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Legionella sampling: Part 2 - technique & chain of custody

by Water Hygiene Centre, on 15-02-2018
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Legionella sampling: Part 2 - technique & chain of custodyLast week we looked at the planning process required before any samples are taken. This week we get down to the ‘nuts and bolts’ of bottle filling…

Sampling Technique

Our tongue-in-cheek headline belies the importance of good sampling technique. As with poor planning, poor execution can also undermine the entire sampling exercise.

With so many possibilities, such as single pillar, monoblock, thermostatic mixing valves (TMV), sequential mixer, blending taps, showers outlets etc. your water sampling technique will vary depending on the type of outlet sampled and specific methodologies described (as per standards such as BS8554:2015 and BS 7592:2008).

  • Single Pillar & Monoblock (non-TMV) Outlets - Hot and cold supply should be sampled separately.TMV 1 Moreover, sampling blended water - from a mixer tap not comprising a TMV is not indicated.

  • Thermostatic Mixing Valve (TMV) Taps - When sampling from a TMV blended tap; water samples should be obtained from the outlet with the outlet lever set to the ‘mid-point’.

  • Showers - Consideration should be given to ensuring shower waters are captured accurately and safely (when systems are contaminated with aquatic pathogens), as such food-grade bags may be used to funnel water into the sampling container whilst minimising the dissemination of aerosolised water droplets.

Sampling should be completed by a suitably trained individual, this maybe your in-house ‘competent person’ or an appointed contractor. With either individual you should ensure they are competent to take samples i.e. where is the evidence they have been trained!  The individual should have received suitable Legionella training to ensure samples are taken aseptically and with no occurrence of contamination when samples are taken.


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Water / Legionlla Sample bottlesWhen filling sample containers it is important to use bottles dosed with sodium thiosulphate pentahydrate (STP), in order to neutralise any residual chlorine that may have been collected in the sample. Failure to do this may result in the receipt of false negative test results - as residual chlorine may continue to disinfect the water sample whilst in transit to the laboratory.


This consideration also supports the recommendation to sample from (chlorine) disinfected water systems no sooner than two days and no later than seven days after disinfection (HSG274 Part 2 paragraph 2.132). This ensures that the samples taken are an accurate representation of the water system and not confirmation of biocide strength.


When to take Pre or Post-Flush Samples?

To determine the need for pre or post-flush water samples, consideration must also be given to the part of the water system in question:


  • Pre-flush samples aim to capture potential contaminants within or local to the outlet;

  • Post-flush samples are used to identify potential ‘systemic’ contaminants within the wider distribution system. Obtaining accurate post flush samples can be slightly more involved due to the requirement to locally disinfect and flush the outlet before it’s sampled – to mitigate the capture of ‘local’ contaminants (e.g. particulate matter and biofilm from within the outlet).

Guidance and standards on Legionella sampling outlines pre and post flush sampling. There is no preference to be drawn on which sample should be taken. When sampling is conducted moreover it is a part of an ongoing investigation, the benefits of completing a post flush sample really do help with a detailed investigation.

In the case of Pseudomonas aeruginosa, only pre flush samples will be collected from compliant or previously unaffected systems – in accordance with the requirements of HTM 04-01 Part B – Appendix D.

How should you store and transport your water samples?

Once water samples have been collected, it’s imperative that these samples are handled, stored and transported appropriately with consideration for the differing requirements of the microorganisms of interest.

  • Legionella samples - should be stored at ambient temperature and returned to the laboratory for processing to commence no later than 48 hours from when the sample was taken.

  • Pseudomonas samples – if they cannot be processed by the laboratory within 2 hours of being taken they should be refrigerated and should be processed by the laboratory within 24-hours to ensure the samples remain within stability.

Any deviation from these prescribed storage & transportation criteria may impact upon the accuracy of the final result being reported by the laboratory.

Chain of Custody

The ‘chain of custody’ associated with water samples for microbiological analyses must be understood - to ensure that all samples are suitably received and processed by the test laboratory and that the results reported by the laboratory are correctly cross-referenced to the outlets sampled.


Chain of custody therefore refers to the paperwork trail to be completed on the water samples sent to the laboratory. Upon request, UKAS accredited laboratories will provide such paperwork in advance, so that sampling points/locations identified in the organisational sampling plan can be entered onto this paperwork along with details of the microbiological test(s) to be completed by the laboratory.


Once the paperwork has been prepared, all that remains to do is to update it in the field with such details as time of sampling, temperature of sample etc.


Whilst it’s prudent to keep a copy of this paperwork, a copy must accompany the samples sent to the laboratory so that they can be booked onto their system and processed within the correct timeframe.


The benefit of using clear chain of custody paperwork is that this minimises the need to capture lots of information on the sample bottle(s). Therefore, sample bottles can simply be labelled with a unique identification number sequence and ‘legibly’ cross-referenced against the supporting paperwork.


Next Time…

Upon successful receipt of the samples, the test laboratory will take responsibility for the chain of custody and in Part 3, we discuss the various microbiological diagnostics that can be used for the analysis of water samples – including ‘traditional’ microbiological tests, rapid (molecular) diagnostics and the merits and limitations of both.


NB – any doubt about sampling and technique the reader should refer to the citied guidance and standards documents. This blog is intended to provide a brief explanation and NOT a detailed instruction.


If you have questions regarding the issues raised above or you would like to speak with one of our consultants please click here to get in touch.

Editors Note: The information provided in this blog is correct at date of original publication - February 2018. 

© Water Hygiene Centre 2018


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About the author

Water Hygiene Centre

The Water Hygiene Centre was established in 2009 to address the lack of independent water hygiene consultancy within the industry. From our humble beginnings, we have established ourselves as a market leader, helping clients identify and minimise the risk of waterborne contamination and disease, whilst improving compliance performance.

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