Filter-based PNA in situ hybridization for rapid detection, identification and enumeration of specific micro-organisms

J Appl Microbiol. 2001 Feb;90(2):180-9. doi: 10.1046/j.1365-2672.2001.01230.x.


Aims: A method for rapid and simultaneous detection, identification and enumeration of specific micro-organisms using Peptide Nucleic Acid (PNA) probes is presented.

Methods and results: The method is based on a membrane filtration technique. The membrane filter was incubated for a short period of time. The microcolonies were analysed by in situ hybridization, using peroxidase-labelled PNA probes targeting a species-specific rRNA sequence, and visualized by a chemiluminescent reaction. Microcolonies were observed as small spots of light on film, thereby providing simultaneous detection, identification and enumeration. The method showed 95-100% correlation to standard plate counts along with definitive identification due to the specificity of the probe.

Conclusion: Using the same protocol, results were generated approximately three times faster than culture methods for Gram-positive and -negative bacterial species and yeast species.

Significance and impact of the study: The method is an improvement on the current membrane filtration technique, providing rapid determination of the level of specific pathogens, spoilage or indicator micro-organisms.

Publication types

  • Evaluation Study

MeSH terms

  • Bacteria* / classification
  • Bacteria* / growth & development
  • Bacteria* / isolation & purification
  • Colony Count, Microbial
  • Culture Media
  • Filtration / instrumentation
  • Filtration / methods
  • In Situ Hybridization / methods*
  • Luminescent Measurements
  • Micropore Filters / microbiology*
  • Molecular Probes / genetics
  • Peptide Nucleic Acids / genetics*
  • Peroxidase / metabolism
  • Species Specificity
  • X-Rays
  • Yeasts* / classification
  • Yeasts* / growth & development
  • Yeasts* / isolation & purification


  • Culture Media
  • Molecular Probes
  • Peptide Nucleic Acids
  • Peroxidase