liquid samples was investigated. A particle size spectrometer was used to count six commonly found microbial species suspended in liquid with and without microbiological stains applied: Pseudomonas fluorescens, Micrococcus spp. vegetative cells and Bacillus subtilis var. niger endospores were stained with Acridine Orange and Crystal Violet, while Cladosporium cladosporioides, Penicillium melinii and Aspergillus versicolor fungi were stained with Acridine Orange and Lactophenol Cotton Blue. The counts obtained with the spectrometer were compared with those obtained with a phase-contrast microscope. It was found that the spectrometer counted about 32 % of non-stained B. subtilis endospores and this percentage increased to almost 90 % for stained endospores. Among the investigated species of fungi, the counting efficiency of P. melinii was the only one significantly affected by the application of the stain Lactophenol Cotton Blue: the fraction of counted fungal spores increased from 64 % (non-stained spores) to about 100 % (stained spores). The observed difference in counting efficiency may serve as a basis for differentiating biological from non-biological particles in liquid samples.