INCREASE IN FLUORESCENCE INTENSITY OF 16S RIBOSOMAL-RNA IN-SITU HYBRIDIZATION IN NATURAL SAMPLES TREATED WITH CHLORAMPHENICOL

Despite the numerous advantages of fluorescent in situ hybridization f or the identification of single prokaryotic cells with 16S rRNA probes , use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluor escently labeled cells constitutes the primary problem for in situ hyb ridization of natural samples, probably due to low cellular rRNA conte nt, This study represents an attempt to improve detection of marine pr okaryotes by increasing cellular rRNA content without changing the spe cies composition, Cells from three California coastal sites were treat ed with chloramphenicol, an inhibitor of protein synthesis and rRNA de gradation, at 100 mu g/ml and then were probed with a ''universal'' 16 S rRNA fluorescent probe and viewed by image-intensified video microsc opy, Counts of fluorescent cells increased from ca, 75% for untreated samples to ca, 93 to 99% for chloramphenicol-treated samples, compared to counts produced by DAPI (4',6-diamidino-2-phenylindole) staining, after at least 45 min of exposure to the drug (these percentages inclu de autofluorescent cells, which averaged 6%), This suggests that most cells in these samples were active, We hypothesize that the low fluore scent-cell counts previously reported were probably often due to the f luorescence intensity of labeled cells being below the detection level rather than to high levels of dead cells in marine environments, This method may aid in the characterization of bacterioplankton with fluor escent probes.