🦠 Fecal Enterococci in Water

Microbiology PN-EN ISO 7899-2

Overview

STATUS (as of 29.10.2026). PN-EN ISO 7899-2:2004 — Polish version, published on 14.04.2004, adopts EN ISO 7899-2:2000 [IDT] and ISO 7899-2:2000 [IDT]; it replaced the English version PN-EN ISO 7899-2:2002. The standard is current — no withdrawal note. ISO 7899-2:2000 (second edition) replaced the 1984 edition after technical revision and has the status of a current standard in the catalogue.

SCOPE (clause 1). Detection and enumeration of intestinal enterococci (in the Polish version of the standard: faecal enterococci) in water by membrane filtration — especially in drinking water, water from swimming pools and other disinfected or clean waters. The method can be applied to all types of water, except samples with a large amount of suspended matter or many interfering microorganisms; it is particularly suitable for examining large volumes of water containing only a few enterococci. Part 1 of this series is a miniaturized most probable number (MPN) method for surface and waste water.

WHAT THE METHOD DETECTS (clause 3, introduction). Intestinal enterococci are bacteria which, on the media of 6.3.1 and 6.3.2, reduce 2,3,5-triphenyltetrazolium chloride to formazan and hydrolyse aesculin at 44 °C. The method detects Enterococcus faecalis, E. faecium, E. durans and E. hirae, and occasionally also other Enterococcus species and some Streptococcus species (S. bovis, S. equinus) — the latter do not survive long in water and are probably not enumerated quantitatively. In water examination, enterococci are regarded as indicators of faecal pollution, although some enterococci in water may originate from other habitats.

REGULATIONS. Water intended for human consumption: intestinal enterococci 0 in 100 ml (in water bottled or in containers — in 250 ml) (Journal of Laws 2026, item 748, Annex 1 Part A Table 1); together with E. coli these are “core parameters” whose monitoring frequency is not reduced (Annex 3 part I). PN-EN ISO 7899-2 is the reference method (Annex 4 Part A), and the Enterolert-DW method is an alternative method recognized as equivalent (Annex 4 Part C). Bathing waters: intestinal enterococci in cfu/100 ml — inland waters: excellent quality 200, good 400 (95th percentile), sufficient 330 (90th percentile); coastal and transitional waters: 100, 200 and 185; reference methods ISO 7899-1 or ISO 7899-2 (Directive 2006/7/EC, Annex I).

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends at 6.3.1.2 (TTC solution). Preparation of the complete Slanetz and Bartley medium (addition of TTC), the composition of bile-aesculin-azide agar (6.3.2), sampling (clause 7), the procedure (clause 8), quality assurance (clause 9) and expression of results (clause 10) are beyond it. The incubation time on Slanetz and Bartley medium of (44 ± 4) h and confirmation at (44 ± 0.5) °C for 2 h are given according to the data sheet of a medium manufactured in accordance with the standard — the available text of the standard gives the incubator temperatures and states that intestinal enterococci hydrolyse aesculin within 2 h.

Method principle

A specified volume of the sample is filtered through a membrane filter with a pore size of 0.45 µm, which retains the bacteria. The filter is placed on a solid selective medium containing sodium azide (to suppress the growth of Gram-negative bacteria) and colourless 2,3,5-triphenyltetrazolium chloride (TTC), which intestinal enterococci reduce to red formazan. Typical colonies are raised, red, maroon or pink — in the centre or throughout (4.1). If typical colonies are observed, the filter with all the colonies is transferred onto bile-aesculin-azide agar preheated to 44 °C: intestinal enterococci hydrolyse aesculin on this medium within 2 h, and the end-product — 6,7-dihydroxycoumarin — combines with iron(III) ions to give a tan-coloured to black compound which diffuses into the medium (4.2).

Applications

• Drinking water, water from swimming pools and other disinfected or clean waters (clause 1)
• Large volumes of water containing only a few intestinal enterococci (clause 1)
• Water intended for human consumption — intestinal enterococci 0 in 100 ml, in water bottled or in containers 0 in 250 ml; reference method (Journal of Laws 2026, item 748, Annex 1 Part A and Annex 4 Part A)
• Bathing waters — water quality assessment, reference method alongside ISO 7899-1 (Directive 2006/7/EC, Annex I)

Key parameters

Standard

Standard number

PN-EN ISO 7899-2:2004

Title (PL)

Jakość wody — Wykrywanie i oznaczanie ilościowe enterokoków kałowych — Część 2: Metoda filtracji membranowej

Title (EN)

Water quality — Detection and enumeration of intestinal enterococci — Part 2: Membrane filtration method

Step-by-step procedure

1. Preparing the basal medium

   Dissolve the ingredients of Slanetz and Bartley medium in boiling water, once dissolved heat for an additional 5 min and cool to 50–60 °C; prepare the TTC solution separately and sterilize it by filtration (6.3.1.1–6.3.1.2). Combining the components — 6.3.1.3, beyond the available text of the standard.

2. Filtration

   Filter a specified volume of the sample through a sterile 0.45 µm membrane filter in membrane filtration apparatus according to ISO 8199 (4.1, 5.1–5.2). For water intended for human consumption, the parametric value refers to 100 ml, for water bottled or in containers — to 250 ml (Journal of Laws 2026, item 748, Annex 1 Part A).

3. Incubation on Slanetz and Bartley medium

   Place the filter on the medium and incubate at (36 ± 2) °C (5.3) — for (44 ± 4) h according to the data sheet of a medium manufactured in accordance with the standard.

   🌡 Temperature: 36 ± 2 °C

4. Reading typical colonies

   Typical colonies are raised, red, maroon or pink in the centre or throughout (4.1).

5. Confirmation on bile-aesculin-azide agar

   If typical colonies are present, transfer the filter with all the colonies onto bile-aesculin-azide agar preheated to 44 °C and incubate at (44 ± 0.5) °C. Intestinal enterococci hydrolyse aesculin within 2 h — a tan to black colour forms around the colonies and diffuses into the medium (4.2, 5.4).

   🌡 Temperature: 44 ± 0.5 °C

6. Result

   Number of confirmed colonies in the volume filtered; calculation and expression of the result — clause 10 of the standard and ISO 8199, beyond the available text.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Sodium azide is highly toxic and mutagenic — avoid contact, especially inhalation of fine dust when preparing dehydrated media (6.1)
• Do not mix azide-containing media with strong inorganic acids — toxic hydrogen azide (HN₃) may be produced (6.1)
• Azide solutions in contact with metal pipework (e.g. of sinks) can form explosive compounds; azides are decomposed safely by an excess of saturated nitrite solution (6.1)

Frequently asked questions