1、BRITISH STANDARD BS ISO 10705-4:2001 BS 6068-4.17:2001 Water quality Detection and enumeration of bacteriophages Part 4: Enumeration of bacteriophages infecting Bacteriodes fragilis ICS 07.100.20 BS ISO 10705-4:2001 This British Standard was published under the authority of the Standards Policy and
2、Strategy Committee on 4 November 2003 BSI 4 November 2003 ISBN 0 580 42884 2 National foreword This British Standard reproduces verbatim ISO 10705-4:2001 and implements it as the UK national standard. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, t
3、o Subcommittee EH/3/4, Microbiological methods, which has the responsibility to: A list of organizations represented on this subcommittee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be
4、 found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are
5、responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and k
6、eep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 19 and a back cover. The BSI copyright notice displayed
7、in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsINTERNATIONAL STANDARD ISO 10705-4 First edition 2001-12-15 Reference number ISO 10705-4:2001(E) OSI 2001 Water quality Detection and enumeration of bacteriophages Part 4: Enumerat
8、ion of bacteriophages infecting Bacteroides fragilis Qualit de leau Dtection et dnombrement des bactriophages Partie 4: Dnombrement des bactriophages infectant Bacteroides fragilisBSISO107054:2001ISO -50701:4)E(1002 ii ISO 1002 Ar llithgr seresvde FDP dicslaimre This FDP fam elitnoc yt deddebme niay
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13、Cael 65 G 1121-HCeneva 02 Te.l + 22 14 10 947 11 Fax + 14 47 2290 9 74 E-liam copyrightsio.ch Web wwwsi.oc.h BSISO107054:2001OSI -507011002:4)E( ISO 1002 r llAithgs reservde iii Contents Page 1 Scope . 1 2 Normative references . 1 3 Term and definition . 1 4 Safety precautions 2 5 Principle 2 6 Reag
14、ents 2 7 Apparatus . 2 8 Microbiological reference cultures 3 9 Sampling . 4 10 Preparation of test materials . 4 11 Procedure . 7 12 Expression of results 9 13 Test report 10 Annexes A General description of bacteriophages infecting Bacteroides fragilis 11 B Culture media and diluents . 12 C Altern
15、ative method for preparing inoculum cultures 17 D Culturing of Bacteriophage B56-3. 18 Bibliography. 19 BSISO107054:2001ISO -50701:4)E(1002 iv ISO 1002 Ar llithgr seresvde Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO me
16、mber bodies). The work of preparing International Standards is normally carried out through ISO technical committees. 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, governme
17、ntal and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Dire
18、ctives, 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 casting a vote. Attention is drawn to the possibility that some of the el
19、ements of this part of ISO 10705 may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO10705-4 was prepared by Technical Committee ISO/TC147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 10705
20、 consists of the following parts, under the general title Water quality Detection and enumeration of bacteriophages: Part 1: Enumeration of F-specific RNA bacteriophages Part 2: Enumeration of somatic coliphages Part 3: Validation of methods for concentration of bacteriophages from water Part 4: Enu
21、meration of bacteriophages infecting Bacteroides fragilis Annexes A, B, C and D of this part of ISO 10705 are for information only. BSISO107054:2001ANRETNIITOTS LANDNADRA OSI -507011002:4)E( ISO 1002 r llAithgs reservde 1 Water quality Detection and enumeration of bacteriophages Part 4: Enumeration
22、of bacteriophages infecting Bacteroides fragilis 1 Scope This part of ISO 10705 specifies a method for the detection and enumeration of bacteriophages infecting Bacteroides fragilis by incubating the sample with an appropriate host-strain. The method is applicable to all kinds of water, sediments an
23、d sludge extracts, where necessary after dilution. In the case of low phage numbers, a pre- concentration step may be necessary for which a separate International Standard has been developed. The method is also applicable to shellfish extracts. NOTE It is desirable for International Standards to be
24、adopted as widely as possible. This part of ISO 10705 includes reference to alternative procedures which obviate the need for expensive materials or equipment which may not be readily available in developing countries. Use of these alternatives will not affect the performance of this method. 2 Norma
25、tive references The following normative documents contain provisions which, through reference in this text, constitute provisions of this part of ISO 10705. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based o
26、n this part of ISO 10705 are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently val
27、id International Standards. ISO 3696, Water for analytical laboratory use Specification and test methods ISO 5667-1, Water quality Sampling Part 1: Guidance on the design of sampling programmes ISO 5667-2, Water quality Sampling Part 2: Guidance on sampling techniques ISO 5667-3, Water quality Sampl
28、ing Part 3: Guidance on the preservation and handling of samples ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and de
29、cimal dilutions ISO 8199, Water quality General guide to the enumeration of micro-organisms by culture 3 Term and definition For the purposes of this part of ISO 10705, the following term and definition applies. 3.1 bacteriophage infecting Bacteroides fragilis bacterial virus which is capable of inf
30、ecting selected Bacteroides fragilis host strains by attachment to the bacterial cell wall as the first step of the infectious process NOTE 1 Such bacteriophages produce visible plaques (clearance zones) in a confluent lawn of host bacteria grown under appropriate culture conditions. BSISO107054:200
31、1ISO -50701:4)E(1002 2 ISO 1002 Ar llithgr seresvde NOTE 2 A general description of bacteriophages infecting B. fragilis is given in annex A. 4 Safety precautions The host strain used in this part of ISO 10705 is non-pathogenic to man and animals and should be handled in accordance with the normal (
32、national or international) safety procedures for bacteriological laboratories. Bacteriophages infecting Bacteroides fragilis are non-pathogenic for man and animals, but some types are very resistant to drying. Appropriate precautions shall be taken to prevent cross-contamination of test materials, p
33、articularly when examining or handling cultures of high titre or when inoculating cultures of the host strains. Such procedures shall be carried out in a biohazard cabinet or a separate area of the laboratory. Chloroform is a carcinogenic substance. Observe relevant safety precautions or use an alte
34、rnative method of equal efficacy. It is recommended that personnel using this method have or acquire some experience in handling anaerobic bacteria. 5P r i n c i p l e The sample is mixed with a small volume of semi-solid nutrient medium. A culture of host-strain is added and plated on a solid nutri
35、ent medium. After this, incubation and reading of plaques take place. The results are expressed as the number of plaque-forming particles (also named plaque-forming units, pfu) per unit of volume (pfp/ml, pfp/l, etc.). 6R e a g e n t s 6.1 Basic materials. Use ingredients of uniform quality and chem
36、icals of analytical grade for the preparation of culture media and reagents and follow the instructions given in annex B. For information on storage see ISO 8199, except where indicated in this part of ISO10705. Alternatively, use dehydrated complete media and follow strictly the manufacturers instr
37、uctions. Other grades of chemicals may be used provided they can be shown to lead to the same results. 6.2 Water, for the preparation of media, glass-distilled or deionized, free from substances which might inhibit bacterial growth under the conditions of the test, and complying with ISO 3696. 6.3 D
38、iluent, for making sample dilution, such as peptone-saline solution or another diluent complying with ISO 6887-1. 7 Apparatus Apart from apparatus supplied sterile, sterilize any glassware and other equipment in accordance with ISO 8199. Usual sterile, microbiological laboratory equipment and glassw
39、are or disposable plastics-ware in accordance with ISO 8199 and including the following: 7.1 Hot-air oven, for dry-heat sterilization, and an autoclave. 7.2 Incubator or water bath, thermostatically maintained at . 7.3 Incubator or water bath, thermostatically maintained at with a shaking device. 7.
40、4 Water bath or heating block, thermostatically maintained at . 7.5 Water bath or equivalent device, for melting of agar media. 7.6 pH meter, and pH paper. (36 2) C (36 2) C (45 1) C BSISO107054:2001OSI -507011002:4)E( ISO 1002 r llAithgs reservde 3 7.7 Counting apparatus, with indirect, oblique lig
41、ht. 7.8 Deep freezer, thermostatically maintained at . 7.9 Deep freezer, thermostatically maintained at or liquid nitrogen storage vessel. 7.10 Spectrophotometer, equipped with a filter for the range of to with a maximum bandwidth of , capable of holding cuvettes (7.21) having an optical path length
42、 of or Hungate glass tubes (7.20) with butyl rubber stopper and screw cap or screw-capped glass culture tubes. 7.11 Anaerobic cabinet, or jars or bags, as well as anaerobiosis generators and anaerobiosis indicators. 7.12 Refrigerator, temperature set at . 7.13 Petri dishes, having a diameter of , an
43、d vented. 7.14 Graduated pipettes, having a capacity of , , and and Pasteur pipettes. 7.15 Glass bottles, of suitable volumes. 7.16 Screw-capped glass bottles, of suitable volumes. 7.17 Culture tubes, with caps or suitable alternatives. 7.18 Screw-capped glass culture tubes. 7.19 Measuring cylinders
44、, of suitable capacity. 7.20 Hungate glass tubes, with butyl rubber stopper and screw cap or screw-capped glass culture tubes which can fit in the spectrophotometer (see Figure 1). 7.21 Cuvettes, having an optical path length of . 7.22 Membrane filter units, for decontamination, having a pore size o
45、f , preferably low protein-binding membranes, as for example, those composed of polyvinylidene difluoride. 7.23 Plastics vials, lidded, having a capacity of . 7.24 Glass vials, screw-capped, having a capacity of . 7.25 Syringes and needles. 7.26 Cotton swabs. 8 Microbiological reference cultures The
46、 recommended host strain is Bacteroides fragilis RYC2056 (ATCC 700786). 1 Use bacteriophage B56-3 (ATCC 700786-B1) infecting Bacteroides fragilis RYC2056 for the preparation of reference materials (11.4). NOTE The ATCC strains are available from American Type Culture Collection (ATCC), 10801 Univers
47、ity Boulevard, Manassas, VA 20110-2209, USA. This information is given for the convenience of users of this part of ISO 10705 and does not constitue an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results. (20 5) C (70 10) C 500 nm
48、 650 nm 10 nm 1 cm (5 3) C 9cm 0,1 ml 1 ml 5 ml 10 ml 1cm 0,2 m 3ml 3ml BSISO107054:2001ISO -50701:4)E(1002 4 ISO 1002 Ar llithgr seresvde 9 Sampling Take samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, ISO 5667-2 and ISO 5667-3. 10 Preparation of test materials 1
49、0.1 Culturing and maintenance of host strains 10.1.1 General The culturing and maintenance of host strains involves several stages which are summarized in Figure 2. Bacteroides fragilis is an obligate anaerobe. However, it does not require handling under conditions of strict anaerobiosis. Incubation of cultures in solid media should be carried out in an anaerobic cabinet, or
