🦠 Sampling of water for microbiological testing — bottles, inactivation of the disinfectant, transport (PN-EN ISO 19458)

Microbiology PN-EN ISO 19458

Overview

SCOPE, TRANSLATED FROM THE TEXT OF THE STANDARD: the standard provides guidance on the planning of water sampling programmes, on sampling procedures for microbiological analysis and on the transport, handling and storage of samples until the analysis begins. It focuses on sampling for microbiological testing. General information on sampling from the individual types of water is in the relevant parts of the ISO 5667 series — hence this standard and PN-ISO 5667-5 or PN-EN ISO 5667-6 are read together, not interchangeably.

THE SAMPLING POINT — AND THREE EXAMPLES THAT EXPLAIN WHY. The sampling point must give a representative characterisation and take account of vertical, horizontal and temporal variability; it is determined in accordance with the general recommendations of ISO 5667-1 and ISO 5667-2, with additional considerations specific to microbiology. Points with unstable conditions should be avoided and the heterogeneity of the hydraulic system should be taken into account. The standard gives three examples of this heterogeneity: a subsurface sample, a surface sample and a subsurface sample "contaminated" when withdrawn through the surface film are not the same — in lakes and swimming pools the concentration in the surface film may be A THOUSAND TIMES higher than just below the surface; not all points of a network are equivalent, because there are dead ends and sections with reduced flow in it, especially when the network is supplied from two sources; the quality at the outlet of a well-mixed tank is usually the same as in the bulk of the water, but may differ considerably from the quality at the inlet. When testing the effectiveness of disinfection, the point must be chosen so that the reaction is already complete.

PERSONNEL. The standard requires that formal training, training records and the way competence is established be described for all persons taking samples — and that this information be properly documented. This is a system requirement, not a technical one, and is sometimes omitted in procedures.

CONTAINERS. For routine samples (taps, recreational waters, swimming pool waters) clean, sterile bottles are used with a volume sufficient for all the parameters ordered. For sampling by immersion the bottles must be sterile inside and outside and protected, for example with kraft paper (so that they remain dry after autoclaving), aluminium foil or a plastic bag — the bag is opened just before sampling and can serve as a glove holding the bottle. If bottles cannot be autoclaved, sterilisation by gamma radiation or ethylene oxide is permitted. The external surface of the bottle can also be disinfected immediately before immersion with a suitable agent, for example isopropanol, and left to dry — but this is NOT suitable for testing spore-forming bacteria.

VOLUMES — NUMBERS FROM THE STANDARD. In most cases a 500 ml bottle is sufficient, because fewer than five categories of microorganisms are determined and each requires inoculation of at most 100 ml. Larger volumes are needed for bottled waters (250 ml per parameter), for Legionella spp. or Salmonella spp. (up to 1 l) and for viruses, Giardia cysts, Cryptosporidium oocysts and amoebae in clean waters — there from 10 to several hundred litres or more are tested and concentration is usually carried out on site, on a filter cartridge that is transported to the laboratory. Bottles may be of glass or of plastics (polypropylene, polystyrene, polyethylene, polycarbonate); glass usually reusable, polyethylene as single-use.

INACTIVATION OF THE DISINFECTANT — THE ESSENCE OF THIS STANDARD FOR WATER UTILITIES. When assessing the microbiological quality of water disinfected with an oxidant (chlorine, chloramine, bromine, ozone), the action of the oxidant must be stopped immediately after sampling by adding a reducing agent — sodium thiosulfate — to the bottle. The standard gives a calculation: the theoretical mass of sodium thiosulfate (pentahydrate) needed to neutralise 1 mg of chlorine is 7.1 mg, so 0.1 ml of thiosulfate solution (18 mg/ml) is added for every 100 ml of bottle capacity. This neutralises at least 2 mg/l, and depending on the kinetics even up to 5 mg/l of residual free chlorine — which is sufficient for most samples. At higher chlorine concentrations (paddling pools at swimming pools, Legionella elimination campaigns in water supply networks) the dose must be increased proportionally. Thiosulfate is not destroyed in the autoclave or by dry heat, and the pH of its solution is to be close to neutral, because a low pH causes decomposition. Thiosulfate does not affect the sample and may also be used for non-chlorinated waters. If inactivation is not possible or practicable, this must be recorded in the report.

PRACTICE OF A POLISH WATER UTILITY. The instruction of a water utility citing this standard gives the sequence of operations at a tap: remove the strainer, run the water in a free stream for at least 3 minutes, close the valve, wash the tap with a brush and detergent, rinse and close, sterilise a metal tap with a flame from a blowtorch or with a cotton wool swab soaked in spirit, a plastic tap — immerse in a disinfectant and DO NOT flame it, when sampling from a plastic hose cut off the end 5–8 cm and immerse it in disinfectant liquid, open the valve so that the water does not splash, take the sterile bottle out of the foil, open the cap protecting it from contamination and — without touching the tap — fill the bottle up to the narrowing in the neck, close immediately, label and pack.

WHAT WE DO NOT GIVE. We have a preview of the text of the standard covering the scope, the sampling point, personnel, containers, inactivation of disinfectants and the list of reagents and equipment; clause 5 on transport and storage is in the inaccessible part. We therefore DO NOT GIVE: the permissible transport times and storage temperatures required by this standard or the deadlines for starting the test for individual groups of microorganisms.

Method principle

A microbiological sample must reach the laboratory in the state in which it left the sampling point. Three measures of the standard follow from this: sterility of the path of the sample (sterile containers, a sterilised tap, work without touching surfaces in contact with the water), stopping the action of the disinfectant immediately after sampling (reducing agent in the bottle) and inhibiting changes in numbers over time (cooling and prompt start of the test). The sampling point is chosen so as to take account of the heterogeneity of the system — the surface film, dead ends of the network, the difference between the inlet and outlet of a tank.

Applications

• Sampling of water for human consumption for microbiological testing — from consumer taps and network points
• Recreational and swimming pool waters, including sampling by immersion
• Testing the effectiveness of disinfection — with the sampling point chosen so that the reaction is complete
• Sampling for Legionella spp. and Salmonella spp. (up to 1 l) and bottled waters (250 ml per parameter)
• Sampling for viruses, Giardia, Cryptosporidium and amoebae — with on-site concentration on a filter cartridge
• Documenting the competence of persons taking samples

Key parameters

Standard

Standard number

PN-EN ISO 19458:2007 (wersja polska; wprowadza EN ISO 19458:2006 [IDT] i ISO 19458:2006 [IDT])

Title (PL)

Jakość wody -- Pobieranie próbek do analiz mikrobiologicznych

Title (EN)

Water quality — Sampling for microbiological analysis

Step-by-step procedure

1. Selection of the sampling point

   Representative, taking account of vertical, horizontal and temporal variability and the heterogeneity of the hydraulic system. When assessing disinfection — a point at which the reaction is already complete.

2. Preparation of the bottle

   Sterile, with a volume sufficient for all parameters. For sampling by immersion sterile inside and outside, protected with kraft paper, foil or a bag.

3. Adding the reducing agent

   For water disinfected with an oxidant: 0.1 ml of sodium thiosulfate solution (18 mg/ml) for every 100 ml of bottle capacity. At higher chlorine concentrations increase the dose proportionally. When inactivation is impossible — record it in the report.

4. Preparation of the tap

   Remove the strainer, run the water for at least 3 minutes, close, wash the tap with a brush and detergent, rinse. Sterilise a metal tap with a flame or a swab with spirit; immerse a plastic tap in a disinfectant and DO NOT flame it.

5. Filling

   Open the valve so that the water does not splash. Open the bottle protecting the cap from contamination and — without touching the tap — fill up to the narrowing in the neck. Close immediately.

6. Labelling and packing

   Label permanently: point, date, time, type of water. Pack in a protective bag.

7. Transport

   Under refrigerated conditions, without shocks and exposure to light, to the laboratory on the day of sampling. WE DO NOT GIVE THE SPECIFIC TIMES AND TEMPERATURES REQUIRED BY THE STANDARD — clause 5 is outside the available preview.

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Sampling from lakes, swimming pools and reservoirs by immersion — risk of falling into the water; weight with rope and safety line
• Sterilisation of the tap by flame — open fire in a manhole or a technical room; do not use near flammable materials and vapours
• Samples for Legionella are taken in hot water installations — risk of scalding and exposure to aerosol
• SUBSTANTIVE NOTE: disinfection of the external surface of the bottle with isopropanol is NOT suitable for testing spore-forming bacteria
• SUBSTANTIVE NOTE: metal caps, especially aluminium ones, may show toxicity after autoclaving — a heat-resistant, tight liner prevents this

Frequently asked questions