The direct viable count (DVC) is a procedure for enumerating viable-nonculturable cells. It should be noted, however, that bacteria demonstrating the viable but nonculturable phase have to date included only Gram-negative species, mainly because the DVC procedure does not lend itself to the analysis of Gram-positive bacteria since the DVC procedure is dependent on the bacterium being sensitive to nalidixic acid. The authors report here concerning studies on an analogous procedure for the direct enumeration of viable-nonculturable Gram-positive bacteria. To facilitate a differential DVC for Gram-positive bacteria, ciprofloxacin, enoxacin, norfloxacin or isopropyl cinodine were substituted for nalidixic acid. These antibiotics were chosen because, like nalidixic acid, they are DNA gyrase inhibitors. The concentrations used for each antibiotic were 1000 micrograms ml-1, 100 micrograms ml-1. Pure cultures of Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Listeria monocytogenes and Bacillus subtilis were obtained from the culture collection at the University of Wyoming and a faecal streptococcus was isolated from the Laramie wastewater treatment plant. An antibiotic and optimal concentration thereof was found which gave enlarged cells for all the organisms except the faecal streptococcus isolated from the wastewater plant for which no enlarged cells were ever seen. The antibiotic and concentration thereof which gave the optimal percent enlarged cells in the DVC procedure varied between organisms.

1993. Isopropyl cinodine and nalidixic acid were compared in the direct viable count. With raw water and biofilms, elongated cells were seen in the presence of isopropyl cinodine. Increased incubation time led to an increased direct viable count. Individual bacteria responded differently to isopropyl cinodine. Five organisms grew in the presence of 0.01 p g ml-' of isopropyl cinodine but were inhibited by 0.1 pg ml-'. These values for a sixth organism were 0.1 pg ml-' and 1.0 p g ml-' respectively. T h e direct viable count was done with inocula taken when the cells were in either lag, log or stationary phases of growth. No differences were seen in the percentage of elongated cells within an experiment but there was variation between experiments. T h e effect of nalidixic acid and isopropyl cinodine appeared to be additive with respect to inhibition of growth, but little or no additive effect was seen upon the percent of nutrient responsive cells. Environmental Microbiology 53, 2889. Zelibor, J.L., Doughten, M.W., Grimes, D.J. and Colwell, R.R. (1987) Testing for bacterial resistance to arsenic in monitoring well water by the direct viable counting method. Applied and Environmental Microbiology 53, 292!+2934. Zimmerman, R., Iturriaga, R. and Becker-Brick, J. (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Applied and Environmental Microbiology 36, 926-935.