Let it Flow: How Flow Cytometry Improves Probiotic Testing Reliability

Why perform probiotic enumeration? 

Probiotic enumeration, or the method for counting the number of microbial cells in a probiotic sample, has wide application throughout the probiotic industry. If you're a brand making a product, you need to know how many cells are in a serving size so you can have the correct potency on your label. If you're a manufacturer, you need to know how much of your raw ingredients to put in a product as well as what you're formulating with when you get the final product. Finally, if you're a raw ingredient supplier, it's important to test for probiotic counts to make sure the product you’re selling meets the expected value on your Certificate of Analysis. 

The primary justification for testing is confirmation of actual values at the specific stage in the production process. Processing and packaging conditions, formulations, as well as storage and transportation can impact the number of probiotics that end up in the final product. Ultimately, when making a potency claim, it's critical to have proof of testing to back up those claims to meet the regulations. 

Traditional probiotic enumeration methods 

The traditional approach to probiotic testing is the plate count method. With a plate count method, a probiotic material is put into a solution and then serial diluted to count individual colonies on a plate. Countable plates are then used to back-calculate how many viable units were in the original powder, with results reported as colony forming units (CFU) per gram or per serving.  

While this is the standard approach to probiotic enumeration, there are some limitations. Mainly, it is very time-consuming, and it can take up to five days to get results. The plate count also provides limited cell viability information, as the cells are required to replicate on the plate under ideal growth conditions to form the colonies. 

Flow cytometry 

With flow cytometry, microbial cells are diluted and put into a phosphate-buffered saline solution inside the instrument to create a homogeneous slurry of all cells in the sample. The cells then flow one at a time through a small capillary in the instrument and a laser passes through each cell individually.  The characteristics of that cell, like cell size or cell shape, then cause the light to scatter in different ways. The detector then captures the information related to cell size and complexity based on how the light is scattering, and specific software performs data analysis. Viability dyes can be added to the slurry prior to running on the instrument to separate live, injured and dead cells based on membrane integrity or some other viability marker. 

Flow cytometry will give quantitative, enumeration values reported as active fluorescent units (AFU) and qualitative values such as whether the cell is alive, injured, or dead and the cell type. 

Advantages of flow cytometry 

Through the method validation and onboarding of the ISO 19344 Protocol B flow cytometry method to accreditation according to ISO 17025 standards, we were able to identify several key advantages of flow cytometry for probiotic enumeration and related applications. 

Precision 

Flow cytometry gives superior analytical precision compared to a traditional plate count method, giving approximately 1/2 the percent relative standard deviation (%RSD) compared to both cultural plate counts and a direct microscopic counts¹. Precision is the closeness of agreement or degree of scatter among individual test results obtained from multiple sampling of the same homogeneous preparation, with the acceptance criteria for validation established as less than 15%. 

Table 1. Three different genera of probiotic bacteria were tested by flow cytometry, cultural plate count, and counted directly by microscope and %RSDs were compared. 

^a Mean count units are AFU/g for flow cytometry, CFU/g for plate counts, and cells/g for direct microscopic counts. 

Real-time analysis 

Flow cytometry provides real-time enumeration data versus waiting two to three, sometimes five days for colonies to grow on a plate under incubation conditions. 

Specificity 

Flow cytometry, when used as a viability assay, can discern live and injured cells. This information is valuable in helping inform product formulations and stability, guiding product development. 

In a validation of this method, we demonstrated that the method gives reliable results when challenged with extraneous matter of different states (live/dead) of cells. Using a population of live cells, a portion of that was taken and heat killed. Live and dead cells were then mixed at different ratios, and the mixtures were evaluated using flow cytometry. Plotted charts for each organism evaluated (figure 2) confirmed that the coefficient of determination (R2) obtained was above the threshold established for the acceptance criteria. This experiment demonstrated that no matter what ratio of those cells are in the product, each can accurately be enumerated by flow cytometry. 

Inclusive to injury 

Another key advantage gained from flow cytometry’s unique qualitative evaluation of live/injured/dead cells, is the concept of viable but not culturable cells (VNBC). Cells in this type of viability state are metabolically active yet non-replicating, and non-culturable. A common situation may be processing injury observed for various manufacturing applications depending on the finished format.  

Injury determination is achieved through the way that flow cytometry measures viability within the ISO 19344 protocol B procedure specifically, based on membrane integrity, using a dual nucleic acid stain. In this procedure, one of the stains can only penetrate intact cell membranes, while the other can penetrate both damaged and intact membranes. The stain differences can then be observed by a differentiation in the fluorescence between live and injured cells. 

These VBNC cells, when incorporated into the final viable cell count, can arguably be considered a more representative estimation of the cell population since plate counts need the cells to replicate under ideal growth conditions. Therefore, plate counts are only determining a portion of the viable cells that have the ability to replicate. 

Discerning Bacillus spp. 

An extension of the basic flow cytometry method allows for a unique ability to identify and enumerate spore materials. Within probiotics, this can be applied to differentiating between Bacillus coagulans and Bacillus subtilis mixtures within a blend, based on the way that each takes up a dye and are subsequently displayed on the dot plot as separate concentrations of live cells. 

This is important, because B. coagulans and B. subtilis growth rates on petri plates are drastically different. B. subtilis will grow quickly and may spread depending on the type of growth media used, causing interference with the B. coagulans' ability to grow and difficulty when counting the colonies due to the interference. What is then observed is inaccurate data that tends to skew lower than the theoretical input or true value. Using flow cytometry for this blend, in particular, is a potential solution to the issue of being able to accurately discern and enumerate that blend of spores using cultural methods. 

Probiotic yeast 

We have expanded the application of flow cytometry to probiotic yeast via matrix extension studies. The same validated ISO protocol used to enumerate probiotic bacteria by flow cytometry has been shown to accurately and precisely enumerate probiotic yeast, such as Saccharomyces cerevisiae subspecies boulardii. Also, due to the differences in their size and cellular characteristics, probiotic yeast can typically be distinguished from the probiotic bacteria when incorporated into a blend.  

Exclusion of extraneous materials 

With the addition of a camera to take pictures of the cells as they're passing through the flow cell, flow cytometry can assist in troubleshooting events that may look atypical, such as differentiating between results that appear as a cell versus debris or extracellular particle. 

Limitations of flow cytometry 

Reporting AFU vs. CFU 

One of the primary concerns of flow cytometry is in the standard unit of measure of this method, where flow cytometry provides a different measurement of viability resulting in the reporting of active fluorescing units (AFU) specific to the fluorescence characteristics of presumed live, dead or injured cells. Conversely, the unit of measure for traditional plate count is the colony forming unit (CFU), used to estimate the number of viable and culturable bacteria based on their ability to replicate on a plate under optimal growth conditions. 

There is not a universal correlation between CFU and AFU, with variance due to probiotic strains, species, product, time, and storage temperature. At time zero when a product is manufactured, the results between plate counts and flow cytometry are consistent, but as the product ages specifically under room temperature or stressed temperature situations there tends to be a divergence where the viability of the plate count cells will decrease faster than the flow cytometry assay² (figure 3). 

As the industry standard for potency remains today using CFU, reporting AFU is one of the biggest hurdles with flow cytometry becoming a mainstream technology for probiotic enumerations. While this hurdle exists, adoption of flow cytometry for use in QC applications to confirm potency is growing. Companies are using flow to verify label claims and release lots of material in the United States since evidence exists to tie those results back to the efficacious dose used to verify the health claim of the probiotic itself. 

Matrix interference 

Product matrix can become a concern with flow cytometry, particularly in situations where there is a low concentration of probiotic cells relative to extracellular material (e.g. granola). In this case, it can be very difficult to parse out the probiotic cells when all that extracellular material is present, as accurate measurements by flow cytometry are achieved when the product is diluted to a range where it can be detected on the instrument in a statistically significant way. We have developed extensive sample preparation techniques to minimize debris interference. While the limit of detection for flow cytometry is higher than that of plate count, the LOD is low enough to be able to enumerate most materials containing efficacious doses of probiotics especially in dietary supplements and their respective ingredients. 

Another form of matrix interference can occur when certain ingredients within the product show autofluorescence or themselves have the ability to bind to the flow cytometry dyes that are intended to bind to the target organism, thereby causing debris interference that could cause inaccuracies in these evaluations. 

Due to matrix challenges, flow cytometry tends to have a higher limit of detection compared to plate count, however this should be a conversation to have with the testing laboratory based on the specific product that may be on the lower end of that spectrum or present an opportunity for a validated method adjustment to navigate around the interference.  

Applications for flow cytometry 

Investigation of below and Out of Specification (OOS) Results 

The unique ability for flow cytometry to discern between live, injured, and dead cells provides the opportunity for the technology to aid in investigations for lower-than-expected plate counts. Some situations where this could apply include issues in product handling, method performance, product manufacturing, and formulation. 

Scenario 1 – Product handling issue 

Consider an example for a probiotic capsule, where a customer is having an issue with low counts. Plate count result replicates in this scenario are reported as 4.5, 5.2, 4.1 and 4.8 billion CFU per serving, but the label claim is 10 billion CFU per serving and the product had been formulated to about 12 billion CFU per serving. 

Flow cytometry can be used in this situation to provide greater context to the results and aid in investigating the low counts. Flow cytometry results in this case are 4.8 billion AFU live cells, 1.3 billion AFU injured and 6.1 billion AFU dead. Flow cytometry therefore identifies that the cells are there, and the product was formulated correctly, but there is a die off issue suggesting concern with the shipment of the product or an issue in the manufacture.  

Scenario 2 – Method interference 

Considering a separate scenario where the customer is facing low plate counts, replicate results are reported at 17, 19, 18 and 16 billion CFU per serving but specifications were set at 20 billion and formulated at 24 billion CFU per serving. In this case, however, the results are opposite of flow cytometry, where we are showing that we have met the label claim at 22 billion AFU live cells, 1.3 billion injured and 0.5 billion dead. 

This scenario suggests a likely recovery issue or an issue with the plate count method that is preventing us from hitting the spec. The next step may then be to optimize the methodology or to consider a different methodology if it is necessary to have the CFU plate count results to support the product’s release. There may also be concerns with viable but not culturable cells that should be investigated. 

Scenario 3 – Conflicting organisms 

Are the organisms in my product making enumeration challenging? Is there something in this product that is causing a problem for us and getting those organisms to grow on the plate?  

In another scenario, the customer has formulated the product using a blend of B. subtilis and B. coagulans. Plate counts are reported as 26 billion CFU per serving, but the label claim for the product is 30 billion total CFU with 15 billion CFU of each Bacillus species. The plate count in this case noted fast-growing B. subtilis spreader colony forming units potentially causing inaccuracies in the recovery of the smaller B. coagulans colonies. 

Flow cytometry has been shown to be able to distinguish and accurately enumerate B. subtilis and B. coagulans. In this case the technology was used as a secondary point of reference to support the hypothesis of low recovery from the plate count test due to differing growth rates of the organisms. The flow cytometry results produced a specific enumeration result for each strain that matched the target input. Decisions for further validation and verification of flow cytometry methods for specific matrices can then be made to update to the best procedure for your product.  

Postbiotic enumeration 

Postbiotics as inanimate cells or their components that confer a health benefit to the host can include many aspects of inactivated intact cells, such as cellular components, proteins, and short chain fatty acids. Flow cytometry is a very promising technology to be able to count these inanimate materials, where plate counts are obviously unable to culture the dead microorganisms. Plate count can be used as a quality control function to make sure that there's no viable cells remaining, but how to quantify is an important quality aspect of these materials and flow cytometry is a potential solution for those postbiotic cells that are somewhat intact or are measured based on the cell number and on some other component like a protein for example.  

Label claim verification 

While there are differences in reported units between flow cytometry and tradition plate count, it is still applicable to use flow cytometry in verification of label claims and product release. This may be displayed on a product label using the direct result from flow cytometry, reported as AFU per serving, or supporting other non-CFU claims such as “billions of live cells”. 

When using flow cytometry for claims, it is important to be able to show substantiative proof that the measured dose enumerated by flow cytometry is the same as the measured dose used in the clinical trial associated with any health claims fora particular strain, blend, or finished format. 

Questions on how this information is applicable to your food process?

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References 

1. Benkowski, A.A. & Schoeni, J.L. Evaluation of an acoustic focusing flow cytometer for enumeration of probiotic organisms. IPA World Congress + Probiota Americas, San Francisco, 2017. 
2. Visciglia, A., Allesina, S., Amoruso, A., De Prisco, Annachiara, D.R., Bron, P.A., Pane, M. Assessment of shelf-life and metabolic viability of a multi-strain synbiotic using standard and innovative enumeration technologies, Frontiers in Microbiology, VOLUME=13, 2022, https://www.frontiersin.org/articles/10.3389/fmicb.2022.989563, DOI=10.3389/fmicb.2022.989563, 1664-302X. 

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