In food analyses, it is desirable to obtain multiple properties of food microbial pathogenic cells simultaneously, because this enables the determination of the physiological status of these pathogens, i.e. knowledge of their status as to whether they are viable, dead or stressed. Moreover, in food microbiology, it may be necessary to have techniques capable of rapid enumeration of pathogenic microbes, which can distinguish between viable, metabolically active cells and dead cells as well. This is very practical because cells differ in terms of their metabolic or physiologic patterns and thus by using an appropriate technique one may obtain information regarding the cell type or even establish the presence of different populations within a given matrix based on the structural or physiological patterns of the species that are present. This knowledge is highly desirable for purposes in the food industry where food development is the key activity and also it may provide information on the mechanisms that lead to food spoilage. Techniques capable of rapid analyses and enumeration of microbial pathogens in foods are therefore highly needed and flow cytometry is one of the technologies that can be used to count and analyze cells or other particles in food, particularly in the form of fluids. Unlike other analytical techniques, which provide measurement for a single parameter for the entire population, flow cytometry enables the possibility to get data for every species/particle that has been detected. In flow cytometry the single microbial cells are stained with a specific dye, passed through a light beam and then measurement of the fluorescence pattern on a cell‐by‐cell basis follows (i.e. the interaction of each cell with the beam is measured and correlated with the cell characteristics or cell components).
Flow cytometry combines attractive attributes due to fluidics and optics, as well as multiparametric data analysis (with both data discrimination and data classification possibilities) to enable quick investigation and data correlation for multiple physical and biochemical attributes of either biological systems (cells mainly) or microspheres.
The principle of flow cytometry measurements exploits the differences that exist between the refractive indices of biological cells and their surrounding media, such that when light impinges upon the cells, it becomes scattered (Julià et al., 2000). Generally, the forward scattered light (FSC, light scattered at low angles) in flow cytometry measurements is useful in providing information about the cell size (Julià et al., 2000). In instances where light is scattered either in an orthogonal or near‐orthogonal direction, there is a separate detector, for example the SSC (side scatter) detector, that collects and measures this light. Alternatively, other detectors such as the PMT (photomultiplier tube) are used with flow cytometry instruments to collect and measure fluorescence radiation from macromolecules found within the biological cells. In order to enable the detection of a number of cellular parameters that are associated with functions or structural components of the cell simultaneously, a combination of light‐scattering and fluorescence measurements on either stained or unstained cells has been encouraged. In flow cytometry, fluorescent dyes, such as rhodamine 123, have been used to stain viable eukaryotic cells as well as microorganisms (Diaper et al., 1992; Quiró et al., 2007; Resnick et al., 1985).
Dye staining can be used to gain information on the integrity of cell membranes, depending on their dye exclusion or dye retention characteristics. Examples of exclusion dyes include propidium iodide and ethidium bromide, which normally stains nucleic acids and other macromolecules. If these dyes are used to stain cells, and happen to stain macromolecules and other biological components such as nucleic acids, then it implies that the cell membrane is permeable and therefore the cell is dead.
In order to distinguish dead and live cells, it is required that a combination of a cell‐permeant dye such as those that belong to the class SYTO family in combination with cell impermeant dyes such as propidium iodide be applied in conjunction. In the case of dye retention approaches, nonfluorescent cell‐permeant esterase substrates are normally used to produce fluorescent products, which become trapped inside the cell by mechanisms that can be explained by their electrical charge as well as their polarity.
In order to detect food microbial strains using flow cytometry, immunofluorescent techniques have been devised to serve this purpose, such that it is possible to detect microbial cells singly. However, the process requires the availability of antibodies which are obtained from the appropriate organisms (Álvarez‐Barrientos et al., 2000).
In other developments, a combination of flow cytometry together with biochemical techniques has been reported in applications to investigate the heterogeneity of bacterial cultures, mainly the Bacillus species, where live and dead endospores have been discriminated based on either their distinct cytometric scatter (Stopa, 2000) or cytometric scatter in combination with nucleic acid stains (Comas‐Riu and Vives‐Rego, 2002).
Oxidative metabolism indicator tetrazolium dyes, such as cyanoditolyl tetrazolium chloride (CTC), have been reported to detect the type of respiration in aerobic microorganisms (Kaprelyants and Kell, 1993).
Generally, flow cytometry is attractive in food forensic issues due to its ability to detect microorganisms and microspheres in foods at relatively low concentrations within a short time. For example, there have been cases that involve deliberate contamination of foods and food‐related products by microorganisms such as bacteria and fungi (yeasts and moulds) and the presence of these microbes in food products, even at low counts, can severely affect the quality of the food and may have the potential to tamper with the health status of the consumers. The microbial composition of food products is generally taken as an indication of the types and kinds of normal biota of the raw ingredients used to produce the food product and is reflective of how hygienic were the procedures used during processing of that particular food product.
The possibility of multiple labeling enhances the advantages of flow cytometry, as it makes it possible to detect different organisms or even microorganisms at different stages in the same food sample. Therefore, the availability of methods capable of differentiating Gram‐positive and Gram‐negative bacterial populations is desirable in order to provide the information needed about the source of contamination. These methods include those that are used for Gram‐staining for flow cytometry. An example of such a method combines the use of two fluorescent DNA binding stains, mainly a membrane‐permeable stain SYTO 13 dye and hexidium iodide (HI). The hexidium iodide is normally blocked by the lipopolysaccharide layer of Gram‐negative bacteria and is therefore only permeable to Gram‐positive bacteria which possess a destabilized lipopolysaccharide layer (Mason et al., 1998).
Apart from this, there is another technique that makes use of Oregon Green‐conjugated wheat germ agglutinin (WGA) in combination with hexidium iodide (HI), such that the WGA will bind to the N‐acetylglucosamine that is present in the peptidoglycan layer of the cell wall of Gram‐positive bacteria, while the HI will bind to the DNA of all bacteria (Gram‐negative and Gram‐positive) after EDTA treatment and incubation at an appropriate temperature (Holm and Jespersen, 2003).
Flow cytometry can also be used in cases where there is a need to detect specific pathogenic microbes in food products, in which case either monoclonal or polyclonal antibodies conjugated to fluorochromes such as fluorescein isothiocyanate (FITC) or phycoerythrin are employed (Donnelly and Baigent, 1986; McClelland and Pinder, 1994; Völsch et al., 1990). However, this approach suffers from insufficient sensitivity, as its detection limits for the pathogenic microbes in complex matrices such as foods as per the regulations is very low, where it cannot achieve such low levels of detection limits. To counter this shortcoming, strategies that employ the amplification of the microbial count by culture have been suggested prior to analysis using flow cytometry.
Normally food samples may be regarded as contaminated if the microbial count exceeds 100 counts/g. The whole fixed microbial cells in foods can be identified and counted directly using techniques such as fluorescent in situ hybridization (FISH).
Flow cytometry can be very instrumental in the dosage enumeration verification and evaluation of oral food probiotics, which are living microorganisms known to have health benefits when ingested at an optimal concentration (108–109 living microorganisms), either as a food component or as non‐food preparation (Guarner and Schaafsma, 1998). For food probiotics, flow cytometry can be used to provide the evaluation regarding the viabilities of probiotics under certain treatment procedures such as storage conditions, storage time, membrane integrity, etc. (Lahtinen et al., 2006).
Flow cytometry is a relatively new technique and its application may depend much on the availability of the facility as well as skilled personnel to handle the instruments and correctly interpret the data generated from such techniques.