Rapid microbial detection using fluorescent staining

Rapid identification of the presence of such contamination is essential but well-established laboratory methods for evaluating microbial load by growth in a suitable medium and counting the resulting colonies are effective but time consuming. Typical incubation periods required to produce countable colonies can be of the order of five to seven days. In order to accelerate the identification process, a rapid fluorescence-based technology has been introduced for fast, quantitative detection of microorganisms over a broad range of filterable matrices. This easy-to-use and simple system not only uses industry standard membrane filtration techniques to detect viable and culturable microorganisms down to one colony-forming unit (cfu) per sample, but introduces some industrial vision components into a laboratory instrument. In addition, the fluorescence staining procedure is non-destructive, allowing downstream specific identification following a positive result.

Rapid microbial detection
A rapid microbial detection method is now available using the Milliflex Quantum system, developed by the Millipore. This system is a laboratory instrument which utilises two proven technologies, membrane filtration and fluorescent staining, to reduce by two-thirds the incubation time needed to measure the microbial load on a membrane. The system consists of a reader, camera and fluorescence reagents (Fig 1) used in combination with a pump, filtration devices and media cassettes. The reader features an optical system to allow the fluorescent microcolonies on the entire membrane to be seen with the naked eye. A detachable camera module enables the user to view the membrane on a computer screen, with associated ‘point and click’ colony counting capability. The system can be used in a range of applications including:
* raw materials (media, buffers, pharmaceutical ingredients and water)
* in-process samples (eg bioburden prior to sterilisation, CIP/SIP samples, cell culture/fermentation samples, media for fermentation, buffers for manufacturing, and intermediate process samples)
* final products
* environmental samples.

Sample preparation is a single-stage process for water testing or a two-stage process for bioburden testing which ensures consistent and accurate microbiological results while reducing overall time to result. For water testing, the desired sample volume is filtered through a membrane in a presterilised filter unit. The membrane filter base is then placed on a Milliflex Quantum R2A media cassette, which contains fluorescent dye. For bioburden testing, the desired sample volume is filtered through a membrane in a presterilised, disposable Milliflex filter unit. The membrane filter base is then placed on a prefilled agar cassette and incubated for the required time according to the application. The membrane is then transferred to a pad pre-wetted with fluorescent reagent and incubated for 30 minutes.
 The principle of fluorescence detection is based on an enzymatic reaction. The fluorogenic substrate used is a non–fluorescent viability marker that is cleaved by non-specific intracellular enzymes, resulting in a fluorescent product. Accumulation of this product inside cells is an indicator of microbial metabolic activity and membrane integrity.