Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Wilkinson, Martin G.

doi:10.1016/j.tifs.2018.05.006

Cited by 120 publications

(105 citation statements)

References 56 publications

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“…Previous research on plate count enumeration showed an average CV of 15%, which agrees with our findings (Table 3), however, ISO acceptable levels can vary between 15 and 35%. Although the ISO method for flow cytometry reported high variation, literature reports from 3 to 7% CV (Wilkinson, 2018) similar to our results. Overall, ddPCR had a CV of 2.0 ± 1.0% calculated in scientific notation, as opposed to reporting in logarithmic form which lowers the overall CV.…”

Section: Discussionsupporting

confidence: 90%

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains

et al. 2020

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Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability-up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

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Section: Discussionsupporting

confidence: 90%

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains

et al. 2020

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“…Given the alterations in antibody binding to cells subjected to simulated food processing treatments recorded in this study, a cautious approach to the use of antibodies to detect bacteria in food systems in advised. Significant discrepancies between FCM counts and traditional plate counts (a feature of comparative studies between the two techniques [19]) may arise from the inclusion of nonviable but nonetheless antibody-positive cells in an FCM-derived count (false positives). In this study, we have demonstrated the presence of nonviable cells which give a strong reaction to the antibody (e.g., heat-treated cells), as well as catastrophically damaged cells which showed dramatic, generalized increases in autofluorescence in all channels (e.g., CTAB-treated cells).…”

Section: Discussionmentioning

confidence: 99%

“…Both strains had similar lower MFI/MFI (us) values compared to controls following exposure to 1% NaCl. Overall, there were no significant differences when we compared the fluorescence values for strain 1 and strain 2 (P Ͼ 0.05 [unpaired t test], two-tailed df ϭ 19) and no significant differences between antibody A and antibody B labeling (P Ͼ 0.05). When we compared the treatments, a one-way analysis of variance using Dunnett's multiple-comparison test showed that, overall, five treatments differed significantly from the control (P Ͻ 0.05).…”

Section: Flow Cytometric Analysis Of Bacterial Cells (I)mentioning

confidence: 99%

“…Intracellular esterase activity, which is probably the most common form of metabolic activity detected by FCM (19), was measured using the dye calcein acetoxymethyl ester (calcein-AM), a nonfluorescent substrate that is converted by intracellular esterase activity into the fluorescent calcein and which is subsequently retained intracellularly. Calcein-AM has been used as a viability marker because of its high cell retention and pH-insensitive fluorescence.…”

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confidence: 99%

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Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis

Kennedy

Cronin

Piterina

et al. 2019

Appl Environ Microbiol

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The effects of heat and chemical treatments on Staphylococcus aureus viability and physiology and their subsequent effects on antibody binding ability and cell morphology were measured. Treatments included lethal and sublethal heat; exposure to organic acids, salt, and sodium hydroxide; and freeze-thawing. Strain-related differences in viability were noted depending on treatment and were reflected in changes in physiology as monitored by flow cytometry (FCM) using three different staining protocols: SYTO 9/propidium iodide (PI), DiOC2(3), or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments that resulted in significant losses in viability as measured by plate counting were reflected better by the first two staining combinations, as intracellular calcein-AM uptake may have been impaired by certain treatments. FCM analysis using labeling by commercial anti-S. aureus antibodies indicated that differences in cell physiology as a result of treatments influenced immunofluorescence detection. The ratio of the mean fluorescence intensities of stained cells to those of unstained cells [MFI/MFI(us)] varied with treatment, five of these treatments, including freeze-thaw, citric acid, oxalic acid, NaCl, and NaOH treatments, resulted in significantly lower fluorescence values compared to controls. IMPORTANCE FCM data indicated that cells conventionally considered to be dead and which would not give rise to CFU in a plate count assay, e.g., cells heated to 80°C, were labeled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye, these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physicochemical treatment were strain related, reflecting the complexity of the phenomenon under study and illustrating that substantial validation of any new antibody detection-based method, including physiological staining and cell sorting, should be undertaken. Researchers should be aware of physicochemical treatments causing false-negative results: in this study, freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy carried out on treated cells revealed a range of morphological changes resulting from physicochemical treatments which may have hindered antibody binding.

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“…The brewing industry has long had an interest in applying FCM to the microbiology of the brewing process. The measurement of the DNA content of yeast cells was one of the first major applications of flow cytometry in brewing [19,20,21,22] and is still one of the main applications of this method in this field. Yeasts are widely used to produce commercially important compounds such as pharmaceutical agents, enzymes for food industry, bioethanol, and chemicals, including organic acids.…”

Section: Brewingmentioning

confidence: 99%

Updates on the applications of flow cytometry in microbiology

2019

Lett Appl NanoBioSci

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Flow cytometry (FCM) was first developed for medical and clinical applications such as hematology and oncology. Although these areas still account for the vast majority of publications on this technique, during the past few years it has been also introduced in other areas, such as optimization and monitoring of biotechnological and environmental processes, pharmacology, toxicology, bacteriology and virology. In the food and drinks industries, the time required for conventional microbiology tests can lead to substantial delays in product release to the market. FCM has been used in conjunction with viability markers for rapid counting of yeast, molds and bacterial cells, including foodborne pathogens and microbial contaminants, in food products as well as for monitoring and improving the final products quality. FCM is an excellent tool, still unexplored in clinical microbiology, allowing for detection of cellular and non-cellular components in different clinical specimens, the study of antimicrobial activity, allowing for rapid and direct antimicrobial susceptibility testing and for the investigation of resistance mechanisms. Recent FCM developments important for addressing questions in environmental microbiology include the study of microbial physiology under environmentally relevant conditions, the development of new methods to identify active microbial populations and to isolate previously uncultured microorganisms and of high-throughput autofluorescence bioreporter assays. Moreover, the technological advancements will make possible the miniaturisation and automation of FCM devices, allowing to revolutionize their applications in the near future. The purpose of this minireview is to update the current applications of FCM in different fields of applied microbiology, and to highlight the main advantages and pitfalls for each of them.

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Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

Cited by 120 publications

References 56 publications

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains

Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis

Updates on the applications of flow cytometry in microbiology

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