🦠 Most probable number (MPN) of intestinal enterococci in surface water, bathing water and waste water by the miniaturized method — dilutions of the sample in a special diluent with sea salt, 200 µl each into the wells of 96-well microplates with dehydrated MUD/SF medium, incubation for 36–72 h at 44 °C ± 0,5 °C, wells with blue fluorescence under UV 366 nm positive, result MPN/100 ml with a 95 % confidence interval, PN-EN ISO 7899-1

Microbiology PN-EN ISO 7899-1

Overview

WHAT THE STANDARD COVERS. Scope from the PKN catalogue card of PN-EN ISO 7899-1:2002 (Polish version), in full (our translation of the Polish card text): “A method is described for the detection of most intestinal enterococci in surface water and waste water and for their enumeration by determination of their most probable number — MPN”. We read the text of the standard in the sample of ISO 7899-1:1998: foreword, introduction and Clauses 4–10 (principle, apparatus, sampling, media, procedure, expression of results, test report); the page with Clauses 1–3 (scope, normative references, definitions) is blank in the sample, and we do not have Annexes A–G (programs for calculating the MPN, tables, composition of sea salt, quality criteria for the medium in microplates — E, preparation of calibration microplates — F).

ACCORDING TO THE TEXT OF ISO 7899-1:1998 (foreword, introduction, Clauses 4–10). The standard was prepared by ISO/TC 147 “Water quality”, subcommittee SC 4 “Biological methods”; the second edition replaced the first (ISO 7899-1:1984). The ISO 7899 series: Part 1 — miniaturized MPN method by inoculation in liquid medium, Part 2 — membrane filtration method; Annexes E and F are normative, A, B, C, D and G informative. Introduction: the aim is to determine the main intestinal enterococci — E. faecalis, E. faecium, E. durans and E. hirae — often found in the faeces of humans and warm-blooded animals; occasionally other faecal species (E. avium, E. cecorum, E. columbae, E. gallinarum) and strains of Streptococcus bovis/equinus are detected, which are rare in environmental samples and poorly recovered; the non-faecal species E. casseliflavus and E. mundtii (e.g. from plant material and some industrial waste waters) are counted as intestinal enterococci — they usually produce a yellow pigment on non-selective medium — which has to be taken into account in interpretation. Principle (Clause 4): the diluted sample is inoculated into a row of wells of a microplate with dehydrated medium; after 36–72 h of incubation at 44 °C ± 0,5 °C the plates are examined in the dark under UV light of 366 nm; the presence of enterococci is indicated by fluorescence from the hydrolysis of MUD (4-methylumbelliferyl-β-D-glucoside); the result is expressed as MPN/100 ml. Apparatus (Clause 5; glassware sterilized according to ISO 8199): dry-heat steriliser or autoclave; incubator 44 °C ± 0,5 °C; tunnel dryer or vertical laminar-flow cabinet (preferably class II); UV observation cabinet (Wood’s lamp 366 nm) — warning: UV irritates skin and eyes, gloves and goggles; portable refractometer (optional); pH meter ±0,1; test tubes 16 × 160 mm and 20 × 200 mm; 8-channel multichannel pipette or another system dispensing 200 µl per well and sterile tips; membrane filtration apparatus with 0,2 µm filters for sterilizing media; sterile 96-well microplates of 350 µl, flat-bottomed, non-fluorescent; sterile adhesive tapes for sealing; sterile 90 mm Petri dishes. Sampling (Clause 6): according to ISO 8199 and ISO 5667-1, -2 and -3. Media and diluents (Clause 7): from ingredients of uniform quality or ready-made dehydrated; distilled or demineralized water free from substances inhibiting or promoting growth; if not used immediately — in the dark at (5 ± 3) °C for up to one month. Special diluent SD (7.2.1): synthetic sea salt 22,5 g, optionally 10 ml of bromophenol blue solution (0,04 g in 100 ml of 50 % ethanol — only for colouring, so as not to confuse it with water), water to 1000 ml; autoclaving at 121 °C ± 3 °C for 15–20 min; pure NaCl solution is not suitable (marked inhibition); typical composition of sea salt — Annex C. MUD/SF medium (7.3): solution A — tryptose 40 g, KH2PO4 10 g, D(+)-galactose 2 g, polyoxyethylene sorbitan monooleate (Tween 80) 1,5 ml, water 900 ml, dissolved by heating and brought to the boil; solution B — NaHCO3 4 g, nalidixic acid 250 mg, water 50 ml; solution C — thallium(I) acetate 2 g, 2,3,5-triphenyltetrazolium chloride 0,1 g, water 50 ml; solution D — MUD 150 mg in 2 ml of N,N-dimethylformamide; warning: thallium acetate and N,N-dimethylformamide are toxic — work in a fume cupboard; preparation: mixing A + B + C + D, pH 7,5 ± 0,2, sterilization by 0,2 µm filtration, dispensing 100 µl into the wells of microplates (capacity at least 350 µl) and immediate dehydration in a tunnel dryer or laminar-flow cabinet; production of the medium must meet the quality criteria of Annex E. Choice of dilutions (8.1, Table 1, read from the image of page 4): bathing water — 2 dilutions: 64 wells 1/2 and 32 wells 1/20, measurement limits 15 to 3,5 × 10⁴ bacteria/100 ml; other surface waters — 4 dilutions of 24 wells each (1/2, 1/20, 1/200, 1/2000), 40 to 3,2 × 10⁶/100 ml; waste water and treatment plants — 6 dilutions: 16 wells 1/2 to 16 wells 1/200 000, 60 to 6,7 × 10⁸/100 ml. Dilutions (8.2; preferably in a biological safety cabinet — aerosols): fresh and brackish waters (salinity < 30 g/kg, by refractometer) — 9 ml of SD diluent in each tube, 9 ml of the mixed sample into the first tube (1/2), 1 ml into the second (1/20), then in steps of 1/10; sea water (salinity > 30 g/kg) — in the first tube 9 ml of distilled or demineralized water instead of SD, in the following ones SD. Inoculation (8.3.1): the contents of the tube are poured into a sterile Petri dish (at least 90 mm) and distributed with the 8-channel pipette in 200 µl portions into the wells assigned to that dilution, changing the dish and tips for each dilution; care must be taken to avoid overflow between wells. Incubation (8.3.2): the plate is sealed with sterile tape and incubated at 44 °C ± 0,5 °C for at least 36 h and at most 72 h, without tilting. Reading (8.4): the plates with tape in the UV cabinet; wells with blue fluorescence are positive; reading is possible at any time after 36 h, because the fluorescence does not change. Results (Clause 9): for each dilution the number of positive wells; characteristic number, e.g. bathing water 64+/64 and 5+/32 → 32/5; surface waters 24/24, 18/24, 5/24, 1/24 → 18/5/1; waste water 16, 16, 12, 5, 0, 0 (of 16) → 12/5/0; with three or more dilutions — a three-digit number, ending in 0 where possible, according to ISO 8199. The MPN is a statistical estimate of density assuming a Poisson distribution, with a confidence interval; the programs of Annexes A or B give the MPN per millilitre and the 95 % interval; examples: 32/5 → 7,56/ml, interval 5,42–10,54, i.e. 756/100 ml (542–1054); 18/5/1 → 159,08/ml (101,99–248,11); 12/5/0 → 1724,61/ml (1003,98–2962,50); with no positive wells the result is “< n/100 ml”, where n is the MPN for one positive well in the arrangement used. Test report (Clause 10): data identifying the sample, method and results.

STATUS (card as of 02.10.2026). The card of PN-EN ISO 7899-1:2002 (Polish version) has no “Withdrawn” header — the standard is CURRENT: published 20-12-2002, 45 pages, price group S, Health, Environment and Medicine Sector, KT 120 Water Quality — Microbiological and Biological Testing, ICS 07.100.20, “Introduces: EN ISO 7899-1:1998 [IDT], EN ISO 7899-1:1998/AC:2000 [IDT], ISO 7899-1:1998/Cor 1:2000 [IDT], ISO 7899-1:1998 [IDT]”. The PKN search for “PN-EN ISO 7899-1” (02.10.2026) shows only this card. We do not know the content of corrigendum Cor 1:2000. We did not check whether ISO is working on a new edition (iso.org refuses access from our server).

LEGAL ACT. Directive 2006/7/EC concerning the management of bathing water quality, Annex I (original text and the latest consolidated version in the CELLAR database, 01.01.2014): for intestinal enterococci the reference methods of analysis are “ISO 7899-1 or ISO 7899-2”; values for inland waters: excellent quality 200, good 400 (95th percentile), sufficient 330 (90th percentile) cfu/100 ml; for coastal and transitional waters 100, 200 and 185 cfu/100 ml. We have not read the Polish regulation on supervision of bathing water quality — we do not state how it transposes these methods.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, data up to 17.09.2026, read 02.10.2026). The number 7899-1 appears in 8 records at 8 laboratories: AB 216, AB 313, AB 521, AB 560, AB 565, AB 589, AB 631, AB 680. In the current scopes on the PCA website (read 02.10.2026, issues from 11.07.2025 to 07.08.2026) the number appears at all 8 as “PN-EN ISO 7899-1:2002”, at AB 313 written “PN-EN-ISO 7899-1:2002” (with a hyphen after EN). No row is set in bold italics. The “Laboratories” tab searches entries BEGINNING WITH the text entered: under “PN-EN ISO 7899-1” it shows 7 laboratories and 7 positions, under “PN-EN-ISO 7899-1” — AB 313 — checked with the tab queries on the labcoda.pl production server on 02.10.2026.

WHAT THE LABORATORIES TEST (positions in PCA). Water, water in bathing areas and places occasionally used for bathing (AB 216, AB 313, AB 521), surface water (AB 560, AB 589), water (AB 565, AB 631, AB 680) — “most probable number of intestinal enterococci, miniaturized method (MPN)”; at AB 680 “enterococci (faecal streptococci)”. Everywhere the adjacent position includes PN-EN ISO 9308-3:2002 (MPN of Escherichia coli, miniaturized method).

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. In the diluent: the SD diluent contains sea salt — a pure NaCl solution markedly inhibits growth; with sea water (salinity > 30 g/kg) the first dilution is made with distilled water, not SD. In the dilution arrangement: the number of dilutions and wells depends on the type of water (Table 1) — the arrangement for bathing water (upper limit 3,5 × 10⁴) gives an “above range” result with waste water. In reading: a positive result is blue fluorescence under UV 366 nm, in the dark; non-faecal species (E. casseliflavus, E. mundtii) also give a positive result — with waters containing plant material or industrial waste waters they inflate the count of “intestinal enterococci”. In the calculation: the characteristic number is chosen according to ISO 8199 (with three dilutions — three digits, ending in 0 where possible); the MPN from the program is given per ml — the conversion to 100 ml and the confidence interval have to be given together. In the medium: production of the microplates must meet the criteria of Annex E — plates not meeting the criteria give seemingly correct MPNs. At work: thallium acetate and N,N-dimethylformamide are toxic.

WHAT WE DO NOT GIVE. We do not have Clauses 1–3 (blank page in the sample) or Annexes A–G — we therefore give no MPN tables, calculation programs, quality criteria for the medium in microplates (Annex E), preparation of calibration microplates (Annex F) or precision data.

Method principle

The diluted water sample is dispensed in 200 µl portions into the wells of microplates with dehydrated MUD/SF medium; after 36–72 h at 44 °C enterococci hydrolyse 4-methylumbelliferyl-β-D-glucoside and the wells fluoresce blue under UV light of 366 nm; from the number of positive wells in the successive dilutions the most probable number (MPN) per 100 ml with a 95 % confidence interval is calculated.

Applications

• Water in bathing areas and places occasionally used for bathing — intestinal enterococci; Directive 2006/7/EC names ISO 7899-1 or ISO 7899-2 as reference methods
• Surface waters — MPN of intestinal enterococci (4 dilutions, 40 to 3,2 × 10⁶/100 ml)
• Waste water and treatment plants — up to 6 dilutions (60 to 6,7 × 10⁸/100 ml)
• Sea water and brackish water — separate dilution procedure at salinity > 30 g/kg

Key parameters

Standard

Standard number

PN-EN ISO 7899-1:2002 (wersja polska; EN ISO 7899-1:1998 z AC:2000 IDT, ISO 7899-1:1998 z Cor 1:2000 IDT)

Title (PL)

Jakość wody — Wykrywanie i oznaczanie ilościowe enterokoków kałowych — Część 1: Zminiaturyzowana metoda do badania wód powierzchniowych i ścieków (najbardziej prawdopodobna liczba bakterii)

Title (EN)

Water quality — Detection and enumeration of intestinal enterococci in surface and waste water — Part 1: Miniaturized method (Most Probable Number) by inoculation in liquid medium

Step-by-step procedure

1. Sample and dilutions

   Sampling according to ISO 8199 and ISO 5667; dilution arrangement according to Table 1; fresh and brackish waters — SD from the first tube, sea water (> 30 g/kg) — first tube with distilled water. PN-EN ISO 7899-1:2002 current (card as of 02.10.2026).

2. Inoculation

   Each dilution poured into a sterile Petri dish and distributed with the 8-channel pipette in 200 µl portions into the wells with MUD/SF medium; a new dish and tips for each dilution.

3. Incubation

   Sealing the plate with sterile tape; 44 °C ± 0,5 °C for 36–72 h, without tilting.

4. Reading

   In the UV cabinet at 366 nm, in the dark, plate with tape; wells with blue fluorescence — positive.

5. Calculation

   Characteristic number according to ISO 8199; MPN per ml and 95 % interval from the program (Annex A or B), conversion to 100 ml; no positives — “< n/100 ml”.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The standard warns: thallium acetate and N,N-dimethylformamide are toxic (chemical fume cupboard), UV irritates skin and eyes (gloves, goggles); diluting and pipetting preferably in a biological safety cabinet (aerosols); waste water samples — infectious material
• TECHNICAL NOTE: non-faecal species (E. casseliflavus, E. mundtii) are counted as intestinal enterococci — with waters containing plant material and industrial waste waters this has to be taken into account in interpretation; pure NaCl instead of sea salt inhibits growth

Frequently asked questions