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 978-0-626-22941-2 SANS 11731-2:2009Edition 1ISO 11731-2:2004Edition 1SOUTH AFRICAN NATIONAL STANDARD Water quality Detection and enumeration of Legi
4、onella Part 2: Direct membrane filtration method for waters with low bacterial counts This national standard is the identical implementation of ISO 11731-2:2004 and is adopted with the permission of the International Organization for Standardization. Published by SABS Standards Division 1 Dr Lategan
5、 Road Groenkloof Private Bag X191 Pretoria 0001Tel: +27 12 428 7911 Fax: +27 12 344 1568 www.sabs.co.za SABS SANS 11731-2:2009 Edition 1 ISO 11731-2:2004 Edition 1 Table of changes Change No. Date Scope National foreword This South African standard was approved by National Committee SABS SC 147B, Wa
6、ter Microbiological and biological treatment and assessment of water, in accordance with procedures of the SABS Standards Division, in compliance with annex 3 of the WTO/TBT agreement. This SANS document was published in August 2009. Reference numberISO 11731-2:2004(E)ISO 2004INTERNATIONAL STANDARD
7、ISO11731-2First edition2004-05-01Water quality Detection and enumeration of Legionella Part 2: Direct membrane filtration method for waters with low bacterial counts Qualit de leau Recherche et dnombrement des Legionella Partie 2: Mthode par filtration directe sur membrane pour les eaux faible teneu
8、r en bactries SANS 11731-2:2009This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731-2:2004(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but
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12、tronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.
13、org Web www.iso.org Published in Switzerland ii ISO 2004 All rights reservedSANS 11731-2:2009This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731-2:2004(E) ISO 2004 All rights reserved iiiContents Page Foreword iv 1 Scope 1 2 Normative r
14、eferences . 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 SANS 11731-2:2009This s tandard may only be used and printed by approved subscription and freemailing clients of
15、the SABS .ISO 11731-2:2004(E) iv ISO 2004 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical commi
16、ttees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the I
17、nternational Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft Internationa
18、l Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the
19、 subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11731-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 11731 consists of the following parts, under the general title Water
20、quality Detection and enumeration of Legionella: Part 2: Direct membrane filtration method for waters with low bacterial counts The general method will be the subject of a future Part 1 of ISO 11731. SANS 11731-2:2009This s tandard may only be used and printed by approved subscription and freemailin
21、g clients of the SABS .INTERNATIONAL STANDARD ISO 11731-2:2004(E) ISO 2004 All rights reserved 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 no
22、rmal 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 Scop
23、e 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 fo
24、r 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 indisp
25、ensable 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 8199:1),
26、 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 bacteria
27、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 appear
28、ance 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-cysteine
29、 thereafter. 1) To be published. (Revision of ISO 8199:1988) SANS 11731-2:2009This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731-2:2004(E) 2 ISO 2004 All rights reserved4 Safety The reagents used in this part of ISO 11731 should be sub
30、ject to assessment in accordance with control substances hazardous to health. Legionella species can be handled safely by experienced microbiologists on the open bench in a conventional microbiology laboratory conforming to containment level 2. Infection is caused by inhalation of the organism and i
31、t 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. 5 Principle 5.1 General Bacteria, including Legionella organisms, in the water sample are concentrated by membrane filtration. After filtration, the filte
32、r is treated with acid buffer added directly into the funnel to reduce the growth of non-Legionella organisms. The filter is subsequently transferred onto a plate of agar medium selective for Legionella and incubated. Samples expected to contain sufficient numbers of Legionella need not be subjected
33、 to concentration prior to culture (9.1). 5.2 Enumeration After incubation, morphologically characteristic colonies which form on the selective medium are regarded as presumptive Legionella. 5.3 Confirmation Presumptive colonies are confirmed as Legionella organisms by subculture to demonstrate thei
34、r growth requirement for L-cysteine and iron. Further biochemical and serological tests are needed for species identification. Species identification may not be considered necessary for routine monitoring but is indispensable in outbreak situations. NOTE L. pneumophila serogroup 1 is the causative a
35、gent of most legionellosis cases and is therefore considered the most “critical” type of Legionella to be found in the water system. Since increasing numbers of cases of legionellosis caused by other serogroups of L. pneumophila and other Legionella species are being described, even the presence of
36、other Legionella species in water is considered a potential risk. 6 Culture media and reagents 6.1 General Use chemicals of analytical grade in the preparation of media and reagents unless otherwise stated. Alternatively, use commercially available dehydrated media and reagents. Prepare the media ac
37、cording to the manufacturers instruction and add freshly prepared (or thaw the stored material at room temperature prior to use) selective agents or growth supplements at the concentrations recommended. Prepare media using glass distilled water or water of equivalent quality complying with ISO 3696:
38、1987, Grade 3. Other grades of chemicals may be used providing they can be shown to produce the same results. SANS 11731-2:2009This s tandard may only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731-2:2004(E) ISO 2004 All rights reserved 36.2 Culture media
39、 6.2.1 Buffered charcoal yeast extract agar medium (BCYE) 6.2.1.1 Composition Yeast extract (bacteriological grade) 10,0 g Agar 12,0Activated charcoal 2,0 g -Ketoglutarate, monopotassium salt 1,0 g ACES buffer (N-2-acetamido-2-aminoethane sulfonic acid) 10,0 g Potassium hydroxide (KOH) (pellets) 2,8
40、 g L-cysteine hydrochloride monohydrate 0,4 g Iron(III) pyrophosphate Fe4(P2O7)3 0,25Distilled 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 6.2.1.2.1 Cysteine and iron solutions Prepare fresh s
41、olutions 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 size of 0,2 m. Store in clean, sterile containers
42、 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 and dissolve with gentle shaking. To prepare the
43、 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 -ketoglutarate. Prepare a 0,1 mol/l solution of potassium h
44、ydroxide (KOH) by dissolving 5,6 g in 1 l of distilled water. Prepare a 0,1 mol/l solution of sulfuric acid (H2SO4) by carefully adding 5,3 ml of H2SO4 ( = 1,84, of 95 % to 98 % purity) to 1 l of distilled water. Use the solutions of 0,1 mol/l potassium hydroxide or 0,1 mol sulfuric acid as appropri
45、ate 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 between additions. SANS 11731-2:2009This s tandard may
46、 only be used and printed by approved subscription and freemailing clients of the SABS .ISO 11731-2:2004(E) 4 ISO 2004 All rights reservedDispense 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). Th
47、e pH of the final medium is 6,8 0,2 at 25 C. Allow excess moisture on the plates to dry and store 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
48、 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 BCYE medium. 6.2.3.1 Selective supplements The final concentration
49、s in the GVPC medium shall be: Ammonium-free glycine 3 g/l Polymyxin B sulfate 80 000 IU/l Vancomycin 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 decontaminate by membrane filtration as described in 6.2.1.2. Dispense 5,5 ml volumes into sterile cont
