Probiotics are microorganisms that, when given in sufficient amounts, provide a health benefit to the host. They are commonly referred to as “good” or “friendly” bacteria and play a crucial role in maintaining a balanced microbiota in the human body. Probiotics can be found in various foods, including yogurt, kefir, kimchi, sauerkraut, and dietary supplements. Enumeration involves counting or quantifying the viable probiotic cells in a sample. This enumeration process is essential for manufacturers as it provides data to support the potency and purity of their products.

In the United States, probiotics are classified as dietary supplements by the FDA, and therefore, probiotic supplements must adhere to the guidelines outlined in CFR 21 Part 111. These guidelines ensure that all manufacturing facilities, personnel, and procedures comply with the current Good Manufacturing Practices (cGMP) set by the FDA. The Chromak Research has ISO 17025:2017 certified laboratory with cGMP and FDA-registered services according to the standards. At Chromak Research, we have a highly skilled and experienced team specializing in conducting probiotic enumeration testing

Probiotics are commonly known as bacteria (such as Lactobacillus, Bifidobacterium, and Saccharomyces strains) or yeasts that resemble the advantageous microorganisms naturally present in the human gut. These microorganisms are typically consumed through fermented foods or in the form of supplements. The primary function of probiotics is to restore and sustain a harmonious equilibrium of gut microbiota, which plays a vital role in promoting digestive health, enhancing immune function, and ensuring overall well-being.

In microbiology, the process of enumerating probiotics involves measuring the number of viable cells of probiotic microorganisms present in a sample. Typically, various techniques are used for this purpose.

• Plate Count Method: The sample is diluted and spread onto agar media that promotes the growth of probiotic microorganisms, as part of this standardized procedure. After incubation, the viable colonies are counted and the data is reported in colony-forming units (CFU) per gram or milliliter of the sample. Generally, cell quantification is done through culture-dependent techniques. Although uncomplicated, the plate counting method requires a long incubation time and the careful selection of an appropriate medium.
• Most Probable Number (MPN) Method: The MPN technique is a statistical method used to measure low levels of probiotics in samples by diluting the sample into multiple tubes or wells and observing growth. The MPN value is then calculated using a statistical table.
• Flow Cytometry: Flow cytometry complements plate counting by identifying intact bacteria proportions quickly and efficiently. This method allows for probiotic enumeration based on size, shape, and fluorescent markers. By staining cells with dyes and analyzing them in a flow cytometer, researchers can accurately count and analyze live, damaged, and dead cells. This technique provides insights into cell characteristics and growth on different mediums, offering a deeper understanding of strain responses to environments. Flow cytometry has been effective in assessing LAB and Bifidobacteria viability and activity under stress conditions, resulting in higher microorganism counts in samples analyzed.
• Molecular Techniques: Polymerase chain reaction (PCR) and quantitative PCR (qPCR) are molecular techniques utilized for quantifying probiotics through the amplification of particular DNA sequences. While these methods offer excellent sensitivity and specificity, they necessitate specialized equipment and expertise.
• Viable Cell Count: This technique requires the direct enumeration of viable probiotic cells through the use of microscopy, automated cell counters, or staining methods like viability dyes.
• FISH: Fluorescence in situ hybridization (FISH) is widely regarded as a highly effective method in contemporary microbial ecology due to its unique ability to directly quantify cells in their natural habitats, eliminating the necessity for cultivation.

The choice of enumeration method is determined by factors such as the type of probiotic microorganism, sample matrix, required sensitivity, and available resources. Each method has its own advantages and disadvantages, so it is important to select the most appropriate technique based on the specific requirements of the study or application.

At Chromak Research, our team of experts is thrilled to assist you throughout the entire probiotic formulation, manufacturing, and quality control process. We possess expertise in all the primary enumeration methods used by major probiotic manufacturers. Our team of dedicated microbiologists is well-trained to execute these methods in accordance with the specifications provided by the manufacturers.