Simple detection of small amounts of Pseudomonas cells in milk by using a microfluidic device

Aims: Flow cytometry offers rapid and reliable analyses of bacteria in milk. However, a flow cytometer is relatively expensive and operation is rather complicated for an unskilled operator. We applied flow cytometry using a microfluidic device (on‐chip flow cytometry) in detection of small amounts of milk‐spoiling bacteria. Methods and Results: Pseudomonas cells in milk were in situ hybridized with Cy5‐labelled probe specific for Pseudomonas spp. under optimized condition. Numbers of Pseudomonas cells in the stationary phase and in the starved state determined by on‐chip flow cytometry were compared with those determined by conventional plate counting, and on‐chip flow cytometry detected targeted cells in milk that were undetectable as colony forming units(CFU) on Standards Methods Agar. Conclusions: The contamination in milk with fewer than 10 CFU ml−1 of targeted cells in starved state was detectable with simple procedure (0·5 h milk‐clearing, 1 h fixation, 2 h hybridization and 0·5 h on‐chip flow cytometry following 12 h enrichment of cells). Significance and Impact of the Study: On‐chip flow cytometry following fluorescence in situ hybridization could be applicable to simple detection of milk‐spoiling bacteria.