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The widely accepted view that most bacterial species have yet to be cultivated in vitro has gained support from recent ribosomal DNA-based environmental studies. To enable elucidation of the phenotypes of organisms recognized solely by molecular genetic techniques, we developed and evaluated cytochemical methods which colocalize phenotypic properties with in situ rRNA probe hybridization signals. Application of these methods to artificial mixtures of Pseudomonas putida and Escherichia coli or Vibrio vulnificus showed that biochemical properties, such as the cytochrome oxidase reaction and specific substrate-enhanced tetrazolium salt reduction, can be assigned to cells identified by signals from determinative fluorescent rRNA probe binding. By doing the reactions directly on the stage of an inverted microscope and monitoring reaction product formation with a charge-coupled device video camera, it was possible to determine the kinetics of oxidizable substrate utilization in single cells. Analysis of digitized images permitted quantitative study of the relationship between rRNA signal strength and the rate of tetrazolium salt reduction. The approach used in this study opens up new opportunities to investigate the biochemistry, physiology, and behavior of both culturable and nonculturable bacteria in their natural environments.

Cytochemical colocalization and quantitation of phenotypic and genotypic characteristics in individual bacterial cells