🦠 Validation of methods for concentrating bacteriophages from water (when fewer than 3 plaque-forming particles per ml are expected) — spiking material from treated or raw sewage (centrifugation at 1000 g for 20 min or filtration through 8–12 µm, 60–200 pfp/ml, 5 % glycerol, 10 ml portions frozen at −20 or −70 °C) checked by the dispersion tests T1 (0.01 < T1 < 5.99) and T2 (< 3.84); for each water type at least five samples on different days and seasons, volumes of 0.125, 0.25, 0.5 and 1 × Vmax spiked with 1 ml of material, recovery in the whole final concentrate, Dixon test for outliers; phages enumerated according to ISO 10705-1, -2 or -4; PN-EN ISO 10705-3:2025-06

Microbiology PN-EN ISO 10705-3

Overview

WHAT THE STANDARD COVERS. We read the text of the standard in the iTeh sample of ISO 10705-3:2003 (first edition of 01.10.2003, French version) — PN-EN ISO 10705-3:2025-06 adopts EN ISO 10705-3:2024 and ISO 10705-3:2003 as identical (PKN search), so the ISO text read is the one in force: foreword, clauses 1–8 and clause 9 to the end of 9.2 — the sample ends on page 5. We do not read calculations (10), analytical quality control (11), the test report (12), Annex A (recommended concentration methods depending on volume, turbidity and particle content) or Annex B (example of validation); we know their titles from the table of contents. We did not read the title page of EN ISO 10705-3:2024.

ACCORDING TO THE TEXT OF ISO 10705-3:2003. The standard was prepared by ISO/TC 147 "Water quality", Subcommittee SC 4 "Microbiological methods". The ISO 10705 series: Part 1 — F-specific RNA bacteriophages, Part 2 — somatic coliphages, Part 3 — validation of concentration methods, Part 4 — bacteriophages infecting Bacteroides fragilis. Warning: the user establishes safety and health practices; important: personnel validating concentration methods must be experienced in bacteriophage enumeration methods (ISO/TR 13843). Scope (1): general principles for assessing the performance of methods for concentrating bacteriophages from water; concentration is recommended for samples expected to contain fewer than 3 plaque-forming particles (pfp) per millilitre; the methods can be applied to all types of water provided the amount and nature of suspended or dissolved matter do not interfere with concentration; the standard gives no details of methods, only the principles for evaluating the suitability of a method for a given type and volume of water; examples of methods found satisfactory are given in Annex A. References (2): ISO 3696, ISO 6887-1, ISO 8199, ISO 10705-1, -2, -4, ISO/IEC Guide 2. Definition (3.1): bacteriophages — bacterial viruses capable of infecting selected host strains; note: they produce visible plaques (zones of lysis) in a confluent lawn of the host strain. Principle (4): the sample is treated by a chosen method that concentrates bacteriophages from a large volume (100 ml to several litres) to a small one (a few to 20 ml); the concentrate is examined by a standardized method or another suitable protocol; the concentration method is described precisely in a protocol (as far as possible following the layout of ISO standards) including target phage groups and their detection methods, water types, volume range and exceptions, e.g. turbidity; validation consists of determining the recovery of bacteriophages from a series of samples spiked with naturally polluted water (raw or treated sewage) over a range of volumes, with particular attention to the reproducibility of recovery. Reagents (5): ingredients of uniform quality and analytical-grade chemicals; glass-distilled or deionized water free of substances inhibiting bacterial growth, at least grade 3 (ISO 3696); diluent — peptone saline solution or another according to ISO 6887-1 or ISO 8199; culture media and reference cultures according to the relevant phage enumeration method (ISO 10705-1, -2, -4); glycerol (870 g/l) autoclaved at (121 ± 3) °C for 15 min, stored in the dark at room temperature for at most one year. Apparatus (6): safety precautions — field equipment is disinfected before use; some steps require hydrostatic or pneumatic pressure; usual microbiological equipment according to the enumeration method and the concentration protocol. Sampling (7): samples up to 10 l are easily transported to the laboratory — sampling and transport according to ISO 8199 (also ISO 19458); for larger volumes the first concentration step is preferably done on site (it may take several hours); if bacterial indicators are examined in parallel, a proportional sample is taken, preferably from a side stream of the concentration apparatus; filters, precipitates or other first-step products may be processed further on site or transported; transport and storage conditions of intermediate steps are included in the validation. Spiking material (8): a sample of primary or secondary (biologically treated) sewage is centrifuged at 1000 g for 20 min or filtered through an 8–12 µm membrane; the supernatant or filtrate is kept in melting ice; target phages are counted in 1 ml volumes and the sample diluted if necessary to 60–200 pfp/ml; glycerol is added to 5 % by volume, mixed, dispensed in 10 ml portions into glass or plastic bottles (or tubes, vials) and frozen at (−20 ± 5) °C or (−70 ± 10) °C; two bottles are thawed at room temperature and two 0.5 ml portions from each are examined — the mean count should be within the limits (30–100 pfp); homogeneity within and between bottles is checked with Cochran dispersion statistics: T1 (variation within a vial, I(J−1) degrees of freedom) and T2 (variation between vials, I−1 degrees of freedom), with I = 2 vials and J = 2 replicates; if phages are randomly distributed, T1 and T2 approximately follow a χ² distribution with 2 and 1 degrees of freedom; the material is acceptable if 0.01 < T1 < 5.99 and T2 < 3.84; note: somatic coliphages, F-specific RNA bacteriophages and phages infecting Bacteroides fragilis naturally present in partially treated sewage show no significant inactivation when frozen with 5 % glycerol and can be stored below (−20 ± 5) °C, preferably at (−70 ± 10) °C, for at least one year without significant loss. Batch methods (9.1.1): samples of all water types within the scope of the method, taken on different days, preferably representing different seasons and weather conditions; at least five samples for each water type; Vmax — the maximum volume to be processed by the method; at least 3 × Vmax of sample is needed; containers with 0.125, 0.250, 0.500 and 1 × Vmax, each with 1 ml of spiking material warmed to room temperature, the rest kept in ice. Continuous methods (9.1.2): field studies for each water type on different days, at least five samples, the same volumes; 1 ml of material in about 10 ml of diluent is injected into the inflow of the apparatus (e.g. with a syringe needle through a tube) in four similar portions, each after about one fifth of the volume has passed. Recovery (9.2): spiked samples are processed according to the protocol, including transport and storage simulating real samples; the whole final concentrate is examined in portions of 1 ml or less; where possible phages retained on concentration surfaces, e.g. filters, are also examined; in parallel two 0.5 ml portions of spiking material — to calculate concentration efficiency and T1, T2; if more than 20 % (1 in 5) of spiking material samples fail T1 or T2, the material is discarded; if 20 % or fewer — the result of that analysis is disregarded and repeated; outliers are rejected with the Dixon test; at least five experiments with non-rejected results; if the natural water may contain phages detected by the same host, they are counted — if > 20 % of the phages added, the water is heated and held at 80 °C for 30 min and cooled before use; if < 20 %, they are counted and taken into account in the calculations (10).

STATUS (as of 03.10.2026). The PKN search engine (query "10705-3", 03.10.2026) returns three results: PN-EN ISO 10705-3:2025-06 — Polish version and English version, "Introduces: EN ISO 10705-3:2024 [IDT], ISO 10705-3:2003 [IDT]", without a withdrawal note; and PN-ISO 10705-3:2021-01 (Polish version) — "Withdrawn and replaced by PN-EN ISO 10705-3:2025-06 - English version". The record of PN-ISO 10705-3:2021-01 (read 03.10.2026): publication date 27-01-2021, withdrawal date 13-06-2025, introduces ISO 10705-3:2003 [IDT], KT 120 "Water Quality — Microbiological and Biological Testing", replaced by PN-EN ISO 10705-3:2025-06 (English version). What the withdrawal changes for the result: both standards introduce the same text ISO 10705-3:2003 (the new one via EN ISO 10705-3:2024), so the validation principles remain the same; what changes is the document designation that should appear in the scope and the report.

HOW MANY LABORATORIES AND IN WHAT FORM (copy of the accreditation scope database, load up to 17.09.2026, read 03.10.2026). The number 10705-3 appears in 2 records at 2 laboratories: AB 700 and AB 1095. In the current PCA documents (read 03.10.2026; AB 1095 issue no. 25 of 03.07.2026, AB 700 issue no. 33 of 21.08.2026) the number appears at the same 2 laboratories; in each document the number of page markers equals the number of PDF pages, without duplicates; searching all PCA documents downloaded from the BIP on 03.10.2026 found no laboratories outside the database copy. Forms: "PN-EN ISO 10705-3:2025-06" (AB 700) and "PN-ISO 10705-3:2021-01" (AB 1095 — edition withdrawn on 13.06.2025), at both together with PN-EN ISO 10705-2:2005. None of these items is suspended. The "Laboratories" tab shows AB 700 under the query "PN-EN ISO 10705-3" and AB 1095 under "PN-ISO 10705-3" — checked by tab queries on the labcoda.pl production server on 03.10.2026.

WHAT THE LABORATORIES TEST (PCA items). AB 700: water, water intended for human consumption, sewage — number of somatic coliphages, plate method (pour plate), PN-EN ISO 10705-2:2005 and PN-EN ISO 10705-3:2025-06. AB 1095: water, water intended for human consumption — number of somatic coliphages, plate method (pour plate), PN-EN ISO 10705-2:2005 and PN-ISO 10705-3:2021-01. Enumeration of somatic coliphages according to PN-EN ISO 10705-2 has a separate entry in the Knowledge Base.

WHERE A SEEMINGLY CORRECT RESULT IS EASY TO GET. Nature of the standard: it is not a concentration method but the principles of its validation — the actual method is described in the laboratory protocol, with examples in Annex A (1, 4). Scope of use: concentration is recommended when fewer than 3 pfp/ml are expected (1); validation covers the water types and volume range of the protocol, and exceptions (e.g. turbidity) must be described in it (4). Spiking material: phages naturally present in sewage; homogeneity checked by T1 and T2, and if more than 20 % fail the material is discarded entirely (8, 9.2). Samples: at least five for each water type, on different days and preferably seasons, at four volumes up to Vmax (9.1). Recovery: the whole concentrate is examined and, where possible, phages retained on filters (9.2). Natural phages: if above 20 % of the phages added, the water is heated for 30 min at 80 °C (9.2). Transport: transport and storage conditions of intermediate steps belong to the validation (7). Edition: AB 1095 cites PN-ISO 10705-3:2021-01, withdrawn on 13.06.2025 — the technical text is the same (ISO 10705-3:2003).

WHAT WE DO NOT GIVE. We do not reproduce the formulas for calculating recovery (10), quality control requirements (11), the content of the report (12), or the methods in Annex A and the example in Annex B — they are outside the sample. We do not summarize ISO 10705-1, -2 or -4 in this entry.

Method principle

A concentration method intended to transfer bacteriophages from a large volume of water (100 ml to several litres) into a few millilitres is checked by its recovery. A spiking material is prepared from phages naturally present in sewage, frozen with glycerol and checked for homogeneity (tests T1 and T2). The same 1 ml of material is added to a series of volumes of the water tested (up to the maximum volume of the method), the samples are concentrated according to the protocol and phages counted in the whole concentrate by a standardized enumeration method; the recovery, repeated in at least five samples of each water type, shows whether the method is suitable.

Applications

• Waters with low numbers of bacteriophages (< 3 pfp/ml) that need concentration (1)
• Validation of batch concentration methods (samples in containers) and continuous ones (flow through the apparatus in the field) (9.1)
• Somatic coliphages, F-specific RNA bacteriophages and Bacteroides fragilis phages — enumeration according to ISO 10705-1, -2, -4 (2, 5.3)
• Water, water intended for human consumption and sewage — number of somatic coliphages (with PN-EN ISO 10705-2) — 2 accreditation scope records of 2 laboratories in PCA

Key parameters

Standard

Standard number

PN-EN ISO 10705-3:2025-06

Title (PL)

Jakość wody — Wykrywanie i oznaczanie liczby bakteriofagów — Część 3: Walidacja metod zatężania bakteriofagów z wody

Title (EN)

Water quality — Detection and enumeration of bacteriophages — Part 3: Validation of methods for concentration of bacteriophages from water

Step-by-step procedure

1. Protocol of the concentration method

   Phage groups, detection methods, water types, volumes and exceptions (4). PN-EN ISO 10705-3:2025-06 — no withdrawal note (PKN, 03.10.2026).

2. Spiking material

   Sewage cleared of particles, 60–200 pfp/ml, 5 % glycerol, 10 ml portions frozen; T1, T2 check (8).

3. Spiked samples

   ≥ 5 samples of each water type, four volumes up to Vmax, 1 ml of material each (9.1).

4. Concentration and counting

   According to the protocol including transport and storage; whole concentrate, also phages on filters (9.2).

5. Evaluation

   Material check (T1, T2), Dixon test, natural phages; calculations — outside the sample (9.2, 10).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Field equipment is disinfected before use, with safety precautions appropriate to the disinfectant (6)
• Steps under hydrostatic or pneumatic pressure require appropriate safety precautions (6)

Frequently asked questions