🦠 Clostridium perfringens in Water

Microbiology PN-EN ISO 14189

Overview

STATUS (as of 29.10.2026). PN-EN ISO 14189:2016-10 — Polish version, published on 04.07.2017, adopts EN ISO 14189:2016 [IDT] and ISO 14189:2013 [IDT]. The standard is current — no withdrawal note. ISO 14189:2013 (first edition) has the status of a current standard in the catalogue.

SCOPE (clause 1). Enumeration of vegetative cells and spores of Clostridium perfringens by membrane filtration in samples of water intended for human consumption. The method can be applied to all types of water, provided they do not contain particulate or colloidal matter that interferes with filtration.

SIGNIFICANCE (introduction). C. perfringens is a recognized indicator of faecal pollution. In the intestinal tract of animals and humans these Gram-positive bacteria form spores, which are resistant to heating compared with vegetative cells; spores are also found in environmental samples. They survive in water for months, much longer than vegetative faecal indicator bacteria, so their presence may indicate remote or intermittent faecal pollution. Monitoring of C. perfringens is useful for assessing water resources and for checking the stages of water treatment; the spores are not always inactivated by routine disinfection, such as chlorination.

DEFINITIONS (clause 3). Presumptive C. perfringens — bacteria producing on TSC agar colonies of all shades of black or grey to yellow brown, even if the colour is faint, after anaerobic incubation at (44 ± 1) °C for (21 ± 3) h; colonies on the membrane do not always show distinct blackening, so faint colonies are also counted. Confirmed C. perfringens — bacteria producing characteristic colonies on TSC agar and possessing the enzyme acid phosphatase.

REGULATIONS. Water intended for human consumption: Clostridium perfringens (including spores) 0 cfu/100 ml — an indicator parameter, measured if a risk assessment indicates that it is appropriate (Journal of Laws 2026, item 748, Annex 1 Part C Table 1 item 2 and explanatory note 3); the reference method is PN-EN ISO 14189 (Annex 4 Part A).

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends at 8.3 (sample dilution). Filtration (8.4), incubation and examination (8.5), the confirmation procedure (8.6), expression of results (clause 9), quality assurance (clause 11), the composition of the media and the acid phosphatase reagent (Annex A) and the performance data (Annex B) are beyond it. The composition of TSC agar, the acid phosphatase reagent and the confirmation procedure are given as an example from the British official method for drinking water (The Microbiology of Drinking Water, part 6, 2021), based on the same principle — marked “example” in the steps.

Method principle

A measured volume of the sample, or a dilution of it, is filtered through a membrane filter with a pore size of 0.45 µm, which retains the spores of clostridia. The filter is incubated on a selective/differential medium — tryptose sulfite cycloserine agar (TSC) — anaerobically at (44 ± 1) °C for (21 ± 3) h. C. perfringens usually produces black or grey to yellow brown colonies, because it reduces sulfite to sulfide, which reacts with an iron(III) salt in the medium. Characteristic colonies are counted and confirmed, and the result is calculated as the colony count per sample volume. If a count of spores alone is required, the sample is first heated at (60 ± 2) °C to inactivate vegetative bacteria. Cycloserine inhibits the growth of Bacillus species, and incubation at 44 °C increases the selectivity for C. perfringens (clause 4).

Applications

• Water intended for human consumption and other waters without particulate or colloidal matter that hinders filtration (clause 1)
• Water intended for human consumption — C. perfringens (including spores) 0 cfu/100 ml where a risk assessment indicates it; reference method (Journal of Laws 2026, item 748, Annex 1 Part C and Annex 4 Part A)
• Assessment of water resources and checking the stages of water treatment (introduction)
• Detection of remote or intermittent faecal pollution — spores survive in water for months (introduction)

Key parameters

Standard

Standard number

PN-EN ISO 14189:2016-10

Title (PL)

Jakość wody — Oznaczanie ilościowe Clostridium perfringens — Metoda filtracji membranowej

Title (EN)

Water quality — Enumeration of Clostridium perfringens — Method using membrane filtration

Step-by-step procedure

1. Transport and storage of the sample

   Start the examination as soon as possible, preferably on the day of sampling; cool the samples during transport, ideally to (5 ± 3) °C. Recommended maximum time including transport: 12 h for vegetative bacteria and 24 h for spores; it must not exceed 18 h and 72 h (8.1).

2. Heating — only for counting spores

   Mix the sample well and heat it in a water bath to (60 ± 2) °C for (15 ± 1) min; the volume heated should be greater than the volume analysed. The temperature is monitored with a thermometer in a reference bottle of the same size containing the same volume of water at the same initial temperature; reaching (60 ± 2) °C must not take longer than 15 min — water circulation in the bath shortens it (8.2).

   🌡 Temperature: 60 ± 2 °C

3. Choice of volume and dilution

   Choose the sample volume or dilution (after heating, if required) to yield, if possible, 10–80 colonies on a filter of 47–50 mm diameter; dilutions according to ISO 8199 (8.3).

4. Filtration and incubation

   Filter the portion through a 0.45 µm membrane, place the filter on TSC agar and incubate anaerobically at (44 ± 1) °C for (21 ± 3) h (clause 4). Funnels used for samples with spores must be sterile — boiling is insufficient (5.2).

   🌡 Temperature: 44 ± 1 °C

5. Counting presumptive colonies

   Count colonies of all shades of black or grey to yellow brown, including faint ones — on the membrane they do not always show distinct blackening (3.1).

6. Confirmation — acid phosphatase

   Confirmed C. perfringens are characteristic colonies with acid phosphatase (3.2); blood agar, Columbia agar base or another nutrient-rich agar is used for subculture (6.2.2). Example (British method, 2021): subculture the colonies onto Columbia agar base, incubate anaerobically at 37 °C for (21 ± 3) h, place 2–3 drops of the reagent — a purplish or dark brown colour within 3 min means a positive result; test positive and negative controls at the same time.

7. Result

   The number of confirmed colonies is related to the volume of sample filtered (clause 4); calculation rules — clause 9 and ISO 8199, beyond the available text of the standard.

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