Rapid enumeration of viable bacteria by image analysis

Abstract A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell acounts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually of by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35°C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cell enlarged incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the orginal sample did not influence the efficiency of the method. However, the optimal concentration of nalidixi (2.5–20 μg · ml −1 ) and lenth of incubation (4–8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.