• Confirmation or exclusion of the implicated site as a source of infection
• Risk assessment of the site's water system(s)
• Distinguishing between local or system-wide colonisation of water system(s)
• Identifying critical sites
• Checking the regulation of the temperature, pressure and flows in the plumbing system
• Selecting the right strategy for short term control of Legionella
• Facilitating a proposal for the long term control strategy for the whole facility.

• There are variations in sampling techniques and laboratory methods
• Factors other than Legionella counts such as aerosolisation, strain virulence, and human susceptibility influence the risk of illness
• Legionella in the water will not always cause disease so positive test results could cause a false sense of alarm and lead to unnecessary spending on corrective measures
• Negative test results may cause a false sense of security leading to relaxation of preventive maintenance
• Healthcare facilities might be tempted to use routine sampling as a substitute for appropriate patient surveillance and system maintenance

• Eliminating the house as a source of infection in an individual case for epidemiological purposes only
• Identifying a continuing risk of exposure in situations where there is reason to believe that another occupant of the property might be at increased risk (as opposed to a normal level of risk) of developing illness. HPS also proposed that at least one of the following additional criteria should be fulfilled:
• Evidence that a legionella-like illness, though not necessarily clinically or microbiologically confirmed, has occurred previously amongst occupants of the same house
• Evidence that sampling of the water system would contribute information to inform prevention and control of legionellosis in general terms and which could not otherwise be obtained.

• The quality of the sample(s)
• The location of sampling points in terms of being representative of the water system being tested
• The timing of the sampling in relation to the normal operating conditions and control measures of the system, including the timing and levels of biocide dosing
• Proper transportation and storage of the sample(s) to ensure that the sample(s) should undergo as little change as possible before the analysis begins.

5.4 Safety

Environmental samples for Legionella should be collected by people with knowledge of Legionella ecology and general risk assessment. People taking environmental samples require training to ensure that they select samples containing the highest numbers of bacteria, and that they are aware of the risk to themselves and to others from potentially positive sites. Based on a written risk assessment, in some circumstances, it may be necessary to use respiratory protective equipment (see Chapter 6, Section 6.4 on PPE).3 Individual staff who may be particularly prone to an increased risk of Legionella infection due to underlying conditions or immunosuppression should not be involved in sampling operations.

5.5 Site assessment

The number and types of sites that should be tested to detect Legionella must be determined on an individual system basis because of the diversity of plumbing, heating, ventilation and air-conditioning systems in the various institutions that may be sampled.3 Samples should be representative of each separate water system. They should be taken from the proximal and distal end of the water system and a number of sentinel points in between, the number and location being based on a comprehensive risk assessment. Selection of sampling sites also depends on whether the sampling is for routine monitoring or to investigate an outbreak.

It is essential to undertake a survey of the site to be investigated, prior to taking any sample.26 All surveys follow a basic pattern. The source and the quality of the water should be determined and the site should be examined to establish the location of all systems using water. These systems should then be reviewed and assessed to determine which systems contain water at temperatures likely to support the growth of Legionella bacteria. In addition, areas within the systems where growth of Legionella bacteria may be expected to be greatest should be reviewed, as should locations where potentially contaminated water might produce aerosols or where aerosols might be released into the environment. The route or pathway of the water through the system should be followed from its entry into the site to the point where it is used or discharged. If a schematic diagram does not exist or is not available, or is known to be or is suspected of being out-of-date, then an up-to-date diagram should be prepared indicating, for example locations of:

• The in-coming water supply, whether of mains or private source
• Storage tanks, expansion or pressure vessels, filters, booster vessel pumps and strainers
• Water softening, filters or other storage or treatment facilities
• Calorifiers or water heaters

The type and nature of materials and fittings, for example taps, showers, water closet cisterns, valves, thermostatic mixer valves, pressure release valves, bathroom radiators and towel rails connected to the domestic water supply (and associated pipework) and the presence of metals, plastics, jointing compounds, etc.

• Evaporative cooling towers and condensers or heating circuits
• Air conditioning systems or humidifiers within the building which are supplied with, and store, water and which may produce aerosols
• Other equipment that contains water and which might be a potential risk, such as spa pools, humidified display cabinets, machine tools, fountains, etc.
• Equipment that is used infrequently or might not normally be of concern but presents a risk only when the system undergoes maintenance or repair
• The presence of dead-legs or blind-ends.

When all risk sites have been identified the appropriate samples can be collected. There should be discussion with the laboratory which will analyse the samples on the number and type of samples required.26 Arrangements should also be made for the transportation of the samples to the laboratory.

Aseptic precautions during sampling

It is important to take appropriate precautions to eliminate cross contamination occurring between sampling sites, especially when collecting dip samples from storage tanks, cisterns and cooling towers.

5.6 Sample types

Two primary sample types should be collected when sampling for Legionella - water samples and swabs of biofilm.3
Water samples capture the planktonic form of Legionella or any disturbed biofilm. Generally, a minimum of one litre must be collected.27 Samples should be collected in new, unused capped or pre-sterilised polyethylene or similar containers containing sufficient sodium thiosulphate to neutralise any chlorine or other oxidising biocide. Temperatures should be measured using a calibrated thermometer, placed in the middle of the water stream.

Swabs samples capture the sessile form of Legionella that is associated with biofilms.72 Swab samples must be taken before water samples when collecting both sample types from the same outlet. Swab samples must be kept moistened with sterile water. Multiple samples can be collected from the same site. Sterile absorbent cotton wool swabs should be used.

5.6.1 Pre-flush sample

A pre-flush sample is water collected immediately after the tap, or fitting, is opened. The tap or fitting should not have previously been disinfected, or water run to waste. The pre-flush sample represents water held within the tap or fitting and, ideally, should be taken when the tap has not been used for several hours.

5.6.2 Post-flush sample

A post-flush sample is water collected after the tap, or tap fitting, has been disinfected and water in the fitting has run to waste. The post-flush sample represents the quality of circulating water supplied to the tap or fitting.
Information should be gathered to help interpret the results. As a minimum, the following information should be included on the request form:

• the site and sample point
• the sample references and date
• the reason for sampling
• the temperature of the sample source (e.g. the temperature of a hot-water system at one minute after turning on the tap, and at two minutes after turning on the cold tap)
• any biocide used
• the timing of the dosage in relation to sampling
• the concentration detected at the time of sampling
• any other risk factors of importance (e.g. closed system opened for maintenance)
• high risk of nutrient present, such as in plastics manufacturing plants
• any cases associated with the site.

During the sampling all details that may help the implementation of possible remedial measures should be recorded. For example, obvious pressure and temperature drops or rises in the water circuits, the presence of iron sediment or sludge, the condition of the aerator and taps, the occurrence of scale, and the presence of various rubber and plastic attachments.

5.7 Sample transport and storage

All samples should be transported to the laboratory in dark, insulated containers to protect them from extreme temperatures and from light.

Analysis should begin as soon as possible after the sample has been taken, preferably on the same day. If analysis is delayed, samples should be stored so that concentration and incubation procedures can be commenced within 48 hours of collection. The maximum time from sample collection to culture of the concentrate is 14 days. Samples should be transported and stored at less than 18oC but not less than 6oC.27 Storing the sample in a refrigerator at temperatures below 6oC may reduce subsequent recovery of Legionella bacteria since the bacteria may be induced into a non-culturable state. Although Legionella will not multiply significantly during this period, the organism may be adversely affected by the presence of biocides remaining in the sample. If biocides are likely to be present in the sample and cannot be neutralised prior to storage this information should be recorded, and the transport and storage times kept to a minimum

5.8 Laboratory analysis

Analysis of water samples and swabs for Legionella should be carried out preferably by an accredited laboratory, which takes part in an external quality assessment scheme for the isolation of Legionella from water. It should be a target of all currently testing laboratories to attain accreditation. Laboratory facilities for environmental testing should be available in each HSE area.
To meet international best practice requirements a national legionella reference laboratory should be established for clinical and environmental sample testing, to act as a typing centre and to provide expert opinion on the microbiology of the organism.

For more detailed information on sampling procedures see 'The determination of Legionella bacteria in waters and other environmental samples (2005) - Part 1 - rationale of surveying and sampling' produced by the UK Environment Agency.