The limitations of the colony-forming unit in microbiology
Posted: 6 January 2016 | | 1 comment
The recent revision to USP General Informational Chapter <1223> Validation of Alternative Microbiological Methods that became official on December 1, 2015 contained a section discussing the limitations of the colony-forming unit (CFU) in terms of enumerating only those microorganisms that readily grow on solid microbiological media. The section highlights its inappropriateness 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.
The advent of ribosomal RNA gene sequence analysis thirty years ago has significantly expanded our awareness of microbial diversity in water, air, soil and the human body and reemphasised that, overall, cultivable microorganisms represent less than 1% of the microorganisms observed by direct microscopic examination. This cultivability could range from 0.1 to 0.01% in oceanic microbiota to around 80% for the microbiota on the human forearm. Questions that can be asked include: what does this understanding mean to the pharmaceutical microbiologist responsible for testing and releasing drug products? How does this apply to the challenge of evaluating, validating and implementing alternative methods to the compendial microbiological test methods? As a background to a general discussion of these questions amongst microbiologists, it is useful to review the history of the development of microbiological testing methods.
From Pasteur’s discoveries to the Petri dish
The development of classical microbial methods, broth culture, plate count, serial dilution, enrichment and microbial identification over time has been described in the literature. The method of growing bacteria in a transparent liquid nutrient medium and inoculating fresh medium from turbid medium to isolate a pure culture of the bacterium was commonly employed by the French pioneering microbiologist Louis Pasteur, beginning in 1861. Pasteur used a medium consisting of 100 parts water, 10 parts dextrose, one part ammonium tartrate and one part the ash of yeast cells to grow bacteria. However, before isolation on solid media became a standard practice, it can be said that Pasteur did not truly obtain pure cultures of microorganisms…