Application of a Fluorescence Microscopy Technique for Detecting Viable Mycobacterium avium ssp. paratuberculosis Cells in Milk

Abstract

The aim of the present study was to develop a fast and reliable fluorescence staining technique in order to accurately determine the number of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells in milk. Milk was artificially inoculated with known amounts of four different MAP strains. Different dilutions were tested by the combination of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and auramine orange (AO) staining. To validate this combined fluorescence staining technique, two additional methods were applied: culture and the FASTPlaqueTB™ assay. The detection limit of the combined CTC and AO staining was 102 colony-forming unit (CFU) mL−1 for spiked ultra-high-temperature (UHT) milk and 103 CFU mL−1for spiked raw milk (probability of detection >95 %). Combined CTC and AO staining provides the opportunity to determine the total cell count of acid fast bacteria as well as the number of respiratory active cells in milk within 8 h.