Rapid and simultaneous enumeration of viable Enterobacteriaceae and viable Pseudomonas spp. in milk was achieved by using multicolor fluorescence in situ hybridization (FISH) with oligonucleotide probes based on 16S ribosomal RNA (rRNA) sequences in combination with a microcolony growth method (multicolor microcolony-FISH; MMC-FISH). The procedure of MMC-FISH method is rather simple; that is milk clearing, filtration of cells, incubation, hybridization and enumeration. Enumeration of targeted bacteria in logarithmic growth phase, stationary phase, or in a starved state in milk by MMC-FISH required 5-7 h, while it took 1-3 days to test for Escherichia coil and Pseudomonas putida by the conventional culture method. The numbers of E. coil and P. putida in each phase or in a starved state in milk determined by MMC-FISH were almost the same or greater than the number of colony forming units determined by the plate counting method. The MMC-FISH allows rapid examination of contamination in milk by viable Enterobacteriaceae and Pseudomonas spp. with growth potential. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.