🔴 Legionella in Water

Microbiology PN-EN ISO 11731

Overview

STATUS (as of 29.10.2026). PN-EN ISO 11731:2017-08 — Polish version, published on 25.09.2019, adopts EN ISO 11731:2017 [IDT] and ISO 11731:2017 [IDT]; it replaced PN-EN ISO 11731-2:2008 and PN-ISO 11731:2002; it has the national correction PN-EN ISO 11731:2017-08/Ap1:2019-12 (approved on 05.12.2019), which corrects errors of the Polish version — among others the titles of 8.4.2 and 8.4.3 (both concern samples with a LOW amount of interfering microorganisms), the last sentence of 4.1, the descriptions of media preparation in Annex B and the text and examples of the decision matrix in Annex J. The standard is current — no withdrawal note. ISO 11731:2017 (second edition) replaced, after technical revision, ISO 11731:1998 and ISO 11731-2:2004 (foreword of the standard).

SCOPE (clause 1). Culture methods for the isolation of bacteria of the genus Legionella and estimation of their numbers in water samples of all kinds — drinking water, industrial water, waste water and natural water — and in water-related matrices such as biofilm and sediments. Not all Legionella species are culturable, so the methods do not recover all species.

SIGNIFICANCE (introduction). Legionellae are widely found in natural and artificial aquatic environments, in soil and compost, and can cause legionellosis; they can grow intracellularly in protozoa (Acanthamoeba castellanii, Hartmannella, Naegleria). At least 61 species have been described, in 26 of them — strains infecting humans; Legionella pneumophila has at least 15 serogroups. Monitoring serves to identify sources of legionellosis risk, such as evaporative cooling towers, hot- and cold-water systems in buildings with associated equipment — spa pools, dental units and air conditioning units — as well as validating control measures and verifying that they remain effective.

CHOICE OF PROCEDURE (clause 8, Annex J). The laboratory chooses the procedure for the sample type according to the decision matrix of Annex J. In most cases concentration by membrane filtration is needed; direct plating of the unconcentrated sample can be done when more than 10⁴ cfu/l are expected, and highly contaminated samples are diluted and plated directly before and after pre-treatment. The standard distinguishes samples: with a high concentration of Legionella and a low concentration of interfering microorganisms, with low concentrations of both, with a high and with an extremely high concentration of interfering microorganisms — and for each case gives a set of treatments and media (8.4.2–8.4.5). Examples from the matrix (Annex J as worded in correction Ap1): water with a low amount of interfering microorganisms, e.g. drinking water — 8.4.2 or 8.4.3, depending on the expected number of Legionella; water with a high amount, e.g. cooling tower water, industrial water, water from air washers and dental units — 8.4.4; water with a very high amount, e.g. waste water and surface water — 8.4.5 (heat and acid treatment). Example detection limit: 500 ml of filtered sample, elution in 5 ml of water and plating of 0.1 ml — 100 cfu/l; if overgrowth occurs on the plates with the untreated and heated portions and no Legionella is detected on the acid-treated plate, the limit increases tenfold (Annex J, Table J.1, as worded in correction Ap1).

REGULATIONS. Water intended for human consumption in domestic distribution systems: parametric value for bacteria of the genus Legionella below 1000 cfu/l, and in buildings of healthcare providers with inpatient services where patients with reduced immunity stay — below 50 cfu/l (Journal of Laws 2026, item 748, Annex 1 Part E). Assessment of the contamination level of domestic hot water (in brackets — for such healthcare buildings): low below 1000 cfu/l (below 50), medium from 1000 to below 10 000 cfu/l (from 50 to below 100), high from 10 000 to below 100 000 cfu/l (from 100 to below 1000), very high from 100 000 cfu/l (from 1000); from the medium level — review of the system and control measures, and on repetition — cleaning and disinfection; at the high level — also shutting off showers and aerosol-generating devices; at the very high level — the same, immediately (Annex 5). PN-EN ISO 11731 is the reference method; for risk-based verification monitoring and to supplement culture methods, ISO/TS 12869, rapid culture methods, non-culture methods and molecular methods, in particular qPCR, may also be used (Annex 4 Part A). Swimming pools: Legionella sp. 0 in 100 ml in water supplied to the basin, in basins with devices generating a water-air aerosol and in basins for teaching swimming to infants and small children, and in water in the basin — when its temperature is at least 30 °C; in shower water (hot water systems) — below 100 in 100 ml (Journal of Laws 2022, item 1230, Annex 1).

WHAT WE DO NOT GIVE HERE. The publicly available text of the standard ends in 8.4.7 (examination of the plates). The confirmation procedure (8.5), expression of results (clause 9), test report (clause 10), quality assurance (clause 11), the compositions of the media, diluents and acid solution (Annexes B–D), alternative techniques (Annexes E–F), identification of species and serogroups (Annex G), performance data (Annex H) and treatment of biofilm and sediments (Annex I) are beyond it; we know the decision matrix (Annex J) from the fragments quoted in correction Ap1. The composition of BCYE agar, the acid solution and the confirmation procedure are given as an example from the British official method (The determination of Legionella bacteria in waters and other environmental samples, part 2, 2020), based on this standard — marked “example” in the steps.

Method principle

Legionellae in the water sample are concentrated by membrane filtration, diluted or plated directly — depending on the origin and characteristics of the sample. To reduce the growth of concentrated non-target bacteria, portions of the sample are subjected to heat treatment, acid treatment or a combination of both. Samples with high numbers of Legionella or other bacteria are diluted, and separate portions of the diluted sample are treated, one with heat and a second with acid solution (with extremely contaminated samples — with acid solution and heat), before culturing on selective media. Treated and untreated portions are transferred onto plates of the chosen culture medium selective for Legionella and incubated (4.1). After incubation, morphologically characteristic colonies are regarded as presumptive Legionella (4.2) and confirmed by subculture (4.3); by the definition of the standard, bacteria of the genus Legionella grow on buffered charcoal yeast extract (BCYE) agar containing L-cysteine and iron(III) salts (3.1).

Applications

• Hot- and cold-water systems in buildings, evaporative cooling towers, spa pools, dental units, air conditioning units (introduction)
• Water intended for human consumption in domestic distribution systems — below 1000 cfu/l, in healthcare buildings with patients with reduced immunity below 50 cfu/l (Journal of Laws 2026, item 748, Annex 1 Part E and Annex 5)
• Swimming pools — water supplied to the basin, basins with aerosol, water in the basin at a temperature of at least 30 °C, showers (Journal of Laws 2022, item 1230, Annex 1)
• Outbreak investigations — with samples expected to have many interfering bacteria, the plates are checked already on day 2 (8.4.7)
• Validation of control measures and verification of their effectiveness (introduction)
• Drinking water, industrial water, waste water, natural water, biofilm and sediments (clause 1)

Key parameters

Standard

Standard number

PN-EN ISO 11731:2017-08

Title (PL)

Jakość wody — Oznaczanie ilościowe bakterii z rodzaju Legionella

Title (EN)

Water quality — Enumeration of Legionella

Step-by-step procedure

1. Sampling, transport and storage

   According to ISO 19458; do not expose the samples to adverse temperatures (freezing, overheating) — insulated containers help (clause 7). Example (British method, 2020): transport and storage at 6–20 °C, analysis as soon as possible, not later than within two days, preferably within 24 h.

2. Choice of procedure

   The procedure is chosen for the sample type using the decision matrix (Annex J). Usually — concentration by filtration; when more than 10⁴ cfu/l are expected, the unconcentrated sample can be plated; dilute highly contaminated samples and plate directly before and after pre-treatment. Record the volumes and the pre-treatments applied (8.1).

3. Concentration — filter on the medium

   Filter the sample (untreated, after acid treatment and, if required, after heat treatment) through a cellulose nitrate or mixed cellulose esters filter; record the volume; with disinfected forceps place the filter right-side up on the medium, with no air bubbles underneath. The acid treatment can be done on the filter in the funnel (8.2.2).

4. Concentration with elution

   Filter the sample through a polycarbonate or polyethersulfone filter, record the volume, place the filter right-side down in a sterile container (with or without glass beads), add 5–10 ml of sterile diluent or sample and shake vigorously on the vortex mixer for at least 2 min or in the ultrasonic bath for a verified time. Divide the concentrate into three portions: untreated, for heat treatment and for acid treatment (8.2.3).

5. Heat treatment

   Keep the sample or concentrate in a sterile container in a water bath at (50 ± 1) °C for (30 ± 2) min — the time counts from reaching the temperature; preferably volumes up to 5 ml; with many samples or large volumes, monitor the temperature in a separate container (8.3.1).

   🌡 Temperature: 50 ± 1 °C

6. Acid treatment

   Dilute one volume of the sample or concentrate with nine volumes of the acid solution, mix and leave for (5.0 ± 0.5) min; take the dilution into account in the calculation. On the filter: about 30 ml of acid solution for (5 ± 0.5) min, filter off and wash the filter with at least 20 ml of diluent, without rinsing the part of the funnel that had no contact with the acid (8.3.2).

7. Plating

   Plate and spread 0.1–0.5 ml of each portion on plates according to the case: many Legionella and little flora — BCYE and BCYE+AB agar; few Legionella and little flora — untreated filter on BCYE, acid-treated filter on BCYE+AB, GVPC or MWY (or portions of the concentrate on BCYE and selective media); much flora — untreated, heat-treated and acid-treated portions on GVPC or MWY; very much flora — heat treatment, cooling, acid treatment, dilutions 1:10 and 1:100 on GVPC or MWY. Record the inoculated volumes (8.4.2–8.4.5).

8. Incubation

   After the inoculum has been absorbed, invert the plates and incubate at (36 ± 2) °C for 7–10 days in a humid atmosphere, e.g. in an enclosed container. Natural samples with wild strains can require the full 10 days (8.4.6).

   🌡 Temperature: 36 ± 2 °C

9. Examination of plates

   First examination on day 2, 3, 4 or 5 (to detect overgrowth), last at the end of incubation; a dissection microscope with oblique illumination is recommended. Legionella colonies are usually white-grey, smooth, with an entire edge and a ground-glass appearance; under the UV lamp the colonies of several species (among others L. anisa, L. bozemanii, L. dumoffii, L. gormanii) autofluoresce brilliant white, L. erythra and L. rubrilucens — red, and L. pneumophila — dull green, often tinged with yellow. Record the number of presumptive colonies of each type (8.4.7).

10. Confirmation

    Presumptive colonies are confirmed by subculture (4.3; procedure in 8.5, beyond the available text of the standard). Example (British method, 2020): subculture at least three colonies of each type onto BCYE agar and BCYE agar without L-cysteine (or blood or nutrient agar), incubate for 2–3 days; colonies growing only on BCYE with L-cysteine are regarded as Legionella. Identification of species and serogroups requires further tests and is not part of the methods of the standard (4.3, Annex G).

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 and competent staff (warning of the standard)

Frequently asked questions