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The use of rapid microbiological methods (RMM) is increasing in industrial microbiology laboratories. Pharmaceutical, personal care and animal health companies are replacing their traditional agar plate methods with rapid technologies. Companies are implementing rapid methods for their routine microbiological tests, such as purified water monitoring; in-process bioburden analysis; raw ingredient and final product testing; and enumeration of microbial fermentation cultures. There are many different RMM technologies available in the marketplace, such as ATP bioluminescence, colorimetric, impedance, scanning laser cytometry, and flow cytometry. Due to the significant number of technologies available, the focus of this article will be flow cytometry.
Flow cytometry is an established method commonly used for rapid analysis of single cells in microbial suspensions (Brehm-Stecher and Johnson 2004). In general, samples are prepared for analysis by labeling the microorganisms with fluorescent stains. Once the reaction with the dye is completed, the sample is injected. The sample enters the sheath fluid flow which is traveling at a much higher velocity than the sample. The high velocity sheath flow serves to hydrodynamically focus the sample into a very fine stream of monodispersed cells. The fine stream is delivered to the detection region where each cell is illuminated with a laser. Each cell emits laser-induced fluorescent light and scatters some of the laser light for relative size determination.
Examples of two systems currently on the market utilizing the principles of flow cytometry are the D-Count® distributed by AES Chemunex (Rennes, France) and the Micro PRO™ Detection System manufactured by Advanced Analytical (Iowa, USA). These systems differ from traditional flow cytometry in their automation, ease-of-use, and proprietary labeling compounds. Because these systems use the basic concepts of flow cytometry they provide analysis and enumeration of individual cells in a sample. They allow for a count per milliliter result that correlates to the traditional agar plate count method. Additionally, viable but non-culturable microorganisms can also be detected and enumerated.
The Micro PRO™ is capable of differentiating between various types of microorganisms including bacteria, yeast, and mold, as well as bacterial and fungal spores and vegetative cells. Thus, the system is able to enumerate different types of microbes in a single sample. The system also quantifies of a wide range of sizes (0.1- to 15-microns) and concentrations (1 to 6 log10 CFU/mL) of microorganisms in a sample.
Flow cytometry-based systems can be used for quantitative and qualitative measurements. Specifically, quantitative analysis includes direct enumeration of microorganisms in purified water, in-process samples, fermentation cultures and stock lab cultures. Qualitative applications incorporate an enrichment step for raw ingredients, in-process samples and final products. Following enrichment, samples are analyzed for the presence or absence of microbial growth (bacteria, yeast or mold).
The primary benefit of rapid microbiological methods is a faster time to result. These methods are generally less labor-intensive than traditional methods and offer near real-time results. Using flow cytometry as a rapid method provides the added benefit of enumerating individual microorganisms of various sizes across a broad range of concentrations. Companies are increasing their use of rapid technologies as replacements for current traditional methods. Rapid microbiological methods present the opportunity for corporations to be proactive, instead of reactive, allowing corrective action to be taken.
References:
Brehm-Stecher, B.F. and E.A. Johnson. 2004. “Single-cell microbiology: Tools, technologies, and applications.” Microbiol. Mol. Biol. Rev. 68(3), 538-559.
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