Enhanced tetrazolium violet reduction of Salmonella spp. by magnesium addition to the culture media.

Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities.

Optimization of the reactional medium and a food impact study for a colorimetric in situ Salmonella spp. detection method.

Foodborne pathogens are still a major concern for public health authorities. In this paper, we describe the optimization of a previously reported method which combines a highly specific capture of targeted food pathogens with an intracellular staining method. The reaction medium was optimized to simultaneously allow specific enrichment of Salmonella and maximize the staining of the target pathogen. This in situ colorimetric concept was evaluated with a broad range of food samples artificially contaminated with low levels of stressed Salmonella to mimic natural contamination conditions. This direct detection method compared favorably to a commercially available immunoassay system (Vidas® UP Salmonella), for cooked meat, dry milk powder and egg products. Globally 88% agreement was obtained between the two methods with a sensitivity of 80% and a specificity of 100% for the tested method. Main discordances were obtained with food matrices having high levels of competitive Gram negative microflora. These observations show that the design of an adapted culture medium is necessary to enhance the specific in situ capture and revelation system.

Diminution of 2,3,5-triphenyltetrazolium chloride toxicity on Listeria monocytogenes growth by iron source addition to the culture medium.

Tetrazolium salts (TTZ) such as 2,3,5-triphenyltetrazolium chloride (TTC) are readily reduced by bacterial populations of various genus. The reduced form of these redox indicators is conspicuously colored allowing a quick and easy detection of growth. The studies are mainly confined to Gram negative bacteria because of an important toxic effect of tetrazolium salts on Gram positive bacteria. Indeed, we observed an important impact of different tetrazolium salts on Listeria monocytogenes growth, curiously limited to an increase in the duration of the lag phase. In this study, we demonstrate that increasing the iron concentration in a medium containing TTC leads to a significant decrease of the lag phase. L. monocytogenes growth was kinetically measured and growth parameters were estimated using the Baranyi model. While lag phase diminution was found to be iron concentration dependent, growth rate was not affected. Addition of iron enables growth of some strains totally inhibited by a 0.4 g/l of TTC and for the other a significant reduction of the latency is observed. The nature of the mechanism resulting in a decrease of the observed lag phase remains unclear. Then, the use of iron supplementation may be proposed to overcome the inhibitory effect of TTC on L. monocytogenes.

Simplified detection of food-borne pathogens: an in situ high affinity capture and staining concept.

Food industries need simple, rapid and cost-effective solutions for pathogen detection in food and environmental samples. In this paper, we describe a simple but novel detection concept combining an affinity capture surface and intracellular metabolic marker to visualize the bacterial presence on the affinity surface. The surface of a Solid Phase Support (SPS) is functionalized with specific phage tail proteins targeted to the bacterial pathogen of interest. The SPS is placed directly into the primary food enrichment bag after stomaching. Following incubation, the captured bacteria are visually detected in situ as a result of the bacterial reduction of the colorless soluble substrate triphenyltetrazolium chloride (TTC) (present in the primary culture medium) to an intracellular red insoluble formazan product. Detection on the SPS is observed as an intense red color after 22 to 40 hours of enrichment. This is not impaired by the presence of food particles and the natural background microflora. The in situ method significantly simplifies pathogen detection by eliminating any post-enrichment intervention that is necessary in the traditional methods of analysis. We have demonstrated the application of this new approach for the detection of Escherichia coli O157: H7, Listeria spp. and Salmonella spp. in artificially contaminated food samples.