1、 Collection of SANS standards in electronic format (PDF) 1. Copyright This standard is available to staff members of companies that have subscribed to the complete collection of SANS standards in accordance with a formal copyright agreement. This document may reside on a CENTRAL FILE SERVER or INTRA
2、NET SYSTEM only. Unless specific permission has been granted, this document MAY NOT be sent or given to staff members from other companies or organizations. Doing so would constitute a VIOLATION of SABS copyright rules. 2. Indemnity The South African Bureau of Standards accepts no liability for any
3、damage whatsoever than may result from the use of this material or the information contain therein, irrespective of the cause and quantum thereof. ISBN 978-0-626-22071-6 SANS 11731:2008Edition 1ISO 11731:1998Edition 1SOUTH AFRICAN NATIONAL STANDARD Water quality Detection and enumeration of Legionel
4、la This national standard is the identical implementation of ISO 11731:1998 and is adopted with the permission of the International Organization for Standardization. Published by SABS Standards Division 1 Dr Lategan Road Groenkloof Private Bag X191 Pretoria 0001Tel: +27 12 428 7911 Fax: +27 12 344 1
5、568 www.sabs.co.za SABS SANS 11731:2008 Edition 1 ISO 11731:1998 Edition 1 Table of changes Change No. Date Scope National foreword This South African standard was approved by National Committee SABS SC 147B, Water Biological treatment and assessment of water, in accordance with procedures of the SA
6、BS Standards Division, in compliance with annex 3 of the WTO/TBT agreement. This SANS document was published in November 2008. AReference numberISO 11731:1998(E)INTERNATIONALSTANDARDISO11731First edition1998-5-01Water quality Detection and enumerationof LegionellaQualit de leau Recherche et dnombrem
7、ent des LegionellaSANS 11731:2008This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731:1998(E) ISO 1998All rights reserved. Unless otherwise specified, no part of this publication may be reproducedor utilized in any form or by any means,
8、electronic or mechanical, including photocopying andmicrofilm, without permission in writing from the publisher.International Organization for StandardizationCase postale 56 CH-1211 Genve 20 SwitzerlandInternet centraliso.chX.400 c=ch; a=400net; p=iso; o=isocs; s=centralPrinted in SwitzerlandiiConte
9、nts Page1 Scope 12 Normative reference 13 Definition. 14 Safety 15 Principle 26 Culture media and reagents 27 Apparatus . 68 Sampling. 79 Procedure . 710 Expression of results. 1011 Test report 11Annex A: Scraping the bacteria from filter membranes 12Annex B: Colony identification on BCYE - Cys. 13A
10、nnex C: Indirect immunofluorescent assay for identificationof L. pneumophila . 14Annex D: Bibliography 16SANS 11731:2008This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO ISO 11731:1998(E)iiiForewordISO (the International Organization for St
11、andardization) is a worldwidefederation of national standards bodies (ISO member bodies). The work ofpreparing International Standards is normally carried out through ISOtechnical committees. Each member body interested in a subject for whicha technical committee has been established has the right t
12、o be representedon that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISOcollaborates closely with the International Electrotechnical Commission(IEC) on all matters of electrotechnical standardization.Draft International S
13、tandards adopted by the technical committees arecirculated to the member bodies for voting. Publication as an InternationalStandard requires approval by at least 75 % of the member bodies castinga vote.International Standard ISO 11731 was prepared by Technical CommitteeISO/TC 147, Water quality, Sub
14、committee SC 4, Microbiological methods.Annexes A, B, C and D of this International Standard are for informationonly.SANS 11731:2008This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .INTERNATIONAL STANDARD ISO ISO 11731:1998(E)1Water quality Det
15、ection and enumeration of Legionella1 ScopeThis International Standard describes a culture method for the isolation of Legionella organisms and estimation oftheir numbers in environmental samples.This method is applicable to all kinds of environmental samples including potable, industrial and natura
16、l waters andassociated materials such as sediments, deposits and slime.2 Normative referenceThe following standard contains provisions, which, through reference in this text, constitute provisions of thisInternational Standard. At the time of publication, the edition indicated was valid. All standar
17、ds are subject torevision, and parties to agreements based on this International Standards are encouraged to investigate thepossibility of applying the most recent edition of the standard listed below. Members of IEC and ISO maintainregisters of currently valid International Standards.ISO 3696:1987,
18、 Water for analytical laboratory use Specification and test methods.3 DefinitionFor the purposes of this International Standard, the following definition applies:3.1 Legionellagenus of Gram-negative organisms normally capable of growth in not less than 2 days on Buffered Charcoal YeastExtract agar c
19、ontaining L-cysteine and iron(III), and forming colonies, often white, purple to blue or lime green incolourNOTE Some species fluoresce under long-wavelength UV light. The colonies have a ground-glass appearance whenviewed with a low power stereomicroscope. With a very few exceptions, growth does no
20、t occur in the absence of L-cysteine.4 SafetyThe reagents used in this International Standard should be subject to assessment in accordance with Control ofSubstances Hazardous to Health.Legionella species can be handled safely by experienced microbiologists on the open bench in a conventionalmicrobi
21、ology laboratory conforming to Containment Level 2. Infection is caused by inhalation of the organism and itis advisable therefore to assess all techniques for their ability to produce aerosols. If in doubt, carry out the work ina safety cabinet.SANS 11731:2008This s tandard may only be used and pri
22、nted by approved subscription and freemailing clients of the SABS .ISO 11731:1998(E)ISO25 Principle5.1 GeneralBacteria, including Legionella organisms, in the water sample are concentrated by membrane filtration or bycentrifugation. Turbid samples can be centrifuged. To reduce the growth of unwanted
23、 bacteria, a portion of theconcentrated specimen is subjected to treatment with acid and another portion with heat. Treated and untreated testportions are then inoculated onto plates of agar medium selective for Legionella and incubated. Samples containingsufficient numbers of Legionella need not be
24、 subject to concentration prior to culture.5.2 EnumerationAfter incubation, morphologically characteristic colonies which form on the selective medium are regarded aspresumptive Legionella.5.3 ConfirmationPresumptive colonies are confirmed as Legionella organisms by subculture to demonstrate their g
25、rowth requirementfor L-cysteine and iron. Further biochemical and serological tests are needed for species identification.6 Culture media and reagents6.1 GeneralUse chemicals of analytical grade in the preparation of media and reagents unless otherwise stated (see note 1).Alternatively, use commerci
26、ally available dehydrated media and reagents. Prepare the media according to themanufacturers instruction and add freshly prepared selective agents or growth supplements (or thaw the storedmaterial at room temperature prior to use) at the concentrations recommended. Prepare media using glass-distill
27、edwater or water of equivalent quality complying with ISO 3696 Grade 3.NOTE 1 The use of chemicals of other grades is permissible providing they are shown to be of equal performance in the test.Use diagnostic serological reagents of known specificity from a known source. Do not use a reagent for whi
28、ch thisinformation is not available.NOTE 2 The possibility of cross-reactions with other organisms in environmental samples should be considered.6.2 Culture media6.2.1 Buffered Charcoal Yeast Extract agar medium (BCYE)6.2.1.1 CompositionYeast extract (bacteriological grade) 10,0 gAgar 12,0 gActivate
29、d charcoal 2,0 gAlpha-ketoglutarate, monopotassium salt 1,0 gACES buffer(N-2-acetamido-2-aminoethanesulfonic acid) 10,0 gPotassium hydroxide (KOH) (pellets) 2,8 gL-cysteine hydrochloride monohydrate 0,4 gIron(III) pyrophosphate Fe4(P2O7)3 0,25 gDistilled water to 1000 mlNOTE Check manufacturers reco
30、mmendations for concentration of agar to be added to provide adequate gelling strength.SANS 11731:2008This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISOISO 11731:1998(E)36.2.1.2 Preparationa) Cysteine and iron solutions.Prepare fresh solutio
31、ns of L-cysteine hydrochloride and iron(III) pyrophosphate by adding 0,4 g and 0,25 grespectively to 10-ml volumes of distilled water. Decontaminate each solution by filtration through a membrane filterwith an average pore size of 0,22 m. Store in clean sterile containers at 2(20 + 3)oC for not more
32、 than 3 months.b) ACES buffer.Add the ACES granules to 500 ml of distilled water and dissolve by standing in a water bath at (45 to 50) oC. To aseparate 480 ml of distilled water, add all the potassium hydroxide pellets and dissolve with gentle shaking. Toprepare the ACES buffer, mix the two solutio
33、ns.NOTE ACES buffer can cause denaturation of the yeast extract if the following sequence is not followed.c) Final medium.Add sequentially to the 980 ml of ACES buffer, the charcoal, yeast extract and a-ketoglutarate. Prepare a 0,1 mol/lsolution of potassium hydroxide (KOH) by dissolving 5,6 g in 1
34、litre of distilled water. Prepare a 0,1 mol/1 solution ofsulfuric acid (H2SO4) by carefully adding 5,3 ml of H2SO4to 1 litre of distilled water. Use the solutions of 0,1 mol/lpotassium hydroxide or 0,1 mol/l sulfuric acid as appropriate to adjust the pH to 6,9 + 0,2. Add the agar, mix andautoclave a
35、t (121 + 1) oC for (15 + 1) min (see 6.2.4, first paragraph). After autoclaving, allow to cool to (50 + 2) oCin a water bath.Add the L-cysteine and the iron(III) pyrophosphate solutions aseptically, mixing well between additions.Dispense in 20 ml volumes into Petri dishes of 90 mm to 100 mm diameter
36、. The pH of the final medium is 6,9 + 0,4at 25C. Allow excess moisture on the plates to dry and store at (4 + 2)oC in airtight containers in the dark for up to4 weeks.6.2.2 Buffered Charcoal Yeast extract medium without L-cysteine (BCYE - Cys)Prepare this medium in an identical manner to BCYE (6.2.1
37、) but omit the L-cysteine.6.2.3 Selective medium: Buffered Charcoal Yeast Extract medium with selective supplements(GVPC medium)NOTE This medium is identical to BCYE except that three antibiotic supplements and glycine are added to the BCYEmedium.6.2.3.1 Selective supplementsThe final concentrations
38、 in the GVPC medium shall be:Ammonium-free glycine 3 g/lPolymyxin B sulfate 80 000 iu/lVancomycin hydrochloride 0,001 g/lCycloheximide 0,08 g/l6.2.3.2 Preparation of antibiotic supplementsAdd the appropriate amount (usually 200 mg) of polymyxin B sulfate to 100 ml of distilled water to achieve aconc
39、entration of 14 545 iu/ml. Mix and decontaminate by membrane filtration as described in 6.2.1.2. Dispense5,5 ml volumes into sterile containers and store at -(20 + 3) oC. For use, thaw at room temperature.Add 20 mg of vancomycin hydrochloride to 20 ml of distilled water, mix and decontaminate by mem
40、brane filtration(6.2.1.2). Dispense in 1 ml volumes in sterile containers and store at 2(20 + 3) oC. For use, thaw at roomtemperature.SANS 11731:2008This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731:1998(E)ISO4Add 2 g of cycloheximide
41、 to 100 ml of distilled water and decontaminate by membrane filtration as described in6.2.1.2. Dispense in 4 ml volumes in sterile containers and store at 2(20 + 3) oC. For use, thaw at roomtemperature.NOTE Antibiotic supplements may be stored for up to 6 months when frozen.WARNING Cycloheximide is
42、hepatotoxic. Wear gloves and dust mask when handling this chemical inpowder form.6.2.3.3 Preparation of GVPC mediumFollow the instructions for preparation of BCYE medium given in 6.2.1.2, but add 3 g of ammonium-free glycine afterthe addition of the a-ketoglutarate and then adjust the pH to 6,9 + 0,
43、4.After the addition of the L-cysteine and iron, add one volume of each of the above three antibiotic supplements(6.2.3.2) to the final medium. Mix well.6.2.4 Quality control of mediaProlonged heating during sterilization or heating at too high a temperature shall be avoided, as it can affect thenut
44、ritional qualities of BCYE medium. Batch-to-batch variation of the ingredients of the medium (particularlya-ketoglutarate) can also affect its performance. Therefore it is essential to check the quality of each newlyprepared batch of media for its ability to support the growth of L. pneumophila sero
45、group 1 within three days ofincubation.For most bacteria, it is usual to assess the suitability of culture media to support their growth by using cultures ofpreviously isolated organisms, maintained in the laboratory. For Legionellas this method may be misleading, as theycan easily adapt to grow on
46、culture media that would not support the primary isolation of wild strains. The followingprocedure is therefore recommended for assessing the suitability of GVPC selective agar medium for Legionellaorganisms.Eithera) use plates of a previous batch of GVPC medium known to support the growth of Legion
47、ella together with platesfrom the new batch of medium and inoculate them with a water sample known to contain Legionella organisms, orb) from a nationally recognized source of reference cultures, obtain a lyophilized strain of Legionella pneumophilaserogroup 1. Reconstitute and recover as recommende
48、d, and subculture onto BYCE (6.2.1) for purity. If a typeculture is not available, use a freshly isolated and confirmed strain of L. pneumophila serogroup 1. Stock strains ofL. pneumophila shall be replaced after not more than 10 subcultures. After incubation, make a suspension fromthe resulting gro
49、wth just visible to the naked eye and dispense in 1 ml volumes in sterile glycerol broth (6.3.3.4) forstorage at 2(20 + 3)oC, or alternatively in Pages Saline (6.3.2.1) or distilled water for storage at 2(70 + 5) oC.Plate out one suspension of each isolate onto BCYE medium for subsequent identification and recording of theLegionella species and serogroup (see 9.3). For use, allow a stock suspension of one (or more) isolates to thaw atroom temperature. Shake thoroughly, wait 5 min to 10 min to allow aerosols to
