Estimation of Microbial Viability Using Flow Cytometry

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Type Article
Original languageEnglish
Article numbere72
Number of pages13
JournalCurrent Protocols in Cytometry
Issue number1
Publication statusPublished - 14 Apr 2020
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For microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold-standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so-called viability stains and to develop protocols for more routine assessments of microbial viability. This unit provides a commentary and several protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.
One of the most basic questions that a microbiologist might ask of a microorganism is whether it is alive or not, and in microbiology, it is often necessary to determine the number of living (viable) cells in a sample or culture of interest. However, perhaps surprisingly, this is a question that is not always easily answered (Davey, 2011). The gold standard for determining the number of viable microbial cells in a sample is usually achieved by plating a 0.1- to 1 ml sample of cells (diluted as required) onto an agar plate (Hattori, 1988; Postgate, 1969) and scoring as viable (a posteriori) those cells that were able to form visible colonies. The culture viability is then defined as the ratio of these cells to the total cell count in the original sample, which is determined microscopically. However, there are several problems associated with this technique, not the least of which is the length of time required to obtain the results. For some slowly growing organisms (e.g., mycobacteria), it may take several weeks to determine how many cells were viable (as defined by the above technique) in the original sample. Even when the sample contains quickly growing organisms and the plates are incubated under optimal growth conditions, a minimum of overnight growth is usually required before the resulting colonies can be counted. In clinical situations and for economic reasons, such a delay is often unacceptable. Thus, many rapid methods have been developed to allow a speedier assessment of the viable microbial load in a sample.
These alternative, rapid viability measurements include a variety of stain-based methods. The so-called vital stains that have been used in attempts to estimate microbial viability fall into three broad categories. (1) Some dyes are excluded by intact membranes of viable cells but enter freely into cells where the permeability barrier has been lost. Therefore, the presence of the dye within the cell may be expected to be correlated with cell death. (2) Other dyes are actively accumulated by viable cells; thus, the stained cells are deemed to be viable. (3) Membrane-permeant nonfluorescent precursors can be converted by the activity of intracellular enzymes of living cells to membrane-impermeant fluorescent molecules; again, fluorescent cells are deemed viable. Each of these dye-based approaches is discussed in more detail below.
Although usually considered to be the gold-standard measure of viability, a plate count actually only indicates how many of the cells can replicate under the conditions provided for growth. In the case of environmental samples, the laboratory media, the temperature, and other factors may differ substantially from those in the original sample, thus, the proportion of cells that can divide and form colonies may be much lower than the number of cells that would score as viable using the dye-based rapid methods. Nevertheless, the plate count method has remained the gold standard, in part due to the fact that traditional microscopic analyses of stained cells are time consuming and can lead to operator fatigue; thus, conclusions are normally drawn from the analysis of at best a few hundred cells. Furthermore, microscopic examination is largely a qualitative technique, wherein a judgment of alive or dead is all that is possible, and the interpretation of the extent of a cell's staining may vary among operators.
Flow cytometry offers an alternative method of determining the amount of fluorescent dye taken up by each cell in a population. Since quantitative measurements can be made very rapidly on a large number of individual cells, an accurate picture of the distribution of dye uptake by many thousands of cells is possible within a few minutes.
This unit begins with a discussion of the various advantages and disadvantages of classical (proliferative) versus cytochemical (dye-based) viability assays. It discusses the three classes of cytochemical methods in greater detail, and provides instructions for three protocols. Finally, it discusses the use of cell sorting in conjunction with tests for microbial viability.