Direct Viable Count Procedure Research Articles

Single‐cell analysis of cell viability after a biocide treatment unveils an absence of positive correlation between two commonly used viability markers

Discrimination among viable/active or dead/inactive cells in a microbial community is a vital question to address issues on ecological microbiology or microbiological quality control. It is commonly assumed that metabolically active cells (ChemchromeV6 [CV6] procedure) correspond to viable cells (direct viable count procedure [DVC]), although this assumption has never been demonstrated and is therefore a matter of debate. Indeed, simultaneous determination of cell viability and metabolic activity has never been performed on the same cells. Here, we developed a microfluidic device to investigate the viability and the metabolic activity of Escherichia coli cells at single-cell level. Cells were immobilized in a flow chamber in which different solutions were sequentially injected according to different scenarios. By using time-lapse microscopy combined with automated tracking procedures, we first successfully assessed the ability of cells to divide and their metabolic activity at single-cell level. Applying these two procedures on the same cells after a hypochlorous acid (HOCl) treatment, we showed that the ability of cells to divide and their metabolic activity were anticorrelated. These results indicate that the relation between CV6 uptake and cell viability may be partially incorrect. Care must be taken in using the terms “CV6-positive” and “viable” synonymously.

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

We have developed a direct viable count (DVC)-FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC-FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. This technique was successfully applied to detect viable cells in inoculated faeces. Results showed that this DVC-FISH procedure is a quick and culture-independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.

The application of viable count procedures for measuring viable cells in the various marine environments

To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony-forming units and the contribution of capsule-bearing bacteria to the total number of bacteria and esterase-active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0-30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l(-1) (at 35 degrees C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l(-1) for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.

Direct Viable Count Combined with Fluorescence in situ Hybridization (DVC-FISH) for Specific Enumeration of Viable Escherichia coli in Cow Manure

A direct viable count procedure combined with fluorescence in situ hybridization (DVC-FISH) was developed for the specific detection and enumeration of viable Escherichia coli in cow manure. The DVC method was performed by trapping bacterial cells, extracted from cow manure samples, onto Nucleopore filters followed by incubation on a DVC medium containing yeast extract and four gyrase inhibitors. E. coli cells were identified by using the probe ES445. The DVC method efficiently promoted the elongation of E. coli cells and allowed for the recognition of individual cell division events, by observing microcolonies. Cell recovery by DVC-FISH together with bacterial extraction, was 53% with an inoculum of 10(7) to 10(10) cells g(-1) dry weight, when the manure samples were inoculated with a fresh culture of E. coli and determinations were made immediately. An examination of the survival of E. coli in a cow manure microcosm showed that an increasing fraction of E. coli became non-culturable but were still detectable by DVC-FISH. All these results suggest that DVC-FISH is useful for enumerating viable, even non-culturable, E. coli in cow manure.

Combining direct viable count (DVC) and fluorescent in situ hybridisation (FISH) to enumerate viable E. coli in rivers and wastewaters

In this study, a combination of the direct viable count procedure (DVC) and the FISH method was used to monitor by epifluorescence microscopy the abundance of viable E. coli in river water and wastewater samples. The DVC procedure consisted of exposing bacterial cells to a resuscitation medium containing antibiotics preventing cellular division and, thus, inducing an elongation of the viable cells. The FISH was performed using the "Colinsitu" probe specific for E. coil 16S r-RNA. Accuracy and detection limit of the epifluorescence microscopic DVC-FISH procedure were investigated. The method was then applied to river-water and wastewater samples. A good correlation was found in a log-log plot between the abundance of E. coil enumerated by a classical culture-based method (MPN method) and the DVC-FISH procedure. However, the DVC-FISH procedure gave consistently higher numbers. The ratio between both enumerations (DVC-FISH/MPN), which also indicated the ratio between viable and culturable E. coli, ranged between 2 and >30. It increased with decreasing abundance of culturable E. coli.

Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes.

A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in filtered, sterilized distilled water. We used different concentrations of ciprofloxacin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and ciprofloxacin. The mixture was incubated at different incubation times at 37 degrees C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solution or orange acridine. The filters were prepared and examined by epifluorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.

Problems associated with the direct viable count procedure applied to gram-positive bacteria

Despite the numerous advantages of fluorescent in situ hybridization (FISH) for identifying a single bacterial cell with 16S rRNA probes, problems are encountered with starving bacteria in natural samples. The original direct viable count procedure (DVC) includes a revivification step in the presence of an antibiotic inhibiting cell division. Cells elongate and accumulate ribosomes. This results in a natural amplification of 16S rRNA molecules (target of FISH). However, it is limited to gram-negative bacteria which are sensitive to nalidixic acid. The objective of this study was to develop a procedure for estimating the number of metabolically active gram-positive Staphylococcus aureus and Enterococcus faecalis cells by the use of a method which combines the number of substrate-responsive cells and their identification by FISH. It was observed that no single published DVC method could apply to taxonomically different gram-positive bacteria. Since cells were not counted, the revivification step in presence of nalidixic acid will be referred to as revivification without cell division. For each species, different low-nutrient media and complex media, different fluoroquinolones and beta-lactam antibiotics, concentrations of antibiotics, combinations of antibiotics, temperature and time were evaluated using bacteria in different physiological states and in natural samples. Enumeration of bacteria by plate counts and direct FISH were compared. The improved procedure should yield information about the physiological state, the taxonomic identity, and the enumeration of viable gram-positive bacteria. The application of DVC to an entire ecosystem is presently still a challenge.

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

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.

BACTERIAL ADHERENCE AND VIABILITY ON CUTTING BOARD SURFACES

ABSTRACTThe adherence and viability of Escherichia coli inoculated onto the surfaces of plastic cutting boards and new and used wood cutting boards were evaluated. Most of the inoculum was recovered from all surfaces after resident drying times of 5 min and from plastic surfaces at 24 h. When the exposure time was extended to 2 h, > 90% of the cells placed on new and used dry wood surfaces were not recovered after vigorous rinsing. Scanning electron microscopy showed that the bacteria resided within the structural xylem fibers and vegetative elements of the wood. After resident drying times of up to 2 h, almost 75% of the adherent bacteria on the wood surfaces were viable, as defined by a nalidixic acid direct viable count procedure. Microcosm studies showed no intrinsic growth‐supporting or toxic properties of the cutting board materials. Bacteria that adhered to plastic surfaces were more easily removed by low‐temperature washing than were cells that adhered to wood surfaces. These studies demonstrated that bacteria adhering to wood surfaces resided within the structural and vegetative elements of the wood's xylem tissues and were viable; wood was more retentive than plastic; penetration of the inoculum liquid promoted cell adherence to the wood matrix; and conditioning of wood with water before inoculation interfered with bacterial adherence.

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.