Use of multiple 16S rRNA-targeted fluorescent probes to increase signal strength and measure cellular RNA from natural planktonic bacteria

Fluorescent oligonucleotide probes can be used to identify bacterial cells by hybridizing to specific rRNA sequences that are unique to a taxon. It has been suggested that single-cell RNA content could be estimated from the resulhng cell fluorescence, which is directly proportional to rRNA content However, this possibility was never assessed quantitatively for use in field studies, where most natural bacteria, due to their small size and slow growth, do not have enough rRNA to yield sufficient fluorescence for detection. One approach for increasing the fluorescence per rRNA molecule IS the use of multiple probes targeted to independent sites in the rRNA molecule. In this study, we hybridized n~ultiple probes (3 universal and 4 bacterial) to cultured marine bacterial isolates, and to natural bacterial assemblages from coastal water. For S of the 7 probes (2 probes did not hybridize to some of the isolates), cell fluorescence increased linearly with the number of probes hybridized. The natural bacterial assemblages showed an asymptotic increase of the probe-labeled fraction from ca 20% (1 probe) to 75% (5 probes) of the total population. The estimated detection limit was 0.3 fg RNA cell-' with S probes. RNA contents estimated from the fluorescence of probe-labeled cells agreed well to those determined separately by ethidium bromide fluorometry (differed by average 16 %, n = 3). Calibration of the photometer against fluorescence standards was critical for accurate measurements and comparisons.