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Probiotics or live biotherapeutic products (LBPs), when consumed, are expected to survive at sufficient dose levels until their expiration date to have an impact on health or treatment. At present, the number of bacterial cells is considered the most important parameter affecting the efficacy and quality of probiotics or LBPs. Many regulators rely solely on plate counts or similar plating analyses to assess "viability" and ensure the accuracy of label claims. Probiotic bacteria enumeration can be considered the main evaluation tool for product feasibility, formulation, stability, quality control, and the first parameter used for commercial agreements.
Cell counts are usually performed by culture-dependent methods. Cultivation methods, such as plate counting, are the gold standard. The plate counting method is simple to operate, but it requires a long incubation time and the selection of an appropriate medium.
FC can complement plate counting as it detects a ratio of intact versus total bacteria. It is a culture-independent methodology having the potential to enumerate selectively live and damaged or dead cells. FC can provide data on different structural and functional properties of cells, allowing quantification of the proliferative capacity of cells on agar and other microbial media, thereby providing further insight into the response of functional strains to various applications. It was used successfully for the assessment of viability and physiological activity of LAB and Bifidobacteria subjected to different stress conditions. The number of microorganisms in the analyzed products was greater when FC was used for most of the analyzed strains.
Fluorescence in situ hybridization (FISH) is considered one of the most powerful techniques in modern microbial ecology because it has the distinct advantage of directly counting cells in natural environments without the need for culture.
Fig.1 Bacteria enumeration.
Different methods can be used to assess cell viability, and they have been extensively evaluated elsewhere. Selective culture techniques do not always provide an accurate representation of all species. In recent years new culture-independent methods have been used to accurately enumerate probiotic strains based on viability. A more comprehensive definition of live probiotics, using culture-independent techniques, has the potential to provide direct, rapid enumeration methods for researchers and industry-based scientists facing the challenge of providing usable doses of final products. These techniques either use dyes to distinguish live from dead cells, measure the integrity of cell membranes, or characterize some aspect of metabolic activity. Both traditional cell culture methods, as well as alternative techniques (direct imaging and visual enumeration, nucleic acid-based enumeration methods, and FC and cell sorting), offer advantages and limitations for enumerating probiotic microorganisms.
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For Research Use Only. Not intended for use in food manufacturing or medical procedures (diagnostics or therapeutics). Do Not Use in Humans.