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PhAST Blue - Non viable Cell staining
The evaluation of microorganism survival is currently based on cultivation assays or more recently with viable PCR methods. Plate counts are time-consuming and biased by the fact that some microorganisms remains viable but non cultivable. On the other hand, a viable PCR method is a promising approach, but requires a good background in molecular biology. Methods based on fluorescence microscopy, have been successfully used for many years and one of the most used reagents to stain dead cells has been Propidium Iodide. Nowadays their azide derivative, Propidium Monoazide, combines the specificity of staining dead cells with the strong capability to an irreversible bound to DNA by the means of photo -reactive process. Now is possible to stain dead cells prior to fixation and store the sample until the analysis.
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PhAST Blue - Flow Cytometry
Often, for safety reasons or for convenience, it is frequently necessary to fix cells prior to analysis. Later data analysis will be less ambiguous if nonviable or damaged cells can be eliminated. Flow cytometry determination of viable and non-viable cells in fixed samples can be accomplished by using Ethidium monoazide (EMA) and Propidium monoazide (PMA). They are photo-reactive derivatives from Ethidium Bromide and Propdium Iodide, andboth are positively charged molecules which are excluded by cell with intact membranes, but enter cells with damaged membrane,. These dyes can be photo- chemically cross-linked with short exposure to visible light after the excess dye is washed away, the cells are fixed.