🦠 E. coli and Coliform Bacteria in Water

Microbiology PN-EN ISO 9308-1

Overview

STATUS (as of 29.10.2026). PN-EN ISO 9308-1:2014-12 — Polish version, published on 14.10.2015, adopts EN ISO 9308-1:2014 [IDT] and ISO 9308-1:2014 [IDT]; it replaced PN-EN ISO 9308-1:2004 with corrections Ap1:2005 and AC:2009. Amendment PN-EN ISO 9308-1:2014-12/A1:2017-04 — English version only, published on 04.04.2017, adopts EN ISO 9308-1:2014/A1:2017 [IDT] and ISO 9308-1:2014/Amd 1:2016 [IDT]. Neither has a withdrawal note. ISO 9308-1:2014 (third edition) replaced the 2000 edition after technical revision and incorporated its corrigendum Cor.1:2007.

AMENDMENT A1 (ISO 9308-1:2014/Amd 1:2016, the full text of the amendment — one page). It changes the incubation time in clause 4 and 8.3 from (21 ± 3) h to 21–24 h at (36 ± 2) °C and replaces Table 1 for performance testing of CCA (11.2): productivity — Escherichia coli WDCM 00013 or WDCM 00012 (dark-blue to violet colonies) and Enterobacter aerogenes WDCM 00175 or Citrobacter freundii WDCM 00006 (pink to red colonies), quantitatively against TSA, criterion PR ≥ 0.7; selectivity — Enterococcus faecalis WDCM 00087 or WDCM 00009 (total or partial inhibition of growth); specificity — Pseudomonas aeruginosa WDCM 00024 (colourless colonies). The times in this description follow the amendment.

SCOPE (clause 1). Enumeration of E. coli and coliform bacteria: membrane filtration, culture on chromogenic coliform agar (CCA) and calculation of the number of target organisms in the sample. The medium has low selectivity, so background growth interferes with reliable enumeration — the method is not suitable for surface waters or water from shallow wells. It is intended for waters with low bacterial numbers that will give fewer than 100 total colonies on CCA: drinking water, disinfected pool water and finished water from drinking water treatment plants. Strains of E. coli without β-D-glucuronidase, such as E. coli O157, will not be detected as E. coli — being β-D-galactosidase positive, they grow on this medium like coliform bacteria.

DEFINITIONS AND SIGNIFICANCE (clause 3, introduction). Coliform bacteria are members of the Enterobacteriaceae that express β-D-galactosidase, and E. coli — members of the Enterobacteriaceae that express β-D-galactosidase and β-D-glucuronidase. E. coli normally inhabits the bowel of humans and other warm-blooded animals, so it indicates faecal pollution. Some coliform bacteria live in soil and surface water — their presence is not proof of faecal contamination, but may indicate failure in treatment, storage or distribution.

CCA MEDIUM (Annex B — composition according to the ISO 9308-1:2014 specification quoted in the data sheet of a medium manufactured in accordance with the standard). Per 1 l: enzymatic digest of casein 1.0 g, yeast extract 2.0 g, sodium chloride 5.0 g, sodium dihydrogen phosphate dihydrate 2.2 g, disodium hydrogen phosphate 2.7 g, sodium pyruvate 1.0 g, sorbitol 1.0 g, tryptophan 1.0 g, Tergitol-7 0.15 g, 6-chloro-3-indoxyl-β-D-galactopyranoside 0.2 g, 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid (cyclohexylammonium salt, monohydrate) 0.1 g, IPTG 0.1 g, agar 9–18 g; pH 6.8 ± 0.2. The standard allows commercially available media and reagents with the composition of Annex B, prepared strictly according to the manufacturer's instructions (clause 6).

REGULATIONS. Water intended for human consumption: E. coli 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 intestinal enterococci these are “core parameters” whose monitoring frequency is not reduced (Annex 3 part I). Coliform bacteria: 0 in 100 ml; single ones below 10 cfu or MPN in 100 ml are allowed if E. coli and intestinal enterococci are excluded at the same time — E. coli and enterococci are then tested (Annex 1 Part C Table 1, explanatory note 1). The reference method for both parameters is PN-EN ISO 9308-1 or PN-EN ISO 9308-2 (Annex 4 Part A). Swimming pools: E. coli 0 in 100 ml in water supplied to the pool basin and in the basin (Journal of Laws 2022, item 1230, Annex 1).

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends in 8.3 (in the description of the confirmation by the oxidase test). Expression of results (clause 9), the test report (clause 10), quality assurance apart from Table 1 (clause 11), the recipe of the oxidase reagent (Annex B.2) and the performance characteristics (Annex C) are beyond it.

Method principle

A test portion of the sample is filtered through a membrane filter, which retains the bacteria, and the filter is placed on a plate of chromogenic coliform agar (CCA). After incubation at (36 ± 2) °C for 21–24 h, colonies giving a positive β-D-galactosidase reaction (pink to red) are counted as presumptive coliform bacteria other than E. coli — to avoid false-positive results from oxidase-positive bacteria, such as Aeromonas, they are confirmed by a negative oxidase reaction. Colonies giving a positive β-D-galactosidase and β-D-glucuronidase reaction (dark-blue to violet) are counted as E. coli. Total coliform bacteria are the sum of oxidase-negative pink to red colonies and all dark-blue to violet colonies (clause 4 as amended by A1).

Applications

• Drinking water, disinfected pool water and finished water from treatment plants (clause 1)
• Water intended for human consumption — E. coli 0 in 100 ml (water bottled or in containers — 0 in 250 ml); coliform bacteria 0 in 100 ml (Journal of Laws 2026, item 748, Annex 1)
• Reference method for E. coli and coliform bacteria in water intended for human consumption, alongside PN-EN ISO 9308-2 (Journal of Laws 2026, item 748, Annex 4 Part A)
• Swimming pools — E. coli 0 in 100 ml in water supplied to the basin and in the basin (Journal of Laws 2022, item 1230, Annex 1)
• Bottled water — with filtration of 250 ml (8.2)

Key parameters

Standard

Standard number

PN-EN ISO 9308-1:2014-12 (Polish version) together with AMENDMENT A1:2017-04 (English version)

Title (PL)

Jakość wody — Oznaczanie ilościowe Escherichia coli i bakterii grupy coli — Część 1: Metoda filtracji membranowej do badania wód o małej ilości mikroflory towarzyszącej

Title (EN)

Water quality — Enumeration of Escherichia coli and coliform bacteria — Part 1: Membrane filtration method for waters with low bacterial background flora

Step-by-step procedure

1. Sampling and storage

   Samples are taken and delivered according to ISO 19458; transport and storage at (5 ± 3) °C. In exceptional circumstances the sample may be kept at (5 ± 3) °C for up to 24 h before examination — the storage time is stated in the test report (clause 7, 8.1).

2. Filtration

   Preparation of the sample, filtration and inoculation follow ISO 8199. Filter 100 ml of the sample (or another volume, e.g. 250 ml of bottled water) through a membrane filter; the minimum volume is 10 ml of sample or its dilution — it ensures even distribution of the bacteria on the filter (8.1–8.2).

3. Transfer to the medium

   Place the filter on CCA so that no air is trapped underneath; invert the plate (8.3).

4. Incubation

   Incubate at (36 ± 2) °C for 21–24 h (8.3 as amended by A1).

   🌡 Temperature: 36 ± 2 °C

5. Counting colonies

   Count pink to red colonies (β-D-galactosidase) as presumptive coliform bacteria other than E. coli; dark-blue to violet colonies (β-D-galactosidase and β-D-glucuronidase) — as E. coli (8.3).

6. Oxidase test

   Test all or at least 10 pink to red colonies, selected according to ISO 8199 — with a commercial test or by placing 2–3 drops of fresh oxidase reagent on a filter paper in a Petri dish and transferring the colony onto it with a plastic or platinum loop. A dark-blue colour within 30 s means a positive reaction — coliform bacteria are oxidase negative. With dense growth or a colony adjacent to others, first make a subculture so that the test is done on a pure culture (8.3).

7. Result

   E. coli — dark-blue to violet colonies; total coliform bacteria — oxidase-negative pink to red colonies and all dark-blue to violet colonies (clause 4). Calculation per sample volume — clause 9, beyond the available text of the standard.

8. Medium control

   The performance of CCA is tested with the reference strains of Table 1 as amended by A1 (productivity, selectivity, specificity; incubation (36 ± 2) °C, 21–24 h); each batch of membrane filters is tested according to ISO 7704 (5.5, 11.2).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Persons using the standard should be familiar with normal laboratory practice; the standard does not address all safety problems — establishing safety and health practices and compliance with national regulations is the responsibility of the user (warning of the standard)
• Tests according to the standard must be carried out by suitably qualified staff (warning of the standard)

Frequently asked questions