ABSTRACT
Few methods are available to evaluate the distribution of activity among marine bacterial cells. Two approaches are the Iodonitrotetrazolium (INT)-formazan method and microautoradiography. Both methods require incubation of bacteria with specific substrates. Sample manipulations associated with such incubations have been shown to perturb the composition, activity, and growth rate of bacteria. This chapter describes a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. Fluorescently labeled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labeled cells which are also hybridized with oligonucleotide probes. Oligonucleotide probes are inherendy taxon-specific at some level, which depends on the degree to which the probe sequence is evolutionarily conserved, and on the experimental conditions used to control hybridization stringency.
