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. ICS 07.100.20 ISBN 0-626-13974-0 SANS 10705-2:2002 Edition 1 ISO 10705-2:2000 Edition 1 SOUTH AFRICAN NATIONAL STANDARD Water quality Detection and enume
4、ration of bacteriophages Part 2: Enumeration of somatic coliphages This national standard is the identical implementation of ISO 10705-2:2000 and is adopted with the permission of the International Organization for Standardization. Published by Standards South Africa 1 dr lategan road groenkloof pri
5、vate bag x191 pretoria 0001 tel: 012 428 7911 fax: 012 344 1568 international code + 27 12 www.stansa.co.za Standards South Africa 2002 SANS 10705-2:2002 Edition 1 ISO 10705-2:2000 Edition 1 Table of changes Amdt No. Date Scope National foreword This South African standard was approved by National C
6、ommittee STANSA SC 5140.19B, Biological treatment of water, in accordance with procedures of Standards South Africa, in compliance with annex 3 of the WTO/TBT agreement. Reference numberISO 10705-2:2000(E)ISO 2000INTERNATIONALSTANDARDISO10705-2First edition2000-04-01Water quality Detection and enume
7、rationof bacteriophages Part 2:Enumeration of somatic coliphagesQualit de leau Dtection et dnombrement des bactriophages Partie 2: Dnombrement des coliphages somatiquesISO 10705-2:2000(E)PDF disclaimerThis PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file
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10、ile is suitable for use by ISO member bodies. In the unlikely eventthat a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2000All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form
11、or by any means, electronicor mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member bodyin the country of the requester.ISO copyright officeCase postale 56 Gb7 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 734 10 79E-
12、mail copyrightiso.chWeb www.iso.chPrinted in Switzerlandii ISO 2000 All rights reservedISO 10705-2:2000(E) ISO 2000 All rights reserved iiiContents Page1 Scope12 Normative references13 Terms and definitions .14 Safety precautions.25 Principle26 Diluent, culture media and reagents26.1 Basic materials
13、.26.2 Diluent.27 Apparatus and glassware.28 Microbiological reference cultures49 Sampling.410 Preparation of test material510.1 Culturing and maintenance of host strains 510.2 Calibration of absorbance measurements for counts of viable microorganisms611 Procedure.611.1 Preparation of inoculum cultur
14、es 611.2 Standard procedure.711.3 Method for samples with high bacterial background flora .711.4 Samples with low phage counts711.5 Presence/absence test811.6 Quality assurance812 Expression of results912.1 Plaque count procedures (11.2 to 11.4).912.2 Presence/absence test (11.5)913 Test report10Ann
15、ex A (normative) Culture media, reagents and diluent.11Annex B (informative) General description of somatic bacteriophages .14Annex C (informative) Culturing of bacteriophage G66X174.15Bibliography16ISO 10705-2:2000(E)iv ISO 2000 All rights reservedForewordISO (the International Organization for Sta
16、ndardization) is a worldwide federation of national standards bodies (ISOmember bodies). The work of preparing International Standards is normally carried out through ISO technicalcommittees. Each member body interested in a subject for which a technical committee has been established hasthe right t
17、o be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International ElectrotechnicalCommission (IEC) on all matters of electrotechnical standardization.International Standa
18、rds are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.Draft International 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 casti
19、ng a vote.Attention is drawn to the possibility that some of the elements of this part of ISO 10705 may be the subject ofpatent rights. ISO shall not be held responsible for identifying any or all such patent rights.International Standard ISO 10705-2 was prepared by Technical Committee ISO/TC 147, W
20、ater quality,Subcommittee SC 4, Microbiological methods.ISO 10705 consists of the following parts, under the general title Water quality Detection and enumeration ofbacteriophages:Gbe Part 1: Enumeration of F-specific RNA bacteriophagesGbe Part 2: Enumeration of somatic coliphagesGbe Part 3: Concent
21、ration methodsGbe Part 4: Enumeration of bacteriophages infecting Bacteroides fragilisAnnex A forms a normative part of this part of ISO 10705. Annexes B and C are for information only.INTERNATIONAL STANDARD ISO 10705-2:2000(E) ISO 2000 All rights reserved 1Water quality Detection and enumeration of
22、 bacteriophages Part 2:Enumeration of somatic coliphages1 ScopeThis part of ISO 10705 specifies a method for the detection and enumeration of somatic coliphages by incubating thesample with an appropriate host strain. The method is applicable to all kinds of water, sediments and sludge extracts,wher
23、e necessary after dilution. The method is also applicable to shellfish extracts.In the case of low phage numbers, a preconcentration step may be necessary for which a separate InternationalStandard will be developed.NOTE It is desirable for International Standards to be adopted as widely as possible
24、. This part of ISO 10705 includesreference to alternative procedures which obviate the need for expensive materials or equipment which may not be readilyavailable in developing countries. Use of these alternatives will not affect the performance of this method.2 Normative referencesThe following nor
25、mative documents contain provisions which, through reference in this text, constitute provisions ofthis part of ISO 10705. For dated references, subsequent amendments to, or revisions of, any of these publicationsdo not apply. However, parties to agreements based on this part of ISO 10705 are encour
26、aged to investigate thepossibility of applying the most recent editions of the normative documents indicated below. For undatedreferences, the latest edition of the normative document referred to applies. Members of ISO and IEC maintainregisters of currently valid International Standards.ISO 31-0:19
27、92, Quantities and units Part 0: General principles.ISO 3696:1987, Water for analytical laboratory use Specification and test methods.ISO 5667-1:1980, Water quality Sampling Part 1: Guidance on the design of sampling programmes.ISO 5667-2:1991, Water quality Sampling Part 2: Guidance on sampling tec
28、hniques.ISO 5667-3:1994, Water quality Sampling Part 3: Guidance on the preservation and handling of samples.ISO 6887:1983, Microbiology General guidance for the preparation of dilutions for microbiological examination.ISO 8199:1988, Water quality General guide to the enumeration of micro-organisms
29、by culture.ISO/IEC Guide 2, Standardization and related activities General vocabulary.3 Terms and definitionsFor the purposes of this part of ISO 10705, the terms and definitions given in ISO/IEC Guide 2 and the followingapply.ISO 10705-2:2000(E)2 ISO 2000 All rights reserved3.1somatic coliphagebact
30、erial virus which is capable of infecting selected Escherichia coli host strains (and related strains) byattachment to the bacterial cell wall as the first step of the infection processNOTE Somatic coliphages produce visible plaques (clearance zones) in a confluent lawn of host bacteria grown undera
31、ppropriate culture conditions.4 Safety precautionsThe host strain used in this standard is non-pathogenic to man and animals, and should be handled in accordancewith the normal (national or international) safety procedures for bacteriological laboratories. Somatic coliphages arealso non-pathogenic t
32、o man and animals, but some types are very resistant to drying. Appropriate precautions shouldtherefore be taken to prevent cross-contamination of test materials, particularly when examining or handling culturesof high titre or when inoculating cultures of the host strain. Such procedures shall be c
33、arried out in a biohazard cabinetor a separate area of the laboratory.Chloroform is a carcinogenic substance. Observe relevant safety precautions or use an alternative method of equalefficacy.5PrincipleThe sample is mixed with a small volume of semi-solid nutrient medium. A culture of host strain is
34、 added and platedon a solid nutrient medium. After this, incubation and reading of plates for visible plaques takes place. The results areexpressed as the number of plaque-forming particles, pfp (also termed plaque-forming units, pfu), per unit of samplevolume.6 Diluent, culture media and reagents6.
35、1 Basic materialsUse ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media and reagentsand follow the instructions given in annex A. For information on storage see ISO 8199, except where indicated in thispart of ISO 10705. Alternatively, use dehydrated
36、 complete media and follow strictly the manufacturers instructions.For the preparation of media, use glass-distilled water or deionized water free from substances which might inhibitbacterial growth under the conditions of the test, and complying with ISO 3696.NOTE Use of other grades of chemicals i
37、s permissible providing they are shown to be of equal performance in the test.6.2 DiluentFor making sample dilutions, use peptone-saline solution (A.7) or another diluent complying with ISO 6887.7 Apparatus and glasswareUsual microbiological laboratory equipment, including7.1 Hot-air oven for dry-he
38、at sterilization and an autoclave. Apart from apparatus supplied sterile, glasswareand other equipment shall be sterilized according to the instructions given in ISO 8199.7.2 Incubator or water bath, thermostatically controlled at (36 Gb1 2) C. ISO 10705-2:2000(E) ISO 2000 All rights reserved 37.3 I
39、ncubator or water bath, thermostatically controlled at (36 Gb1 2) C and equipped with a shaking device, forexample a rotating platform at (100 Gb1 10) r/min.7.4 Water bath or heating block, thermostatically controlled at (45 Gb1 1) C.7.5 Water bath or equivalent device for melting of agar media.7.6
40、pH meter.7.7 Counting apparatus with indirect, oblique light.7.8 Deep freezer, thermostatically controlled at (G2d20 Gb1 5) C.7.9 Deep freezer, thermostatically controlled at (G2d70 Gb1 10) C or liquid nitrogen storage vessel.7.10 Spectrophotometer, capable of holding cuvettes of 1 cm optical path l
41、ength or side-arm of nephelometricflasks (7.17) and equipped with a filter for the range 500 nm to 650 nm with a maximum bandwidth of Gb1 10 nm.Usual sterile, microbiological laboratory glassware or disposable plasticsware according to ISO 8199 and including7.11 Petri dishes of 9 cm or 14 cm to 15 c
42、m diameter, vented.7.12 Graduated pipettes of 0,1 ml, 1 ml, 5 ml and 10 ml capacity and Pasteur pipettes.7.13 Glass bottles of suitable volume.7.14 Culture tubes with caps or suitable alternative.7.15 Measuring cylinders of suitable capacity.7.16 Conical flasks of 250 ml to 300 ml capacity, with cot
43、ton wool plugs or suitable alternative.7.17 Cuvettes of optical path length 10 mm or nephelometric conical flasks with cylindrical side-arms which fitin the spectrophotometer (7.10) (see Figure 1); capacity 250 ml to 300 ml with cotton wool plugs or suitablealternative.7.18 Membrane filter units for
44、 decontamination, pore size 0,2 m.7.19 Plastics vials, lidded, of 1,5 ml to 3 ml capacity.7.20 Refrigerator, temperature set at (5 Gb1 3) C.ISO 10705-2:2000(E)4 ISO 2000 All rights reservedFigure 1 Nephelometric conical flask for culturing the host strain8 Microbiological reference culturesFor sampl
45、es with low bacterial content (drinking water, unpolluted natural waters), use Escherichia coli strain C,ATCC 13706. Samples containing large numbers of bacteria (polluted natural waters, wastewater) should beexamined using the nalidixic acid resistant mutant E. coli strain CN (ATCC 7000781), also k
46、nown as WG52.Use bacteriophage G66X174 (ATCC 13706-B1) for the preparation of reference material (11.6.1).NOTE The ATCC strains are available from the American Type Culture Collection, 10801 University Boulevard, Manassas,VA 20110.9 SamplingTake samples and deliver them to the laboratory in accordan
47、ce with ISO 8199, ISO 5667-1, ISO 5667-2 andISO 5667-3.ISO 10705-2:2000(E) ISO 2000 All rights reserved 510 Preparation of test material10.1 Culturing and maintenance of host strains10.1.1 GeneralThe culturing and maintenance of host strains involves several stages which are summarized in Figure 2.F
48、or culturing of the host strains in the several stages, it is best to gently shake the cultures. In addition to increasingthe growth rate of bacteria, shaking ensures that all the cells are actively growing and no stationary-phase cellsdevelop, which could decrease the efficiency of plating. Therefo
49、re, inoculum cultures should be repeatedly shakedby hand if a shaker is not available.Figure 2 Scheme for culture and maintenance of host strains10.1.2 Preparation of stock culturesRehydrate the contents of a lyophilized ampoule of the reference culture of the host strains in a small amount (ca.3 ml) of Modified Scholtens Broth (M.S.B.) (A.1) using a Pasteur pipette (7.12). Transfer the suspension to a 300 mlconical flask (7.16) containing (50 Gb1 5) ml of MSB. Incubate for (20 Gb1 4) h at (36 Gb1 2)
