387
utilizing epifluorescence microscopy of filter-concentrated samples showed that these
chemoautotrophic organisms are present in seawater in much greater abundance than was
reviously estimated by culturing techniques. These cells are still quite rare, (typically
10 cell ml- I ), which would make them difficult to sample with present flow cytometers.
However, it has been shown that if present in sufficient concentration,
immunofluorescently-Iabelled bacteria could be detected in the presence of auto fluorescent
phytoplankton by flow cytometry (Ward 1990), as demonstrated by diluting cultured cells of
the ammonia oxidizer Nitrosococcus into whole seawater (Fig. 22).
The pico- and nanophytoplankters, whose autofluorescence is routinely analyzed by flow
cytometry, are also subjects of immunofluorescence studies. Antibodies to several serotypes
of Synechococcus (Campbell et al. 1983; Campbell and Iturriaga 1988) and several small
eukaryotic phytoplankters (Campbell et at. 1989) have been developed, and it has been shown
that flow cytometry is capable of detecting these fluorescently-Iabelled probes (Campbell
1990). These polyclonal antibody probes, which have been chosen to reflect specificity at
roughly the species or genus level, should make it possible to quantify the contributions of
various components of the natural phytoplankton assemblage to biomass and productivity.
Oligonucleotide probes (e.g., to rRNA) offer another approach, in which the taxon specificity
of the probe can be tailored to the aims of the study, from kingdom down to species. The
sequence of the rRNA for the target organisms must be known, but as this database becomes
available for more organisms, probes can be custom designed to be as specific or general as
desired (Giovannoni et al. 1988).
As in immunofluorescence, fluorescently-tagged oligonucleotide probes are usually detected
by epifluorescence microscopy (e.g., DeLong et al. 1989), but the potential for flow
cytometric detection of individual bacteria labelled with rRNA probes has been recently
demonstrated (Amann et al. 1990). In this study, a probe specific to sulfate reducers was
shown to bind to Desulfovibrio gigas but not to E. coli, while a kingdom-specific probe to
eubacteria labelled both species (Fig. 23). It was also possible to enumerate cells of the sulfate
reducing species in mixtures of the two cultures. By hybridizing additional probes to different
utilizing epifluorescence microscopy of filter-concentrated samples showed that these
chemoautotrophic organisms are present in seawater in much greater abundance than was
reviously estimated by culturing techniques. These cells are still quite rare, (typically
10 cell ml- I ), which would make them difficult to sample with present flow cytometers.
However, it has been shown that if present in sufficient concentration,
immunofluorescently-Iabelled bacteria could be detected in the presence of auto fluorescent
phytoplankton by flow cytometry (Ward 1990), as demonstrated by diluting cultured cells of
the ammonia oxidizer Nitrosococcus into whole seawater (Fig. 22).
The pico- and nanophytoplankters, whose autofluorescence is routinely analyzed by flow
cytometry, are also subjects of immunofluorescence studies. Antibodies to several serotypes
of Synechococcus (Campbell et al. 1983; Campbell and Iturriaga 1988) and several small
eukaryotic phytoplankters (Campbell et at. 1989) have been developed, and it has been shown
that flow cytometry is capable of detecting these fluorescently-Iabelled probes (Campbell
1990). These polyclonal antibody probes, which have been chosen to reflect specificity at
roughly the species or genus level, should make it possible to quantify the contributions of
various components of the natural phytoplankton assemblage to biomass and productivity.
Oligonucleotide probes (e.g., to rRNA) offer another approach, in which the taxon specificity
of the probe can be tailored to the aims of the study, from kingdom down to species. The
sequence of the rRNA for the target organisms must be known, but as this database becomes
available for more organisms, probes can be custom designed to be as specific or general as
desired (Giovannoni et al. 1988).
As in immunofluorescence, fluorescently-tagged oligonucleotide probes are usually detected
by epifluorescence microscopy (e.g., DeLong et al. 1989), but the potential for flow
cytometric detection of individual bacteria labelled with rRNA probes has been recently
demonstrated (Amann et al. 1990). In this study, a probe specific to sulfate reducers was
shown to bind to Desulfovibrio gigas but not to E. coli, while a kingdom-specific probe to
eubacteria labelled both species (Fig. 23). It was also possible to enumerate cells of the sulfate
reducing species in mixtures of the two cultures. By hybridizing additional probes to different