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A particularly useful feature of ribosomal RNAs is their cellular abundance. Because of their
central involvement in protein synthesis, the rRNAs account for 70% or more of total cellular
RNA in both prokaryotes and eukaryotes. Due to this high concentration of intracellular target
sequence, in situ hybridizations with rRNA-specific probes are quite sensitive. Phylogenetic
identification of individual microbial cells using rRNA specific probes was first demonstrated
by Giovanonni and coworkers, using radioactively labeled oligonucleotides (Giovanonni et al.,
1988). However, this approach is time consuming and technically demanding, and not well
suited for routine clinical or environmental studies. An extension of this technique uses
fluorescently labeled oligonucleotide probes, for phylogenetic identification of individual
microbial cells by epifluorescence microscopy (DeLong et al., 1989a; Amann et al., 1990a;
Tsien et al., 1990). The method is technically straightforward and, with fluorescent probes
in hand, may be performed in a few hours.
Internal positive and negative controls may be included in parallel samples, to ensure that cells
contain intact, accessible rRNA, and that background noise is minimal. A universal probe,
which binds to the rRNA of all organisms studied so far, serves as a positive control.
Simultaneous hybridization is performed with this internal control probe, labeled with a
fluorochrome which excites and emits at a different wavelengths than the specific probe
(DeLong et al., 1989a). Bright, uniform, cellular fluorescence obtained with the positive
control demonstrates that the cells are intact, and that ribosomal RNA binding sites are
accessible. A negative control, in which no probe is added to the hybridization buffer, serves
as a check for excessive autofluorescence. Another negative control involves the use of a
fluor-labeled probe which does not bind to rRNA sequences. This control detects any
nonspecific binding of nucleic acid probes.
Methods that have been used to detect and quantitate the fluorescence of cells tagged with
fluorescently labeled rRNA probes include confocal microscopy (DeLong et al., 1989a) and
flow cytometry (Amann et al., 1990b). Confocal microscopy allows optical sectioning of the
sample, so that fluorescently labeled cells can be localized three dimensionally in tissue
sections. Video images obtained with the confocal microscope are stored on disk for
documentation, and subsequent image analysis can provide quantitative estimates of
fluorescence (DeLong et al., 1989a). Image analysis of cells labelled with fluorescent,
A particularly useful feature of ribosomal RNAs is their cellular abundance. Because of their
central involvement in protein synthesis, the rRNAs account for 70% or more of total cellular
RNA in both prokaryotes and eukaryotes. Due to this high concentration of intracellular target
sequence, in situ hybridizations with rRNA-specific probes are quite sensitive. Phylogenetic
identification of individual microbial cells using rRNA specific probes was first demonstrated
by Giovanonni and coworkers, using radioactively labeled oligonucleotides (Giovanonni et al.,
1988). However, this approach is time consuming and technically demanding, and not well
suited for routine clinical or environmental studies. An extension of this technique uses
fluorescently labeled oligonucleotide probes, for phylogenetic identification of individual
microbial cells by epifluorescence microscopy (DeLong et al., 1989a; Amann et al., 1990a;
Tsien et al., 1990). The method is technically straightforward and, with fluorescent probes
in hand, may be performed in a few hours.
Internal positive and negative controls may be included in parallel samples, to ensure that cells
contain intact, accessible rRNA, and that background noise is minimal. A universal probe,
which binds to the rRNA of all organisms studied so far, serves as a positive control.
Simultaneous hybridization is performed with this internal control probe, labeled with a
fluorochrome which excites and emits at a different wavelengths than the specific probe
(DeLong et al., 1989a). Bright, uniform, cellular fluorescence obtained with the positive
control demonstrates that the cells are intact, and that ribosomal RNA binding sites are
accessible. A negative control, in which no probe is added to the hybridization buffer, serves
as a check for excessive autofluorescence. Another negative control involves the use of a
fluor-labeled probe which does not bind to rRNA sequences. This control detects any
nonspecific binding of nucleic acid probes.
Methods that have been used to detect and quantitate the fluorescence of cells tagged with
fluorescently labeled rRNA probes include confocal microscopy (DeLong et al., 1989a) and
flow cytometry (Amann et al., 1990b). Confocal microscopy allows optical sectioning of the
sample, so that fluorescently labeled cells can be localized three dimensionally in tissue
sections. Video images obtained with the confocal microscope are stored on disk for
documentation, and subsequent image analysis can provide quantitative estimates of
fluorescence (DeLong et al., 1989a). Image analysis of cells labelled with fluorescent,