1、BRITISH STANDARD BS EN ISO 11731-2:2008 BS 6068-4.18:2004 Water quality Detection and enumeration of Legionella Part 2: Direct membrane filtration method for waters with low bacterial counts ICS 07.100.20; 13.060.70 BS EN ISO 11731-2:2008 This British Standard was published under the authority of th
2、e Standards Policy and Strategy Committee on 20 July 2004 BSI 2008 ISBN 978 0 580 60246 7 National foreword This British Standard is the UK implementation of EN ISO 11731-2:2008. It is identical with ISO 11731-2:2004. It supersedes BS ISO 11731-2:2004 which is withdrawn. The UK participation in its
3、preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/4, Microbiological methods. A list of organizations represented on this subcommittee can be obtained on request to its secretary. Additional information This British Standard is not suitable for cooling tower
4、waters. In addition, laboratories using this method should satisfy themselves that the method works as well in their laboratory for their samples as BS 6068-4.12:1998 (ISO 11731:1998). This publication does not purport to include all the necessary provisions of a contract. Users are responsible for
5、its correct application. Compliance with a British Standard cannot confer immunity from legal obligations. Amendments/corrigenda issued since publication Date Comments 31 July 2008 This corrigendum renumbers BS ISO 11731-2:2004 as BS EN ISO 11731-2:2008. Also, additional information added to nationa
6、l forewordEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO117312 March2008 ICS07.100.20 EnglishVersion WaterqualityDetectionandenumerationofLegionellaPart2: Directmembranefiltrationmethodforwaterswithlowbacterial counts(ISO117312:2004) QualitdeleauRechercheetdnombrementdes LegionellaPartie2:Mtho
7、deparfiltrationdirectesur membranepourleseauxfaibleteneurenbactries(ISO 117312:2004) WasserbeschaffenheitNachweisundZhlungvon LegionellenTeil2:DirektesMembranfiltrationsverfahren mitniedrigerBakterienzahl(ISO117312:2004) ThisEuropeanStandardwasapprovedbyCENon24February2008. CENmembersareboundtocompl
8、ywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCENManagementCentreortoanyCENmember. ThisEuropeanS
9、tandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCENManagementCentrehasthesamestatusas the officialversions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Bulgaria,C
10、yprus,CzechRepublic,Denmark,Estonia,Finland, France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,P ortugal, Romania,Slovakia,Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION
11、EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2008CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENISO117312:2008:EForeword The text of ISO 11731-2:2004 has been prepared by Technical Committee ISO/TC 147 “Water quality”
12、of the International Organization for Standardization (ISO) and has been taken over as EN ISO 11731-2:2008 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an
13、identical text or by endorsement, at the latest by September 2008, and conflicting national standards shall be withdrawn at the latest by September 2008. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not
14、 be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia,
15、Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 11731-2:2004 has been approved by CEN as
16、 a EN ISO 11731-2:2008 without any modification. BS EN ISO 11731-2:2008 iii Contents Page 1 Scope 1 2 Normative references . 1 3 Terms and definitions. 1 4 Safety 2 5 Principle . 2 6 Culture media and reagents. 2 7 Apparatus 6 8 Sampling 7 9 Procedure 7 10 Expression of results 9 11 Test report . 9
17、Annex ZA (informative) A-deviations.10 BS EN ISO 11731-2:2008 blankI SO 4002 All irthgs ersedevr 1Water quality Detection and enumeration of Legionella Part 2: Direct membrane filtration method for waters with low bacterial counts WARNING Persons using this part of ISO 11731 should be familiar with
18、normal laboratory practice. This part of ISO 11731 does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. 1 Sc
19、ope This part of ISO 11731 describes a monitoring method for the isolation and enumeration of Legionella organisms in water intended for human use (e.g. hot and cold water, water used for washing), for human consumption and for treated bathing waters (e.g. swimming pools). It is especially suitable
20、for waters expected to contain low numbers of Legionella. As the growth of Legionella may be inhibited by overgrowth of other bacterial colonies on the membrane, the method is only suitable for waters containing low bacterial counts. 2 Normative references The following referenced documents are indi
21、spensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 8199:
22、1) , Water quality General guidance on the enumeration of micro-organisms by culture ISO 11731:1998, Water quality Detection and enumeration of Legionella 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 Legionella genus of Gram-negative bacte
23、ria normally capable of growth in no less than 2 days on buffered charcoal yeast extract agar containing L-cysteine and iron(III), and forming colonies, often white, purple to blue or lime green in colour NOTE Some species fluoresce under long wavelength UV light. The colonies have a ground-glass ap
24、pearance when viewed with a low power stereomicroscope. Growth does not occur in the absence of L-cysteine with the exception of L. oakridgensis and L. spiritensis. L. oakridgensis and L. spiritensis require L-cysteine and iron for primary isolation but can grow weakly in the absence of added L-cyst
25、eine thereafter. 1) To be published. (Revision of ISO 8199:1988) BS EN ISO 11731-2:20082 4 Safety The reagents used in this part of ISO 11731 should be subject to assessment in accordance with control substances hazardous to health. Legionella species can be handled safely by experienced microbiolog
26、ists on the open bench in a conventional microbiology laboratory conforming to containment level 2. Infection is caused by inhalation of the organism and it is advisable therefore to assess all techniques for their ability to produce aerosols. If in any doubt, carry out the work in a safety cabinet.
27、 5 Principle 5.1 General Bacteria, including Legionella organisms, in the water sample are concentrated by membrane filtration. After filtration, the filter is treated with acid buffer added directly into the funnel to reduce the growth of non- Legionella organisms. The filter is subsequently transf
28、erred onto a plate of agar medium selective for Legionella and incubated. Samples expected to contain sufficient numbers of Legionella need not be subjected to concentration prior to culture (9.1). 5.2 Enumeration After incubation, morphologically characteristic colonies which form on the selective
29、medium are regarded as presumptive Legionella. 5.3 Confirmation Presumptive colonies are confirmed as Legionella organisms by subculture to demonstrate their growth requirement for L-cysteine and iron. Further biochemical and serological tests are needed for species identification. Species identific
30、ation may not be considered necessary for routine monitoring but is indispensable in outbreak situations. NOTE L. pneumophila serogroup 1 is the causative agent of most legionellosis cases and is therefore considered the most “critical” type of Legionella to be found in the water system. Since incre
31、asing numbers of cases of legionellosis caused by other serogroups of L. pneumophila and other Legionella species are being described, even the presence of other Legionella species in water is considered a potential risk. 6 Culture media and reagents 6.1 General Use chemicals of analytical grade in
32、the preparation of media and reagents unless otherwise stated. Alternatively, use commercially available dehydrated media and reagents. Prepare the media according to the manufacturers instruction and add freshly prepared (or thaw the stored material at room temperature prior to use) selective agent
33、s or growth supplements at the concentrations recommended. Prepare media using glass distilled water or water of equivalent quality complying with ISO 3696:1987, Grade 3. Other grades of chemicals may be used providing they can be shown to produce the same results. BS EN ISO 11731-2:2008I SO 4002 Al
34、l irthgs ersedevr 36.2 Culture media 6.2.1 Buffered charcoal yeast extract agar medium (BCYE) 6.2.1.1 Composition Yeast extract (bacteriological grade) 10,0 g Agar 12,0g Activated charcoal 2,0 g -Ketoglutarate, monopotassium salt 1,0 g ACES buffer (N-2-acetamido-2-aminoethane sulfonic acid) 10,0 g P
35、otassium hydroxide (KOH) (pellets) 2,8 g L-cysteine hydrochloride monohydrate 0,4 g Iron(III) pyrophosphate Fe 4 (P 2 O 7 ) 3 0,25g Distilled water 1 000 ml NOTE Check manufacturers recommendations for concentration of agar to be added to provide adequate gelling strength. 6.2.1.2 Preparation 6.2.1.
36、2.1 Cysteine and iron solutions Prepare fresh solutions of L-cysteine hydrochloride and iron(III) pyrophosphate by adding the 0,4 g and 0,25 g respectively to 10 ml volumes of distilled water. Decontaminate each solution by filtration through a cellulose ester membrane filter with an average pore si
37、ze of 0,2 m. Store in clean, sterile containers at (20 5) C for no more than 3 months. 6.2.1.2.2 ACES buffer Add the ACES granules to 500 ml of distilled water and dissolve by standing in a water bath at 45 C to 50 C. To a separate 480 ml of distilled water, add all the potassium hydroxide pellets a
38、nd dissolve with gentle shaking. To prepare the ACES buffer mix the two solutions. NOTE ACES buffer can cause denaturation of the yeast extract if the following sequence is not followed. 6.2.1.2.3 Final medium Add sequentially to the 980 ml of ACES buffer, the charcoal yeast extract and -ketoglutara
39、te. Prepare a 0,1 mol/l solution of potassium hydroxide (KOH) by dissolving 5,6 g in 1 l of distilled water. Prepare a 0,1 mol/l solution of sulfuric acid (H 2 SO 4 ) by carefully adding 5,3 ml of H 2 SO 4 ( = 1,84, of 95 % to 98 % purity) to 1 l of distilled water. Use the solutions of 0,1 mol/l po
40、tassium hydroxide or 0,1 mol sulfuric acid as appropriate to adjust the pH to 6,8 0,2. Add the agar, mix and autoclave at (121 3) C for (15 1) min (6.2.4). After autoclaving allow to cool to (50 2) C in a water bath. Add the L-cysteine and the iron(III) pyrophosphate solutions aseptically, mix well
41、between additions. BS EN ISO 11731-2:20084 Dispense in 20 ml volumes into Petri dishes of 90 mm to 100 mm diameter. Petri dishes of 60 mm may also be used for incubating the membranes (see 9.1 and 9.2). The pH of the final medium is 6,8 0,2 at 25 C. Allow excess moisture on the plates to dry and sto
42、re at (5 3) C in airtight containers in the dark for up to 4 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) but omit the L-cysteine. 6.2.3 Selective medium: buffered charcoal yeast extract medium with selec
43、tive supplements (GVPC medium) NOTE This medium is identical to BCYE except that three antibiotic supplements and glycine are added to the BCYE medium. 6.2.3.1 Selective supplements The final concentrations in the GVPC medium shall be: Ammonium-free glycine 3 g/l Polymyxin B sulfate 80 000 IU/l Vanc
44、omycin hydrochloride 0,001 g/l Cycloheximide 0,08 g/l Natamycin may be used instead of cycloheximide. 6.2.3.2 Preparation of antibiotic supplements Add the appropriate amount (usually 200 mg) of polymyxin B sulfate to 100 ml of distilled water to achieve a concentration of 14 545 IU/ml. Mix and deco
45、ntaminate by membrane filtration as described in 6.2.1.2. Dispense 5,5 ml volumes into sterile containers and store at (20 5) C. For use, thaw at room temperature. Add 20 mg of vancomycin hydrochloride to 20 ml of distilled water, mix and decontaminate by membrane filtration (6.2.1.2). Dispense in 1
46、 ml volumes in sterile containers and store at (20 5) C. For use, thaw at room temperature. Add 2 g of cycloheximide to 100 ml of distilled water and decontaminate by membrane filtration as described in 6.2.1.2. Dispense in 4 ml volumes in sterile containers and store at (20 5) C. For use, thaw at r
47、oom temperature. Antibiotic supplements may be stored for up to 6 months when frozen. WARNING Cycloheximide is hepatotoxic. Wear gloves and dust mask when handling this chemical in the powder form. 6.2.3.3 Preparation of GVPC medium Follow the instructions for preparation of BCYE medium given in 6.2
48、.1.2 but add 3 g of ammonium free glycine after the addition of the -ketoglutarate and then adjust the pH to 6,8 0,2. 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. BS EN ISO 11731-2:2008 ISO 2004 All rights reserved 56.2.4 Quality control of media Prolonged heating during sterilization or heating at too high a temperature
