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How to Measure CFU - It's Good to know!

CFU - What is it?

CFU (colony-forming units) is a unit of measurement used to estimate the number of viable bacterial colonies that form on a petri dish.

How to measure CFU

To measure the quantity of CFU for a given probiotic strain, we use MRS agar as the growth media (food source). The petri dishes, also known as plates, are incubated at body temperature (37 C) for 2-3 days or until visible colonies develop.

Typically, a sample of a probiotic product is diluted multiple times in sterile buffer or peptone to achieve an estimated strength of 30-300 CFU per milliliter. One milliliter of this diluted sample is then pipetted into a sterile petri dish where liquid MRS agar is poured over the sample, mixed, and allowed to solidify before incubation. After incubation, the colony count is determined, multiplied by the dilution factor, and the total CFU per serving is reported. This process is called plating the sample or making a plate count.

Video from How to Calculate CFU

Are all CFU created equal?

A CFU may represent one cell or several or more cells depending on the probiotic being evaluated. For example, a particular strain may form in short chains containing several cells, say 3 cells on average, and a single CFU would develop on a petri dish from 3 cells not one. In this case, the actual number of live cells represented would be approximately 3 times the CFU count.

In another example, a particular strain of the probiotic, Bifidobacterium lactis, forms as both single cells and clumps of 2-5 cells producing measurable CFU from as few as one cell or as many as 5 cells. The determined number of CFU is partly a function of how a sample is prepared before analysis because samples mixed in a stomacher machine (simulates stomach motility) will usually test higher than samples shaken by hand.

Are all colonies created equal?

It is important to mention that it is not possible to differentiate different species or strains of probiotic bacteria from the appearance of their colonies. Probiotic colonies are usually round or disc shaped, 1-5 mm in diameter, and tan or cream colored on MRS agar. When a multi-species probiotic supplement gets plated, only the total CFU count can be obtained.

That said, we have discovered that an approximate determination of Lactobacillus count to Bifidobacteria count can be made in a mixed sample be incubating duplicate sets of plates aerobically versus anaerobically. Because both the Lactobacilli and the Bifidobacteria grow anaerobically  (without oxygen) while only the Lactobacilli grow aerobically (in the presence of oxygen), we can subtract the amount of Lactobacilli CFU grown aerobically from those grown anaerobically to determine Bifidobacteria CFU.

Why is it so important to take billions of probiotic CFU per serving?

There are about 100 trillion live microorganisms in the human intestinal tract. Most congregate in the lower ileum of the small intestine and the upper colon and many have not been identified or characterized. Some are beneficial, some are neutral, while others are outright harmful or potentially harmful. Candida yeast, for example, is present in most of us but is held back from becoming problematic by billions of bacterial competitors.

When antibiotics arrive, the overall competition is reduced and the Candida cells morph into infective agents. Stifiling this nasty scenario and creating a positive balance between probiotics and other microorganisms takes billions of probiotic CFU per day. Furthermore, candida and antibiotics aren't the sole offenders: diet, activity level, and water intake can all influence the bacterial balance in your digestive tract for better or for worse.

Why take a probiotic supplement?

Taking a probiotic supplement can help restore balance and promote a beneficial environment for probiotic bacteria; but, eating a diet rich in fiber, drinking plenty of water, and exercising regularly will help promote a healthy digestive environment too. When combined with a healthy lifestyle, probiotics can do amazing things for you and your gut!

Most available probiotic literature is filled with studies employing less than 10 billion CFU per day. Summarily, significant positive results have been hard to achieve in many of these studies. Good strains or not, this is simply not enough dosage to consistently effect positive results. The researchers behind these studies always write, “More research is needed”; however, in many of these cases, all that was needed was a higher dosage (specifically, a higher dosage delivered alive through stomach acid into the small intestine)!

How much should I take?

So what determines an appropriate dosage? First, you have to be sure that you're purchasing a product that protects the bacteria through the stomach.  This is not an easy trick as stomach pH can be as low as pH 1.5 and residence time can range from 60-90 minutes which is long enough to kill most probiotic strains. If 99% of 300 billion CFU die in the stomach before reaching the intestinal tract, that 300 billion effectively becomes a 3 billion CFU dose.

Consumers need to look for high strength probiotic products that can ensure live delivery through the stomach. What is high strength? 10 billion CFU/day is now accepted by industry leaders as the minimum effective dose but, in our experience, any dose between 10 – 40 billion CFU will work well.

If you would like more information, or have any questions, Ask Us Anything!

Randolph S. Porubcan, MSc
Consulting Biochemist

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