Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes

Appl Environ Microbiol. 1992 Sep;58(9):3007-11. doi: 10.1128/aem.58.9.3007-3011.1992.

Abstract

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaea / cytology
  • Archaea / genetics
  • Archaea / isolation & purification
  • Bacteria / cytology
  • Bacteria / genetics
  • Bacteria / isolation & purification*
  • Base Sequence
  • Cell Membrane Permeability
  • Horseradish Peroxidase
  • Molecular Sequence Data
  • Nucleic Acid Hybridization / methods
  • Oligonucleotide Probes*
  • Prokaryotic Cells / microbiology*
  • RNA Probes*
  • RNA, Ribosomal / genetics*
  • RNA, Ribosomal / isolation & purification

Substances

  • Oligonucleotide Probes
  • RNA Probes
  • RNA, Ribosomal
  • Horseradish Peroxidase