FLOW CYTOMETRIC DISCRIMINATION BETWEEN ACINETOBACTER-CALCOACETICUS 69-V AND ALCALIGENES-EUTROPHUS JMP134 BY FLUORESCENTLY LABELED RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES AND DNA STAINING

In situ hybridization with fluorescently monolabelled rRNA-targeted ol igonucleotide probes (17 to 18 nucleotides) was used to discriminate b etween Alcaligenes eutrophus JMP134 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 intens ity of fluorescence after rRNA staining depended on the content of rib osomes, which correlated with the growth rate of bacteria. Therefore e xponentially 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 probe s under an epifluorescent microscope. Using a single laser cytometer, rRNA based identification was possible but not ideal. Better discrimin ation between the two strains of the mixed population was achieved by DNA staining, combined with the different forward light scatter signal s. Due to the significantly different cellular DNA and GC content of b oth strains, the fluorescent dye DAPI (4',6-diamidino-2-phenylindole), preferring AT-rich regions of DNA, was found to be a supplementary to ol for population analysis. The abundance ratios of the two strains in mixed culture determined by DNA or rRNA staining were similar.