Measuring Probiotics in CFUs Only Gives Part of the Story, Pharmachem Says of Move to Flow Cytometry

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Pharmachem Laboratories (Kearny, NJ) is now using flow cytometry to measure the viable cells in its probiotic formulas-a method than it says is faster, less expensive, and more accurate than traditional plating techniques. What’s more, while plating only measures probiotic formulas for colony forming units (CFUs), flow cytometry also reveals the viable, but non-culturable, cells (VBNCs), which can also be key in determining a formula’s activity potential.

Alexis Collins, director of scientific affairs for Pharmachem, says that “at least one-third” of Pharmachem’s probiotic customers are already request flow cytometry be used on their unique formulations, and Probiotical (Novara, Italy), Pharmachem’s partner, was the first to adapt the method. Flow cytometry tags probiotic cells with fluorescent markers, producing a total viable cell count in active fluorescent units (AFUs), rather than the CFU counts from traditional plating.

“You will often see higher numbers of viable cells versus CFUs, because flow cytometry measures all viable cells,” Collins explains, in a press announcement. “At times, a probiotic ingredient can contain up to 50% viable, but non-culturable cells (VBNCs). The total count of viable probiotic cells equals CFUs plus VBNCs.”

Although VBNCs are overlooked in plating, when they are outside of the body, they can still have an enormous impact and show “strong activity” once they reach the gut, even if they are dead probiotic cells, according to Marco Pane, product development specialist for Probiotical.

“When probiotics-and other enteric microorganisms-are outside their native environment they enter into a VBNC state,” Pane tells Nutritional Outlook. “The VBNC state is induced by industrial fermentation, freeze-drying, and stability work because these processes inevitably stress the bacterial cells outside their native environment. Once ingested, these is the possibility of part of the VBNC cells to resuscitate, which is a common mechanism massively reported in scientific literature on enteric pathogen bacteria.”

While Pharmachem will continue to provide counts of CFU/g on its product data sheets, the issue of CFU counts excluding VBNCs is a key reason why it is recommending its customers use flow cytometry results rather than relying only on plating. Probiotical’s Pane adds that flow cytometry is also more accurate than plating, as the variability of plating results can range from 30% to 50%.

“Plating is just too variable to accurately measure probiotics post-formulation,” Pharmachem’s Collins says, in the press announcement.

Ease of Use and Cost

Flow cytometry can also be a much more convenient option than plating, especially in the speed of getting results. Whereas plating can take 48–72 hours to obtain results, flow cytometry can provide results in triplicate in less than an hour, according to Pane. It can also be easier to adopt when working with a variety of different probiotic strains. Plating requires specific protocols for different species of probiotics, even if they belong to the same genus, Pane explains. That’s not the case with flow cytometry, where “one method and three protocols can be used for all probiotic species,” Pane tells Nutritional Outlook.

Then there’s the issue of cost. Pharmachem’s Collins and Art Keegan, quality manager for Pharmachem West, say flow cytometry is actually less expensive than plating once you take into account the time saved and the increased accuracy. Investing in a flow cytometer costs between $60,000 and $250,000, but usually only one technician is required to run samples, Keegan and Collins tell Nutritional Outlook. Plating, on the other hand, typically requires multiple technicians.

Bioform Solutions (San Diego), which has been using flow cytometry to measure probiotics for large companies for the past two years, charges $175 for a sample run in triplicate, according to Keegan and Collins. Meanwhile, plating results cost from between $65 to $120, although that’s only for a single sample analysis, they note. Probiotical’s Pane also says that plating becomes more expensive in the long run because so many plates need to be incubated for just a single sample, making running an analysis in triplicate “basically impossible.”

Read more:

2016 Probiotic Research Update

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A 2016 Update: Promising New Science on Probiotics

Michael Crane
Associate Editor
Nutritional Outlook Magazine
michael.crane@ubm.com