Detecting Contaminants in Mineral Water: An Application Note

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.

Image 1 (click to enlarge): Fluorescent staining. Note: Fluorescence detection is a non-destructive method that enables the microorganisms to continue to grow after they have been stained in order to identify them using standard ID technology. (Image Credit: EMD  Millipore)

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.

1. A filtration unit is installed onto the filtration system.
2. The appropriate volume of sample is poured into the filtration unit.
3. After filtration, the membrane is disconnected from the device and aseptically transferred onto media and incubated.
4. After incubation, the membrane is stained with the fluorogenic reagent for 30 minutes at 32.5 degrees Celsius +/- 2.5 degrees Celsius.
5. The fluorescent micro colonies are counted using the fluorescence reader.
6. 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).

Image 2: The image on the right illustrates a sufficient fluorescent signal intensity translating into an appropriate incubation time. The picture on the left shows that an accurate count is not possible if the intensity of the fluorescence is too low due to an insufficient incubation time. (Image Credit: EMD Millipore)