Detection and identification of lactic acid bacteria in milk and industrial starter culture with fluorescently labeled rRNA-targeted peptide nucleic acid probes

A fast and simple method for whole-cell hybridization using fluorescently l abeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for us e in detection and identification of thermophillic lactobacilli cells growi ng in milk or present in industrial starter cultures. The protocol uses a f iltration technique of the samples and epifluorescence microscopy as a dete ction system, and is completed within 1.5 h. Seven oligonucleotide probes w ith different ranges of specificity have been tested in in situ hybridizati on experiments against a number of collection and industrial strains includ ing those of the species Lactobacillus delbrueckii, Lactobacillus helveticu s, Lactobacillus pentosus, Lactobacillus plantarum, Lactococcus lactis, Leu conostoc sp. and Streptococcus thermophilus. Certain limitations in the use of DNA probes due to non-specific hybridization were encountered and taken into account. Depending on the probe used, a specific and simultaneous det ection of the different species present can be achieved. The equipment used was able to detect between 10(4) and 10(6) cells per mL. The physiological state of a starter cultures of thermophilic lactobacilli can also be evalu ated by in situ hybridization. Thus, this fast method can be used for start er cultures and industrial fermentation quality control. This work constitu tes the first reported use of PNA molecules for in situ detection and ident ification of bacteria in milk or in an industrial medium.