Let it Flow: Flow Cytometry for Probiotic Enumeration – Q&A with the experts
In a recent webinar, Let it Flow: Flow Cytometry for Probiotic Enumeration Part 2 – Q&A with the experts, Andrzej Benkowski and Dave Roth discussed some common questions about flow cytometry for probiotic enumeration. Below is a summary of their informative discussion.
Need a refresher on flow cytometry and probiotic enumeration? Check out this blog, or watch the first instalment of the probiotic webinar series!
Q: Are there expected differences between plate count and flow cytometry results with the same product? How do you expect these differences to change over the shelf life?
A: Flow cytometry and plate count methods are quantifications using different markers of viability. Flow cytometry measures cellular membrane integrity, and results are reported as Active Fluorescent Units (AFUs). Plate counts measure the cell’s ability to replicate, and results are reported as Colony Forming Units (CFUs). Fresh, un-stressed cultures typically have comparable AFU and CFU measurements, but differences can appear as the material degrades or exogenous ingredients are introduced. Over time, certain types of cells may stay alive (viable) based on their membrane integrity but be non-culturable (not replicating), so flow cytometry results may decrease less than that of their cultural enumeration counterpart. Additionally, flow cytometry evaluations, in general, have a lower percent relative standard deviation (%RSD), meaning measurements are more precise than plate counts.
Q: How long does it take to process a sample and receive results?
A: It takes approximately one hour to prepare a single sample, followed by half an hour on the instrument. Overall, it only takes a couple of hours to obtain flow cytometry results. This can be seen as an advantage over plate counts, which can take days for results due to the need for incubation to allow colonies to grow.
Q: What are the challenges associated with different matrices in a flow cytometry study?
A: Our ISO flow cytometry method has been applied to gummies, beverages, dairy, granola bars, and other matrices. If you can think of it, we have tested it! In order to obtain accurate and precise results by flow cytometry, a method requires dilution to obtain a clean sample to put on the instrument. The ability to dilute helps mitigate any potential debris interference. The lower the concentration of probiotic cells relative to the extracellular material in the matrix, the more difficult it is to obtain a clean sample. High concentration samples such as probiotic raw materials, pre-blends, and single ingredient dietary supplements are typically straightforward to test by flow cytometry. Other finished product samples such as fortified foods and beverages, dietary supplements with other active ingredients, and microencapsulated probiotics can pose challenges and may require special preparations to obtain a clean sample.
Q: What is the limit of detection for flow cytometry?
A: Flow cytometry has a higher LOD/LOQ than plate count methods. For probiotics, we rarely measure numbers close to the LOD because a typical efficacious dose is in the millions or billions of cells. Our validation studies have shown detection limits as low 10^5 or 10^6 AFU, but the true limit depends on the matrix and product concentration. Filtering and dilution steps can lower the LOD/LOQ of complex products, and these steps can be optimized on a case-by-case basis.
Q: What type of data is reported by flow cytometry?
A: Flow cytometry provides a count of live, dead, and injured cells. For spores, we provide live spore count and dead/damaged spore count. While only the live cell count is listed on probiotic product labels, having the dead and injured counts provides additional information for future investigations into cell counts, unexpected results, and formulation adjustments. Check out these case studies for more information!
Q: Can flow cytometry be used to quantify a postbiotic product? What are some challenges associated with postbiotic potency measurements by flow cytometry?
A: A postbiotic is a preparation of inanimate microorganisms and/or their components that confers a health benefit on the host (ISAPP, 2021). For an accurate postbiotic quantification by flow cytometry, the dead cells must be somewhat intact because the measurement of viability is dependent on a cellular membrane integrity using the method being routinely employed at Eurofins (ISO 19344 Protocol B). If the lethality process with which a postbiotic is manufactured causes the cells to become completely obliterated into their components, cell count measurements by flow cytometry become incompatible and arguably irrelevant. The quality management of postbiotic materials as it relates to product labeling and dosage is an ongoing endeavor.
Q: Are there complications with probiotic (live cell) enumeration when postbiotics (dead cells) are present in the same product?
A: A typical lyophilized or freeze-dried probiotic product will contain a proportion of dead and injured cells because of the drying process and this proportion will increase over time as the material naturally degrades. Dead cells present in the probiotic can interfere with a postbiotic quantification when a blend of the two materials is measured by flow cytometry. There may be opportunities to look at probiotic spores in conjunction with postbiotics to have each population individually measured in a blend by flow cytometry due to spores’ unique characteristics compared to postbiotics produced from lactic acid bacteria.
Q: Can you differentiate species with flow cytometry?
A: ISO 19344 Protocol B, the flow cytometry enumeration method routinely run by the Eurofins Madison, WI laboratory, provides a count of total live, dead, and injured cells. Standards are being developed to distinguish species by flow cytometry using antibody tagging or fluorescence in situ hybridization (FISH) and will be employed by Eurofins once published.
Q: Can you use flow cytometry for yeast enumeration?
A: Yes, we have shown that ISO 19344 Protocol B can be used to accurately enumerate probiotic yeast such as Saccharomyces cerevisiae subsp boulardii. We can also distinguish yeast from bacteria based on cell size.
Q: Do regulatory bodies allow AFU claims on products?
A: Regulations vary by country, but the US FDA states that dietary supplement ingredients need to be labeled in milligrams. However, the dose of the active ingredients in probiotic supplements – the number of cells of live bacteria – cannot be accurately quantified by weight. For example, 100 mg of Lactobacillus rhamnosus is a different concentration of cells compared to 100 mg of Bifidobacterium animalis subsp lactis. In 2018, the FDA released a draft guidance statement saying products should also list CFUs on labels for a more accurate representation of probiotic content. While CFU plate counts are the “gold standard” for probiotic enumeration, the draft guidance recognizes other methods and units of measure for probiotic quantification exist and have the potential to more accurately and efficiently quantify the number of viable cells in a product.
In the United States, a quantity of probiotics measured in AFU may be used on a label along with the required listing of the probiotic content in milligrams, as long as the labeled AFU dose can be correlated to the way the dose was measured to establish the health claim. In other words, scientific justification is needed if the efficacious dose was measured in a different way during the clinical trial to show a health benefit or a structure-function claim.
Q: Where do you see probiotic testing going in the future?
A: [Benkowski] Long term, in my opinion, I believe there are enough advantages to flow cytometry (and/or other technologies, such as real-time or digital PCR) to create a paradigm shift in the way probiotic enumerations are conducted away from plate count methods. I think we are years away from this shift, but I do believe it is an eventuality.
Learn more about our Probiotic testing capabilities with the two part webinar series:
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