Development of a direct viable count procedure for some Gram-positive bacteria

Abstract

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.

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus

“…In accordance with other authors (Kogure et al. 1979; Servis et al. 1995; Moter and Göbel 2000; Regnault et al.…”

Improved Direct Viable Count Procedure for Quantitative Estimation of Bacterial Viability in Freshwater Environments

“…A. hydrophila is found in freshwater and is widely distributed in aquatic environments (3,14,16,26,31). S. epidermidis is a gram-positive bacterium whose viability is difficult to determine by the original DVC method (24). Strain S1 was isolated from tap water using R2A medium (21).…”

The ecology of Listeria monocytogenes in ready-to-eat (RTE) meats

“…are observed from this DVC method compared to population recovered on nonselective media and significantly more so than selective media (93,252).…”

“…Samples are pre-incubated with a DNA synthesis inhibitor (DNA gyrases), preventing cell division (DNA replication) but allow other synthetic pathways to proceed, resulting in elongated cells (147,233). Examples of these DNA gyrase inhibitors include nalidixic acid for Gram-negatives and novobiocin, ciprofloxacin, enrofloxacin, enoxacin, norfloxacin, and isopropyl cinodine for Gram-positives (18,19,93,147,233,252). Samples are filtered through the preferred nuclepore filters as higher populations are recovered in comparison to cellulose filters.…”