Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 6/3/1999
Publication Date: N/A
Technical Abstract: Species of Brettanomyces (ascosporic state Dekkera) are well recognized as spoilage yeasts in wine and cause 'mousiness', an undesirable odor and taste. Current methods for enumeration and identification rely on culturing on a semi-selective medium followed by final identification from morphology and biochemical testing, procedures that take 1-2 weeks. A new in situ hybridization method using peptide nucleic acid (PNA) probes is described. The sample is filtered, thereby isolating and separating the individual microorganisms onto the filter membrane, which is then placed on a culture medium for up to 44 hrs prior to testing. Micro-colonies of Brettanomyces are detected on the filter membrane using peroxidase-labeled PNA probes targeting Brettanomyces 26S rRNA. Binding of the probe is detected by chemiluminescence. Each Brettanomyces micro-colony is observed as a small dot, thereby allowing simultaneous identification and enumeration of Brettanomyces within 2 days. A 100% sensitivity and 100% specificity of the probes have been assessed using reference strains and wine isolates of Brettanomyces as well as other yeast species potentially found in wine. Furthermore, the method has successfully been used to demonstrate Brettanomyces in wine samples.