What Are Colony-Forming Units and Active-Fluorescent Units?

During a Biotech2050 podcast, Pendulum co-founder and CEO Colleen Cutcliffe, PhD, said there is a challenge in trying to differentiate companies and products that:
  • Are truly founded on science
  • Are saying they're founded on science—but are really just great marketing innovation 

“There's been a big push of just simply looking at the number of [colony forming units] on a probiotic bottle … and more must be better,” says Dr. Cutcliffe. “And that's how many people evaluate probiotics—[by] looking for the one that has the most [colony-forming units]."

On December 1, 2015, the USP General Informational Chapter <1223> Validation of Alternative Microbiological Methods was revised and became official. The revision contained a section discussing the limitations of colony-forming units (CFUs) in terms of identifying only those microorganisms that readily grow on solid microbiological media. 

The section highlights CFU’s shortcomings as a “gold standard” for method validation when there are many signals available—other than CFUs—for the detection, enumeration, and identification of microorganisms in:

  • Water
  • Air
  • Pharmaceutical ingredients, and 
  • Drug products 1
In this article, we will answer the following questions:
  • What are colony-forming units (CFUs)? 
  • How do you calculate CFUs?
  • Is a high CFU a marker for a probiotic’s quality?
  • What are active-fluorescent units (AFUs)?
  • How do you calculate AFUs?
  • What is a safe AFU/CFU level?
  • What role do AFUs and CFUs play in how you eat foods?

What are colony-forming units (CFUs)?

CFUs indicate how many “living and active” beneficial-bacterial cells you are ingesting in one serving of the probiotic.   

A “colony” of bacteria is formed when one bacterial cell replicates into two, and then four, and so on in one spot on a physical surface, thus, resulting in a visible colony that can be counted. 

On the label, some makers list CFUs "at time of manufacture." Because the amount of CFUs may go down as the probiotic product gets older, it's important for the CFU count to tell you how much you're getting when you use the probiotic before it expires. 2

How do you calculate CFUs?
  • When scientists want to know how many microorganisms there are in a solution of bacteria or fungi, they can dilute a sample of microbes and spread it across a petri plate 
  • By doing this, microbiologists can count groups of microbe colonies with the naked eye
  • Each colony is assumed to have grown from a single CFU
  • Scientists can then use the CFU count to determine roughly how many microbes were in the original sample
    • For example, if 200 colonies are counted on a plate made with a 1-milliliter sample of a solution diluted 1,000 times from its original strength, the original solution contains approximately 200,000 CFUs per milliliter 3
Is a high CFU a marker for a probiotic’s quality?

As Dr. Cutcliffe suggested, many companies call it a day after touting their probiotic’s high CFUs. 

This might lead the consumer to think that a higher CFU means better quality.

It might also lead the manufacturer to believe that a huge CFU count justifies a higher price.

While Pendulum actively measures the CFU of its strains throughout its supply-chain and post-production process, it does not believe it is essential to convey the CFU count of its product. 

“We think that the current probiotic-industry practice of highlighting CFUs is mostly a marketing ploy,” says John Eid, PhD, CSO and Co-founder of Pendulum. “CFU is not synonymous with efficacy.” 

Pendulum believes the only way to demonstrate efficacy is to run a double-blinded randomized placebo-controlled clinical trial.

“We did this, and demonstrated that this concentration of our formulation was both safe and beneficial for people with Type 2 diabetes,” says Orville Kolterman. MD, chief medical officer at Pendulum

What are active-fluorescent units (AFUs)?

Active-fluorescent units (AFUs) also represent a count of “living and viable” bacteria in a probiotic. 

When evaluating the strains in Pendulum medical probiotics, CFUs and AFUs differ only in the way they are measured, but are calibrated in order to be in agreement with each other. 

For example:

  • CFUs are measured by plating on an agar plate and 
  • AFUs are measured by a flow cytometer after fluorescent staining
How do you calculate AFUs?

When calculating AFUs  in Pendulum products (e.g. Pendulum Akkermansia and Pendulum Glucose Control), Maggie Stoeva, PhD, senior scientist at Pendulum, says AFUs use flow cytometry to line cells up in a single file, and then blast them with light and measure what comes back. 

“We combine this with dyes that stain cells depending on their membrane integrity,” says Dr. Stoeva. “In other words, live ones will stain one color, and dead ones a different color. This way, when they pass through the flow cytometer one by one, the instrument counts the live ones—and the dead ones.”

AFUs include damaged as well as dead cells. Depending on the degree of severity, Dr. Stoeva says damaged cells are sometimes referred to as viable (potentially alive and metabolically active) but not culturable—meaning they will not form a colony on a plate.

“This is because the flow cytometry can also count damaged and dead cells, since it relies on dyes rather than actual growth/division and colony formation,” says Dr. Stoeva.

There are 2 billion total AFUs for a daily dose of Pendulum Glucose Control—5 billion per pill.

What is a safe and effective AFU/CFU level?

Usually probiotics are taken with the goal of establishing—or increasing—the amount of specific bacterial species in the gut microbiome

So how many AFUs or CFUs does it take to make sure Akkermansia muciniphila is growing in the gut microbiome? 

Or Bifidobacterium infantis

Or Clostridium butyricum? 

Several factors affect how successfully the bacteria will be able to grow in your gut microbiome—such as:

  • The medication you may be taking
  • Your diet
  • Exercise levels
  • Your age, and 
  • Your overall health

If you are trying to figure out how much of a probiotic to take, first talk with your healthcare professional.

Click here to become a Pendulum Akkermansia member and get your first bottle. 

And don't forget to follow Pendulum on social media.


Before you consider any of these gut-microbiome dietary solutions, talk to your healthcare provider. 
The FDA has not approved or evaluated these statements.
Pendulum products are not intended to diagnose, treat, cure, or prevent any diseases.
Results may vary

1. https://www.europeanpharmaceuticalreview.com/article/37416/the-limitations-of-the-colony-forming-unit-in-microbiology/

2.  https://www.webmd.com/digestive-disorders/what-are-probiotics

3.  https://sciencing.com/calculate-cfu-dilution-7806269.html

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