253
rRNA-targeted probes is proving useful for identifying and localizing bacterial endosymbionts
in their host's tissue (Dan Distel, John Waterbury and Ed DeLong, unpublished data).
A recent application of fluorescent rRNA probes is the analysis of labeled cells by flow
cytometry (Amann et al., 1990b). Hybridizations are performed with fixed cells in solution,
rather then with smears or sections. After hybridization with the fluorescent probe, the cells
are diluted in buffer and quantified by flow cytometric analysis. Initial studies show that probe
binding is highly specific, and that the technique readily identifies probe-binding cells, even
when present as a small fraction « 1 %) of the total cell population. The coupling of flow
cytometry with the use of rRNA-targeted probes has great potential. Given adequate
fluorescent signal, specific cell types could be identified and quantified very rapidly (thousands
of cells per second).
The fluorescent signal resulting from in situ hybridization with fluor-labeled rRNA probes is
largely dependent on the rRNA content of the targeted cell. For any given cell type,
fluorescence intensity can vary, and depends in part on the physiological status of the cell at
the time of fixation (Figure 2). Slowly growing cells, which contain fewer ribosomes, yield
proportionally lower fluorescence signal intensities (DeLong et al., 1989a; Figure 2). The low
signal obtained from cells which contain few ribosomes, coupled with the inherent
autofluorescence found in many natural samples, are factors which currently limit the use of
the technique in ecological studies. A number of potential methods for amplifying the signal
obtained with fluorescently labeled rRNA hybridization probes exist. One approach is to use
several different probes of identical specificity, targeted to different regions of the same rRNA
molecule (Amann et al., 1990 a,b). A two or three-fold increase in fluorescent signal intensity
should be readily achievable using this strategy. Another approach involves indirect labelling
with a fluor-labeled reporter groups (E.F. DeLong, unpublished data). An rRNA probe, which
is covalently linked to a poly thymidine tail, is first hybridized with the sample. After
hybridization and washing, the fluorescently labeled reporter group (eg., fluorescently labeled
polyadenosine) is added. Since several fluorescent reporters can bind to each tail, the
fluorescent signal may be significantly enhanced. Preliminary experiments with cultured
bacteria and protozoa suggest that a several fold increase in signal is achievable using this
method.
rRNA-targeted probes is proving useful for identifying and localizing bacterial endosymbionts
in their host's tissue (Dan Distel, John Waterbury and Ed DeLong, unpublished data).
A recent application of fluorescent rRNA probes is the analysis of labeled cells by flow
cytometry (Amann et al., 1990b). Hybridizations are performed with fixed cells in solution,
rather then with smears or sections. After hybridization with the fluorescent probe, the cells
are diluted in buffer and quantified by flow cytometric analysis. Initial studies show that probe
binding is highly specific, and that the technique readily identifies probe-binding cells, even
when present as a small fraction « 1 %) of the total cell population. The coupling of flow
cytometry with the use of rRNA-targeted probes has great potential. Given adequate
fluorescent signal, specific cell types could be identified and quantified very rapidly (thousands
of cells per second).
The fluorescent signal resulting from in situ hybridization with fluor-labeled rRNA probes is
largely dependent on the rRNA content of the targeted cell. For any given cell type,
fluorescence intensity can vary, and depends in part on the physiological status of the cell at
the time of fixation (Figure 2). Slowly growing cells, which contain fewer ribosomes, yield
proportionally lower fluorescence signal intensities (DeLong et al., 1989a; Figure 2). The low
signal obtained from cells which contain few ribosomes, coupled with the inherent
autofluorescence found in many natural samples, are factors which currently limit the use of
the technique in ecological studies. A number of potential methods for amplifying the signal
obtained with fluorescently labeled rRNA hybridization probes exist. One approach is to use
several different probes of identical specificity, targeted to different regions of the same rRNA
molecule (Amann et al., 1990 a,b). A two or three-fold increase in fluorescent signal intensity
should be readily achievable using this strategy. Another approach involves indirect labelling
with a fluor-labeled reporter groups (E.F. DeLong, unpublished data). An rRNA probe, which
is covalently linked to a poly thymidine tail, is first hybridized with the sample. After
hybridization and washing, the fluorescently labeled reporter group (eg., fluorescently labeled
polyadenosine) is added. Since several fluorescent reporters can bind to each tail, the
fluorescent signal may be significantly enhanced. Preliminary experiments with cultured
bacteria and protozoa suggest that a several fold increase in signal is achievable using this
method.