Mineral water manufacturing processes are susceptible to yeast, mold, and bacterial contamination.
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
Hardware:
- Fluorescence reader systems (EZFKIT001WW, MXQUANK01)
- Filtration systems (EZFTIMIC01, MXPPLUS01)
Consumables:
- Fluorescence reagent kit (EZFREAG57, MXQTV0KT1)
- 100 milliliter, 0.45 micrometer mixed cellulose ester filter and funnels (MZHAWG101, MXHAWG124)
Media:
- 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)
Matrices tested:
- Various mineral waters—direct well, storage tank, and final products
Microorganisms:
- 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).
Table 1 summarizes incubation conditions corresponding to each matrix.
Results and Interpretation
Table 2 provides results for mineral water, direct well testing at 22 degrees Celsius using yeast extract agar. Waterborne microorganisms are accurately detected with the EMD Millipore fluorescent technology after 24 hours of incubation at 22 degrees Celsius on yeast extract medium, versus 72 hours with the compendial method.
Table 3 and Image 3 provide results for mineral water from a storage tank at 22 degrees Celsius. Spoilage microorganisms are accurately detected with the EMD Millipore fluorescent technology after 48 hours of incubation at 22 degrees Celsius on yeast extract medium, versus 72 hours with the compendial method.
Table 4 provides results for coliforms that ferment lactose after 24 hours. Conditions were 37 degrees Celsius, lactose TTC with Tergitol 7 medium. The detection of coliforms that ferment lactose after 24 hours using the EMD Millipore fluorescent technology is accurate after 16 hours of incubation on lactose TTC with Tergitol 7 medium, versus 48 hours with the compendial method.
Results for coliforms that ferment lactose after 48 hours are shown in Table 5 (37 degrees Celsius, lactose TTC with Tergitol 7 medium). Coliform strains that ferment lactose after 48 hours are accurately detected with the EMD Millipore fluorescent technology after 14 hours of incubation at 37 degrees Celsius on lactose TTC with Tergitol 7 medium, versus 48 hours with the compendial method.
Table 6 summarizes results for Enterococcus faecalis (37 degrees Celsius, m-Enterococcus agar).
Enterococcus faecalis is accurately detected with the EMD Millipore fluorescent technology after 20 hours of incubation at 37 degrees Celsius on m-Enterococcus Agar medium, versus 48 hours with the compendial method.
Enterococci are classically grown on Enterococcus-selective agar according to Slanetz and Bartley. However, the triphenyl tetrazolium chloride (TTC) contained in the medium interacts with the fluorescent signal, resulting in the observation of dark spots compared to the fluorescent ones that should be observed (see Image 4).
To avoid this phenomenon and to preserve the visual characteristics of this selective medium, the membrane incubation before labelling was done on classical m-Enterococcus agar medium (no TTC). After fluorescent detection of microorganisms, the membrane was re-incubated on Enterococcus-selective agar according to Slanetz and Bartley (with TTC). This allowed the recovery of the colonies with the typical color they would assume if the full incubation were done on the selective medium.
Blue/green Pseudomonas aeruginosa is shown in Table 7 (37 degrees Celsius, cetrimide agar). The detection of blue/green Pseudomonas aeruginosa using the EMD Millipore fluorescent technology is accurate after 18 hours of incubation on cetrimide agar medium, versus 48 hours with the compendial method.
Table 8 provides results for fluorescent Pseudomonas aeruginosa (37 degrees Celsius, cetrimide agar). Fluorescent Pseudomonas aeruginosa is accurately detected with the EMD Millipore fluorescent technology after 18 hours of incubation at 37 degrees Celsius on cetrimide medium. The fluorescence-based technology is fully compatible with the cetrimide agar with nalidixic acid, versus 48 hours with the compendial method.
For microorganisms that grow by proliferation on the membrane, such as Pseudomonas aeruginosa, fluorescent detection is more accurate than a naked eye-reading. When many small colonies in the same area are very close to one another, they often merge during growth, leading to only one colony being counted on the final traditional membrane. When using fluorescent technology, since the detection occurs earlier in the growth phase, the fluorescent count is more accurate in showing the real number of microorganisms present in the tested sample.
This phenomenon accounts for the lower number of colonies obtained with the re-incubated and traditional membranes, as opposed to the fluorescent stained membranes.
Table 9 summarizes results for Aspergillus brasiliensis (28 degrees Celsius, Sabouraud dextrose agar). The detection of Aspergillus brasiliensis using the EMD Millipore fluorescent technology is accurate after 30 hours of incubation on cetrimide agar, versus 7 days with the compendial method.
Candida intermedia results are shown in Table 10 (28 degrees Celsius, Sabouraud dextrose agar). Candida intermedia is accurately detected with the EMD Millipore fluorescent technology after 30 hours of incubation at 28 degrees Celsius on Sabouraud dextrose agar, versus 7 days with the compendial method.
Table 11 presents results for Zygosaccharomyces bailii (28 degrees Celsius, Sabouraud dextrose agar).
Zygosaccharomyces bailii is accurately detected with the EMD Millipore fluorescent technology after 40 hours of incubation at 28 degrees Celsius on Sabouraud dextrose agar, versus 72 hours with the compendial method.
Illustration of Fluorescent Stained Membranes
Table 12 presents some typical images obtained using the EMD Millipore fluorescence-based technology, labeling different microbial species after membrane filtration and incubation on nutritive agar.
Summary of Performances
Table 13 provides an overview of the different time savings observed during this study for rapid microbial detection using the EMD Millipore fluorescence-based technology compared to traditional microbial filtration.
Depending on the matrix challenged, the nutritive medium, and the microorganism growth kinetics, the rapid detection fluorescence based-technology can reduce the time to result by a factor of two to four compared to the compendial microbiological method.
Conclusion
Using fluorescence technology as a microbiology quality-control tool dramatically reduces the time needed to detect yeast, mold, and bacterial contaminations in mineral water. As an example, this article demonstrates that the EMD Millipore fluorescence-based technology can replace the compendial microbiological method with a two to four-fold faster time to result and compatibility with the standard culture media traditionally used for the detection of spoilage microorganisms in mineral water. Moreover, as the method is non-destructive, each fluorescent micro colony detected will continue to grow to yield visible colonies, allowing the identification of contaminants using conventional identification methods.
The faster release of products not only brings logistical advantages for a manufacturer, but in addition there is the financial benefit associated with bringing products to the market faster. A rapid method that enables the release of products faster and results in a reduction in the amount of stock held in the warehouse therefore has a positive improvement in the company cash flow.
Venchiarutti is an application training scientist, BioMonitoring R&D, at Millipore S.A.S. Reach him at [email protected]. Valton El Khoury is also an application training scientist, BioMonitoring R&D, at Millipore S.A.S. Reach her at [email protected].
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