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 bl 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. Conclusions: 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.