Distinguishing between living and nonliving bacteria: Evaluation of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples

• Published in: Journal of microbiological methods Vol. 32; no. 3; pp. 225 - 236
• Main Authors: Williams, S.C., Hong, Y., Danavall, D.C.A., Howard-Jones, M.H., Gibson, D., Frischer, M.E., Verity, P.G.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.05.1998; Elsevier Science; Elsevier
• Subjects: 16S rRNA oligonucleotide probes; Bacteria; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; DAPI; Fundamental and applied biological sciences. Psychology; Live vs. dead; Microbiology; Ocean, Atmosphere; Propidium iodide; Sciences of the Universe; Vital stain; Bacteria; Propidium iodide; DAPI; 16S rRNA oligonucleotide probes; Live vs. dead; Vital stain; Investigation method; Ribosomal RNA; Staining; Molecular probe; Vital staining; Viability; Optimization
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(98)00014-1

Recent studies have suggested that a large fraction of marine bacterioplankton are either dead or moribund and, therefore, new methods are required to distinguish bacteria of different physiological states. A method is described which uses the general cell stain 4′6′-diamidino-2-phenylindole (DAPI), the vital stain propidium iodide (PI), and 16S rRNA-targeted oligonucleotide probes, to quantitatively identify cells with 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 MgSO 4 (pH=6.5) and stained with PI (5 μg/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°C without changes in staining status. Staining of heat-killed cells indicated that PI stained only dead cells. Comparison of PI staining properties and hybridization with 16S rRNA-targeted oligonucleotide probes indicated that there was a strong inverse correlation between hybridization of cells with 16S rRNA-targeted oligonucleotide probes and cells stained by PI. Evidence indicates that this vital stain and probe (VSP) technique differentiates between (1) cells that are dead, (2) cells that are dead but were recently active (<36 h), (3) cells that are living and (4) cells that are inactive but not dead. The VSP protocol comprises a powerful tool to investigate the relative importance of these cell types in situ, and how they change in response to environmental factors.