🦠 Somatic coliphages in water — the plaque method with two agar layers and an Escherichia coli host strain

Microbiology PN-EN ISO 10705-2

Overview

Somatic coliphages are bacterial viruses which infect selected strains of Escherichia coli (and related strains) by attaching themselves to the cell wall as the first step of infection. In a confluent lawn of host bacteria they give visible plaques — zones of clearing. They are more resistant to disinfection than indicator bacteria and survive at a considerable distance from the source of contamination, which is why they are treated as an indicator of the presence of enteric viruses and as a tool for assessing the efficiency of virus removal in treatment processes.

The standard describes the detection and enumeration of somatic coliphages by incubating the sample with a suitable host strain. It is applied to all kinds of water, to sludge and to extracts of sewage sludge (if necessary after dilution), and also to extracts of molluscs. Where the number of phages is small, a preliminary concentration step for the sample is needed — the standard (of 2000) announces in its scope a separate document on the subject, and in the foreword names Part 3 of the series, "Concentration methods"; the Regulation of the Minister of Health permits PN-EN ISO 10705-3 for that purpose.

In Poland somatic coliphages entered the legislation as an operational parameter. The Regulation of the Minister of Health of 22 May 2026 (Journal of Laws 2026 item 748) permits the standards PN-EN ISO 10705-2 and PN-EN ISO 10705-3 for their determination, and in Annex 7, Table 2 sets a reference value of 50 pfu/100 ml for raw water: exceeding this value obliges one to carry out an analysis after the successive stages of treatment, in order to establish the logarithmic removal value and to assess whether the risk of pathogenic viruses getting through is under control. The description in this entry comes from the text of ISO 10705-2:2000 — the first and only edition, confirmed by ISO in a systematic review on 5 August 2021; a further review began on 15 July 2026 and has not yet ended. We cite the Polish adoption of this standard (PN-EN ISO 10705-2, implementing EN ISO 10705-2) without the year of edition: the PKN catalogue card could not be checked while this entry was being prepared, because the PKN shop was then unavailable; in laboratory publications the designation with the year 2005 appears. A DISCREPANCY WITHIN THIS ENTRY, NOTED AND NOT CLOSED: the list of sources contains an item described as the catalogue card of PN-EN ISO 10705-2:2005 with full data (published 22-12-2005, 22 pages, KT 120, implementing EN ISO 10705-2:2001 [IDT] and ISO 10705-2:2000 [IDT], without the "Date of withdrawal" and "Superseded by" fields). We do not decide whether these data come from the card itself or from some other account — until we have seen the card ourselves, the heading keeps the designation without a year, and we treat the year 2005 as unconfirmed. Studies of NIZP–PZH carried out according to PN-EN ISO 10705-2 showed somatic coliphages in 8 % of raw water samples from groundwater intakes and in 89 % of samples from surface intakes, whereby they were not found in any sample of treated water.

THE POLISH TITLE IN THIS ENTRY IS OUR WORKING TRANSLATION, not an official title: the title has not been confirmed on a PKN card, the shop being unavailable; the Regulation of the Minister of Health uses the spelling „colifagi somatyczne" [the regulation spells it "colifagi", not the standard Polish "kolifagi"].

Method principle

The sample is mixed with a small volume of semi-solid growth medium. A culture of the host strain is added and the whole is poured onto a solid medium. Incubation follows and the plates are read, the visible plaques being counted. The result is expressed as the number of plaque-forming particles — pfp, also called plaque-forming units, pfu — per unit volume of sample. The choice of the host strain depends on the bacterial contamination of the sample: for waters with a low bacterial content E. coli strain C is used, for heavily contaminated waters and wastewater — a mutant resistant to nalidixic acid, which makes it possible to suppress the bacterial background of the sample.

Applications

• Operational monitoring of raw water according to Journal of Laws 2026 item 748 — reference value 50 pfu/100 ml
• Assessment of the efficiency of water treatment with regard to the removal of enteric viruses (the logarithmic removal value after successive barriers)
• Surface water and groundwater abstracted by waterworks
• Wastewater and heavily contaminated waters — using a host strain resistant to nalidixic acid
• Sludge and extracts of sewage sludge (if necessary after dilution)
• Extracts of molluscs — the standard names them expressly in the scope

Key parameters

Standard

Standard number

PN-EN ISO 10705-2:2005 (EN ISO 10705-2:2001 [IDT], ISO 10705-2:2000 [IDT])

Title (PL)

Jakość wody — Wykrywanie i oznaczanie ilościowe bakteriofagów — Część 2: Oznaczanie ilościowe colifagów somatycznych

Title (EN)

Water quality — Detection and enumeration of bacteriophages — Part 2: Enumeration of somatic coliphages

Step-by-step procedure

1. Taking and transporting the sample

   Take the samples and deliver them to the laboratory in accordance with ISO 8199 and ISO 5667-1, -2 and -3. In the practice of NIZP–PZH samples were taken into sterile polypropylene bottles with the addition of sodium thiosulfate according to PN-EN ISO 19458, carried chilled and stored at 5 ± 3 °C for not longer than 48 h until examination.

   🌡 Temperature: 5 ± 3 °C

2. Choosing the host strain

   For samples with a low bacterial content (drinking water, uncontaminated natural waters) use E. coli strain C, ATCC 13706. For samples with a large number of bacteria (contaminated natural waters, wastewater) use the mutant resistant to nalidixic acid, E. coli strain CN, ATCC 700078 (WG5).

3. Stock culture of the host strain

   Reconstitute the contents of the lyophilised ampoule of the reference strain in about 3 ml of MSB broth with a Pasteur pipette, transfer to a 300 ml conical flask containing (50 ± 5) ml of MSB and incubate with gentle shaking. Add 10 ml of sterile glycerol (final concentration 15–20 % by volume), mix, dispense about 0.5 ml into vials and store at (−70 ± 10) °C or in liquid nitrogen.

   ⏱ Time: (20 ± 4) h • 🌡 Temperature: (36 ± 2) °C

4. Working cultures and shaking

   Carry out the cultures at the successive stages with gentle shaking — besides faster growth this ensures that all the cells are in the growth phase and that no stationary-phase cells appear, which lower the plating efficiency. Without a shaker, shake the inoculation cultures repeatedly by hand.

5. Calibrating the measurement of the culture density

   Calibrate the absorbance measurements against the number of viable cells (clause 10.2 of the standard), using a spectrophotometer with a filter in the range 500–650 nm and 1 cm cells or nephelometric flasks with a side arm.

6. Dilutions of the sample

   If necessary prepare dilutions of the sample in peptone-salt solution or in another diluent in accordance with ISO 6887. The standard also permits the examination of extracts of sludge and of molluscs after dilution.

7. Double-layer plating

   Mix the sample with a small volume of semi-solid growth medium, add the culture of the host strain and pour onto the solid medium on a Petri dish. Among the equipment the standard names a water bath or a heating block set at (45 ± 1) °C and a separate bath for melting the agar media.

8. Incubation and reading

   Incubate the plates in an incubator or a water bath set at (36 ± 2) °C, and then read them, counting the visible plaques in the confluent lawn of host bacteria. For counting use a counter with indirect, oblique illumination.

   🌡 Temperature: (36 ± 2) °C

9. Samples with a small number of phages

   When the expected number of phages is small, a preliminary concentration of the sample is necessary — the standard refers for this to a separate document (ISO 10705-3, in Poland PN-EN ISO 10705-3, permitted for that purpose by the regulation). NIZP–PZH concentrated 100 ml samples by membrane filtration on cellulose ester filters, after adding magnesium chloride to a final concentration of 0.05 mol/l.

10. Quality assurance and the result

    Prepare the reference material from bacteriophage ΦX174, ATCC 13706-B1 (clauses 8 and 11.6.1 of the standard). Express the result as the number of plaque-forming particles (pfp/pfu) per unit volume of sample; for comparison with the reference value of the regulation it is converted to 100 ml of raw water.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• The host strain used in this standard is non-pathogenic to humans and animals — handle it in accordance with the usual safety procedures of a bacteriological laboratory
• Somatic coliphages too are non-pathogenic to humans and animals, but some types are very resistant to drying — cross-contamination of the material examined must be prevented
• Carry out work with high-titre cultures and the inoculation of host-strain cultures in a biological safety cabinet or in a separate part of the laboratory
• Chloroform is a carcinogenic substance — take the proper precautions or apply an equally effective alternative method
• The samples of surface water and wastewater examined contain faecal flora — the usual rules for work with potentially infectious material apply (gloves, laboratory coat, decontamination of waste)

Frequently asked questions