ABSTRACT
In recent years fluorescent DNA stains, used in conjunction with epifluorescence microscopy, have become standard tools for obtaining quantitative information on microbial communities. The procedure described employs oligonucleotide probes complementary to ribosomal RNA (rRNA) sequences. With proper attention to hybridization conditions, discrimination at the level of one nucleotide mismatch is possible. Detection sensitivity will depend in part on the physiological stale of the target cells. The basic technique described employs fluorescently labeled, rRNA-targeted oligonucleotide probes which bind to specific rRNA target sequences in the ribosomes of fixed, intact cells. One advantage of using single, fluor-labeled oligonucleotides is that labeling and detection is simple and rapid. Attempts to label oligonucleotides with multiple fluors have resulted in poor or nonspecific binding, probably due to the hydrophobicity of the fluorescent moieties. Hybridization of isotopically or nonisotopically labeled probes with bulk nucleic acids fixed to solid supports is convenient for optimizing hybridization and wash temperatures, and screening a large variety of rRNAs.
