Drinking Water Inspectorate: Information Letter 3/2000 - 21 January 2000
To: Board Level contacts of Water and Sewerage Companies and Water Companies in England and Wales
Dear Sir/Madam,
The Water Supply (Water Quality) (Amendment) Regulations 1999 : Cryptosporidium in Water Supplies: Laboratory Analysis
Purpose
1. The purpose of this letter is to:
i) advise water companies of the award of the licence for the inter-laboratory proficiency scheme under the above Regulations;
ii) to issue an update, and provide further advice on, the Standard Operating Protocol For The Monitoring of Cryptosporidium Oocysts In Treated Water Supplies To Satisfy Water Supply (Water Quality) (Amendment) Regulations 1999 (the protocol);
iii) to advise water companies on the laboratories that can validate new methods or parts of methods for sampling and analysis under the protocol;
iv) to give details of currently approved laboratories under the above regulations.
Inter-laboratory proficiency scheme
2. In August this year, the Inspectorate issued an invitation to tender for a licence for three years (extendable to five years) to provide an inter-laboratory proficiency scheme. A licence has been awarded to LGC (Teddington) Limited who will administer the scheme, distribute test solutions, statistically analyse the results and to report the results. LGC will use the Scottish Parasite Diagnostic Laboratory as a sub-contractor for the preparation of the test solutions. The licensed proficiency scheme offered by LGC satisfies part 4 of the protocol on requirements for the inter-laboratory proficiency schemes. Participation in the scheme is mandatory for all approved laboratories undertaking Cryptosporidium analysis under these Regulations. The approved laboratories' representatives on the Steering Board of the proficiency scheme are John Watkins of CREH Analytical Limited (independent) and David Sartory of Severn Trent Water Ltd (Water UK). Contact details for the interlaboratory proficiency scheme are given at Annex 1.
Revision to part 2 of the protocol
3. Part 2 of the protocol dealing with laboratory and analytical procedures has been revised to provide clarity and correct some typographical errors. The revised version of the protocol is available at the end of this letter. A list of the changes, highlighted in bold, is given at Annex 2.
Validation of new methods or parts of methods for sampling and analysis
4. Details of the validation of a new method are given part 3 of the protocol: Validation of New Methods or Parts of Methods for Sampling and Analysis. Any approved laboratory may undertake the phase 1 or phase 2 trials specified in this part of the protocol.
Approved laboratories
5. A list of currently approved laboratories to undertake Cryptosporidium analysis under the Regulations is given at Annex 3. This list and future amendments will be placed on the Inspectorate's website.
Enquiries
6. Any enquiries about this letter should be sent to Mark Smith, Zone 2/A1, telephone 020 7944 5958.
7. Copies of this letter and the attachments are being sent to approved laboratories;the proficiency scheme laboratory, Pamela Taylor, Chief Executive, Water UK; Bob Dinwiddy, Water Supply and Regulation Division, Department of the Environment, Transport and the Regions; Bob Macey, Environment Division, The National Assembly for Wales; Tim Hooton, Water Services Unit, Scottish Executive; Randal Scott, Drinking Water Inspectorate for Northern Ireland; Rowena Tye, Office of Water Services; and David Harper, Department of Health.
8. Please acknowledge receipt of this letter using the attached slip and reply paid envelope.
Yours faithfully
Michael Rouse
Chief Inspector
Annex 1
Contact for the Regulatory Cryptosporidium Inter-Laboratory Proficiency Scheme
Miss J. Peet or Mr S Kippin
LGC (Teddington) Limited
Queens Road
Teddington
Middlesex
TW11 0LYTelephone 0181 943 7500
Facsimile 0181 943 1050
E-mail: jrp@lgc.co.uk
E-mail: sjk@lgc.co.uk
Annex 2
List of amendments to part 2
5.6.2 Centrifuge the 50 ml centrifuge tube containing the membrane disk filter eluate at 1100G or another suitable speed for 15 minutes. Allow the centrifuge to coast to a stop without braking. With a Pasteur pipette or venturi vacuum pump with a disposable micro-pipette tip, and using gentle suction, carefully aspirate off the supernatant to just above the pellet. The speed of the centrifuge must be optimised for maximum recovery of oocysts.
5.6.5 and 5.6.6 (Comment moved to after section 6.3.6)
5.7.2.1 Transfer the water sample concentrate (from section 5.6.3 or section 5.6.6) to the Dynal L10 tube containing the mixed SL-buffer and use a further 1 ml oocyst-free reagent water to rinse out the centrifuge tube. Label the Dynal L10 tube with the sample number and place open tube in a tube rack.
5.7.3.8 Return the microcentrifuge tube in the MPC-M to the vertical position and transfer the entire sample from the tube to the sample well of slide containing the NaOH. Do not disturb the beads at the back wall of the tube. Gently mix sample with the NaOH using the transfer pipette (see section 6.1.2).
NB: Samples may be stored at this stage at room temperature until dry and then in secured refrigerator at +2°C - +8°C in a dry box until ready for stage 6, or incubated at a temperature not exceeding 42°C until the evaporation step is complete.
6.1.1 Use slides which are approved for use with the IMS kit (normally those from the same manufacturer as the kit used, see appendix B.10). Prepare three positive and three negative controls. For each positive control, vortex for approximately 15 seconds and then pipette 25-50 µl of the working oocyst suspension (the exact concentration of which has been established in D.2.2) to the centre of a slide well, to give 80 to 120 oocysts on the control slide. For each negative control, pipette 50 µl of reagent water into the centre of a slide well and allow to spread over the well area. Ensure that each of the positive and negative control slides are labelled. Positive control slides must be prepared outside the area used for preparing sample slides to prevent any risk of cross-contamination. Also, ensure that the sample and the methanol do not spread beyond the non-coated area of the well.
6.1.4 Follow the Immuno-Fluorescent Antibody Test (IFAT) reagent manufacturer's instructions in preparing anti-Cryptosporidium sp. fluorescein-labelled monoclonal antibody (Mab) and overlay the sample slide well, the positive-control slide well, and the negative-control slide well with 50 µl of fluorescein-labelled Mab. Place the slides in a humid chamber and incubate at 37°C for 60 to 90 minutes in the dark. The humid chamber consists of a sealable plastic container containing damp paper towels on which the slides are placed on supports (e.g. swab sticks).
6.2.9 Some commercial mountants contain antifadents that will stabilise fluorescence. However, some may, for reasons that have not yet been identified, cause leaching of stain and loss of fluorescence. It is therefore important to ensure that before the introduction of any new mountant is made, the new mountant is fully checked to ensure that its performance at least matches that of the mountant specified in B.12.
6.3.6 If atypical structures are not observed, then categorise each apple-green fluorescing object as:
(i) A Cryptosporidium oocyst with typical internal structure, or
(ii)A Cryptosporidium oocyst with amorphous internal structure, or
(iii)An empty Cryptosporidium oocyst (which may show an open suture where sporozoites have been released).
The number of Cryptosporidium oocysts to be reported under the Regulations is the sum of these three categories.
(NB: If a number of sub-samples were generated at paragraph 5.6.6 the number of oocysts found in the whole sample is the sum of the number of oocysts found in all the subsamples.)7.1 All results of analysis under the Regulations must be reported to the relevant water company such that its requirements of the Regulations are achieved. Results must include details of the water meter reading at end of sample run and volume of water filtered, timer reading at end of sample run and elapsed sample time, headloss over filter, deposit volume, and concentration of oocysts found for each sample. In addition the report should include the analysis identity number (this is the number on the evidence bag used to transport the sample back to the laboratory), the water company identity number, the sample point identity number, analytical laboratory identity number, the date and time of both the start and finish of the sample run, and the date when the result was reported to the company.
Annex 3
LABORATORIES APPROVED TO UNDERTAKE CRYPTOSPORIDIUM ANALYSIS UNDER THE WATER SUPPLY (WATER QUALITY) (AMENDMENT) REGULATIONS 1999
Annex 3 is available in Adobe Acrobat format for download. The Adobe Acrobat Reader can be freely downloaded.
- Annex 3(5kb)
- Standard Operating Protocols for the Monitoring of Cryptosporidium Oocysts in Treated Water Supplies to Satisfy Water Supply (Water Quality) Amendment Regulations 1999 SI No 1524
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Updated 21 July 2000 Updated 11 July 2001
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