🦠 Intestinal enterococci in water — MPN method with an enzymatic medium (β-D-glucosidase; Enterolert-DW / Quanti-Tray)

Microbiology PN-EN ISO 7899-3

Overview

ISO 7899-3:2025 specifies a method for the enumeration of intestinal enterococci in water — among them Enterococcus faecalis, E. faecium, E. durans, E. avium, E. gallinarum, E. hirae and E. casseliflavus — based on the growth of the target organisms in a liquid medium and the calculation of the most probable number (MPN) from tables or programs. It is applied to water intended for consumption and to bathing water (fresh and sea water) and to other similar waters, including those containing a substantial amount of suspended matter; it makes it possible to detect enterococci at a level of 1 cfu/100 ml with a final result within (26 ± 2) h even where the number of heterotrophic bacteria is high. Bathing waters are best examined after a 1:10 dilution. The test is based on the expression of the enzyme β-D-glucosidase and gives a confirmed result after 24 h without further examination of the positive wells. The standard does not apply to bottled waters, for which the method has not been validated — a laboratory may apply it there after its own validation. The standard is based on a validated product with established characteristics (annex C) and requires the application of ISO 8199, ISO 11133 and ISO 19458 (sampling). The presence of intestinal enterococci serves to assess the effectiveness of treatment in the production of water intended for consumption and indicates the ingress of contamination and leaks in storage and distribution systems.

The manufacturer of the Enterolert-DW medium (IDEXX) describes the test as based on defined substrate technology (DST) with the indicator nutrient substrate ortho-nitrophenyl-β-D-glucoside: when the enterococci metabolise the substrate, the colour of the sample changes from blue to green — any change to green is a positive result, with no need for a UV lamp. Incubation at 41 ± 0.5 °C for 24 h, results valid up to 28 h (unless local regulations provide otherwise); a positive result before 24 h is valid, and a negative one after 28 h is valid too. The medium is stored at 2–25 °C away from light and moisture; the test is intended for drinking water, and for other waters after laboratory validation. In Poland the standard is in force as PN-EN ISO 7899-3:2025-11.

Method principle

Intestinal enterococci grow in a liquid medium containing an indicator nutrient substrate for β-D-glucosidase; the breakdown of the substrate (ortho-nitrophenyl-β-D-glucoside) changes the colour of the medium from blue to green. The sample with the medium is poured into a Quanti-Tray, sealed and incubated; after (26 ± 2) h (the manufacturer: 41 ± 0.5 °C for 24 h, reading up to 28 h) the green wells are counted as positive — without further confirmation — and the most probable number of intestinal enterococci per 100 ml is read from MPN tables or from a program (e.g. the manufacturer's MPN generator). The method detects 1 cfu/100 ml in the presence of a high number of heterotrophic bacteria; bathing waters (fresh and sea water) are diluted 1:10. The medium is ready for use in single-unit packs and contains no toxic components; for comparison when reading, a negative control is used (there is no colour standard). The general principles of cultural examinations (ISO 8199), of the quality of media (ISO 11133) and of sampling (ISO 19458) apply.

Applications

• Water intended for consumption — control of the effectiveness of treatment and of the tightness of the distribution network (a result after 24 h)
• Fresh and sea bathing waters (1:10 dilution), waters with a large amount of suspended matter
• Waters with a high background of heterotrophic bacteria, where membrane filtration (ISO 7899-2) is difficult
• Laboratories of waterworks and of sanitary-epidemiological stations — no confirmation of the positive wells
• Bottled waters — only after the laboratory's own validation (outside the scope of the standard)

Key parameters

Standard

Standard number

PN-EN ISO 7899-3:2025-11 (ISO 7899-3:2025)

Title (PL)

Jakość wody — Oznaczanie ilościowe enterokoków jelitowych — Część 3: Metoda najbardziej prawdopodobnej liczby

Title (EN)

Water quality — Enumeration of intestinal enterococci — Part 3: Most probable number method

Step-by-step procedure

1. Sampling

   Take the sample in accordance with ISO 19458; dilute bathing waters (fresh and sea water) 1:10.

2. Addition of the medium

   To the sample in a sterile vessel add the Enterolert-DW reagent, stopper it and shake to mix the contents until it dissolves.

3. Pouring and sealing

   Pour the sample with the medium into a Quanti-Tray and seal it in the sealer.

4. Incubation

   Incubate for (26 ± 2) h; according to the manufacturer at 41 ± 0.5 °C for 24 h, the reading valid up to 28 h.

5. Reading

   Regard as positive the wells in which the colour has changed from blue to green (any change to green); compare with the negative control. A positive result before 24 h is valid; the absence of green after 28 h is a valid negative result, while a green colour after 28 h requires the examination to be repeated.

6. Calculation and test report

   From the number of positive wells read the MPN of intestinal enterococci per 100 ml from the MPN tables or program; where a dilution was made, allow for the factor. Give the method, the identification of the sample and the result.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Intestinal enterococci indicate faecal contamination — work under the conditions of a microbiological laboratory, autoclaving of the used trays
• The sealer heats up — do not touch the heating elements
• The medium contains no toxic components (according to the manufacturer); observe the general health and safety rules for reagents

Frequently asked questions