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

Abstract

ZR siru hybridization with fluorescently monolabelled rRNA-targeted oligonucleotide probes (17 to 18 nucleotides) was used to discriminate between Alcaligenes eutrophus JMP134 and Acinetobacter cnlcnucericus 69-V by flow cytometry. The strains were grown in batch experiments in a mixed popuhtion. 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 cornelated with the growth rate of bacteria. Thetefore 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 identi!ication was possible but 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 ; I supplementary too1 for population analysis. The abundance ratios of the two strains in mixed culture determined by DNA or rRNA staining were similar.

Modes of cytometric bacterial DNA pattern: a tool for pursuing growth

“…We have observed the pre‐D phase to be independent of the quality of the substrate supplied in both Gram‐negative and Gram‐positive bacteria: Acinetobacter calcoaceticus on phenol and acetate (Fig. 2, also Herrmann et al. 1997; Müller et al.…”

“…We have observed the pre-D phase to be independent of the quality of the substrate supplied in both Gram-negative and Gram-positive bacteria: Acinetobacter calcoaceticus on phenol and acetate (Fig. 2, also Herrmann et al 1997;Müller et al 2000), Ochrobactrum anthropi on glucose, xylitol, 2,4-dichlorophenol and 2,4-dichlorophenoxybutyric acid, R. erythropolis on glucose, xylitol and succinate (Fig. 2, also Müller et al 1999;Müller et al 2002), Sphingomonas sp.…”

Flow cytometric monitoring of Rhodococcus erythropolis and Ochrobactrum anthropi in a mixed culture

“…Furthermore, rRNA techniques have been found to be timeconsuming and to require complex procedures when handling the samples [20]. Bacteria are known to synthesize surface-exposed carbohydrates, which are potential lectin-binding sites.…”

Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities

“…Therefore, the 16S rRNA gene signal can be reliably used to quantify desired groups of organisms growing under wellsupplied microenvironments. In established artificial systems, it is even possible to obtain information on activity states (Herrmann et al, 1997). Gray et al (2000) used the FISH approach to label Achromatium populations with a bright fluorescence, which was verified by simultaneous monitoring of selectively metabolized radiolabelled substrates.…”

“…Therefore, the 16S rRNA gene signal can be reliably used to quantify desired groups of organisms growing under well‐supplied microenvironments. In established artificial systems, it is even possible to obtain information on activity states (Herrmann et al ., 1997). Gray et al .…”