Flow cytometric discrimination between Acinetobacter calcoaceticus 69‐V and Alcaligenes eutrophus JMP134 by fluorescently labelled rRNA‐targeted oligonucleotide probes and DNA staining

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Abstract
In situ hybridization with fluorescently monolabelled rRNA‐targeted oligonucleotide probes (17 to 18 nucleotides) was used to discriminate between Alcaligenes eutrophus JMP 134 and Acinetobacter calcoaceticus 69‐V by flow cytometry. The strains were grown in batch experiments in a mixed population. The forward light scatter and fluorescence of each bacterial cell were measured with a single laser cytometer. The intensity of fluorescence after rRNA staining depended on the content of ribosomes, which correlated with the growth rate of bacteria. Therefore exponentially growing cells could be clearly detected. For other growth phases, signal amplification was necessary using multiple probes. The two bacterial strains were identified with differently labelled probes under an epifluorescent microscope. Using a single laser cytometer, rRNA based identification was possible nut not ideal. Better discrimination between the two strains of the mixed population was achieved by DNA staining, combined with the different forward light scatter signals. Due to the significantly different cellular DNA and GC content of both strains, the fluorescent dye DAPI (4′, 6‐diamidino‐2‐phenylindole), preferring AT‐rich regions of DNA, was found to be a supplementary tool for population analysis. The abundance ratios of the two strains in mixed culture determined by DNA or rRNA staining were similar.

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