Mineral water manufacturing processes are susceptible to yeast, mold, and bacterial contamination.
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A rapid microbiology system that can detect potential contamination three times faster than traditional monitoring methods would result in significant cost savings and consistent, timely release of products to market.
Fluorescence-based technology offers rapid, quantitative detection of microorganisms over a broad range of filterable matrices. These easy-to-use systems employ industry-standard membrane filtration techniques to detect viable and culturable microorganisms down to 1 colony forming unit (CFU) per sample. Test results are comparable to current microbial test results, which facilitate the validation of these rapid systems in any laboratory. The non-destructiveness of these methods also enables the identification of microorganisms detected during the initial fluorescent count using current ID methods.
Materials and Methods
- Fluorescence reader systems (EZFKIT001WW, MXQUANK01)
- Filtration systems (EZFTIMIC01, MXPPLUS01)
- Fluorescence reagent kit (EZFREAG57, MXQTV0KT1)
- 100 milliliter, 0.45 micrometer mixed cellulose ester filter and funnels (MZHAWG101, MXHAWG124)
- Yeast extract agar, powder (1037500500)
- m-Enterococcus agar base (1.05289.0500)
- Lactose TTC with Tergitol 7 agar, powder (1076800500)
- Cetrimide agar (MXSMCET48)
- Cetrimide agar with naladixic acid (MXSMCET24)
- Sabouraud dextrose agar (MXSMSDA48)
- Membrane-filter Enterococcus-selective agar acc. to SLANETZ and BARTLEY (1052620500)
- Various mineral waters—direct well, storage tank, and final products
- All strains challenged were wild types isolated from industrial environments
- Coliforms that ferment lactose after 24 hours
- Coliforms that ferment lactose after 48 hours
- Blue/green Pseudomonas aeruginosa
- Fluorescent Pseudomonas aeruginosa
- Enterococcus faecalis
- Pseudomonas sp.
- Zygosaccharomyces bailii
- Aspergillus brasiliensis
- Candida intermedia
Principle of Detection
The principle of the fluorescence detection is based on an enzymatic reaction. The fluorogenic substrate used is a non-fluorescent viability marker that is cleaved by non-specific ubiquitous intracellular enzymes, resulting in a fluorescent product. Natural amplification of fluorescence by intracellular accumulation is an indicator of microbial metabolism. The dye is diluted in a staining buffer enhancing cell-membrane permeability and thus facilitating the introduction of dye into cells (see Image 1).
Protocol for Rapid Detection
The following is a standard protocol to detect waterborne microorganisms in samples of interest with fluorescence detection.
- A filtration unit is installed onto the filtration system.
- The appropriate volume of sample is poured into the filtration unit.
- After filtration, the membrane is disconnected from the device and aseptically transferred onto media and incubated.
- After incubation, the membrane is stained with the fluorogenic reagent for 30 minutes at 32.5 degrees Celsius +/- 2.5 degrees Celsius.
- The fluorescent micro colonies are counted using the fluorescence reader.
- After detection, the stained membrane can be re-incubated on fresh media for traditional plate count and identification if required.
Definition of a Rapid Incubation Time
An appropriate incubation time is defined as the minimum time necessary to achieve a recovery rate higher than 70 percent compared to the traditional method. The calculation is based on both formulas:
- The fluorescence recovery is the fluorescent count compared to the traditional method count. Fluorescence recovery (percentage) = (average of fluorescence counts/average of traditional method count) x 100.
- The viability recovery is the colony count on stained membranes after re-incubation compared to the traditional method count. Viability recovery (percentage) = (average of CFU counts after re-incubation/average of traditional method counts) x 100.
An optimal incubation time should allow sufficient fluorescent signal intensity, fluorescence, and viability recoveries above 70 percent (see Image 2).