DISTINGUISHING BETWEEN LIVING AND NONLIVING BACTERIA - EVALUATION OF THE VITAL STAIN PROPIDIUM IODIDE AND ITS COMBINED USE WITH MOLECULAR PROBES IN AQUATIC SAMPLES

Recent studies have suggested that a large fraction of marine bacterio plankton are either dead or moribund and, therefore, new methods are r equired to distinguish bacteria of different physiological states. A m ethod is described which uses the general cell stain 4'6'-diamidino-2- phenylindole (DAPI), the vital stain propidium iodide (PI), and 16S rR NA-targeted oligonucleotide probes. to quantitatively identify cells w ith compromised membranes, and those cells containing sufficient rRNA to be considered metabolically active. Validation and optimization of this method was conducted using cultured bacteria. Optimal PI staining was achieved after cells were washed in 10 mM MgSO4 (pH=6.5) and stai ned with PI (5 mu g/ml) for 30 min. Staining of cells with PI appeared to be independent of growth phase and cells could be stored in 25% (v /v) glycerol for at least one month at -20 degrees C without changes i r. staining status. Staining of heat-killed cells indicated that PI st ained only dead cells. Comparison of PI staining properties and hybrid ization with 16S rRNA-targeted oligonucleotide probes indicated that t here was a strong inverse correlation between hybridization of cells w ith 16S rRNA-targeted oligonucleotide probes and cells stained by PI. Evidence indicates that this viral stain and probe (VSP) technique dif ferentiates between (1) cells that are dead, (2) cells that are dead b ut were recently active (<36 h), (3) cells that are living and (4) cel ls that are inactive but not dead. The VSP protocol comprises a powerf ul tool to investigate the relative importance of these cell types in situ, and how they change in response to environmental factors. (C) 19 98 Published by Elsevier Science B.V.