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1、INTERNATIONAL STANDARD ISO 10705-4 First edition 2001-12-15 Reference number ISO 10705-4:2001(E) ISO 2001 Water quality Detection and enumeration of bacteriophages Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis Qualit de leau Dtection et dnombrement des bactriophages Partie 4:
2、Dnombrement des bactriophages infectant Bacteroides fragilis ISO 10705-4:2001(E) ii ISO 2001 All rights reserved PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces wh
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6、 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.ch Web www.iso.ch Printed in Switzerland ISO
7、 10705-4:2001(E) ISO 2001 All rights reserved iii Contents Page 1Scope . 1 2Normative references . 1 3Term and definition . 1 4Safety precautions 2 5Principle 2 6Reagents 2 7Apparatus . 2 8Microbiological reference cultures 3 9Sampling . 4 10Preparation of test materials . 4 11Procedure . 7 12Expres
8、sion of results 9 13Test report 10 Annexes AGeneral description of bacteriophages infecting Bacteroides fragilis 11 BCulture media and diluents. 12 CAlternative method for preparing inoculum cultures 17 DCulturing of Bacteriophage B56-3. 18 Bibliography. 19 ISO 10705-4:2001(E) iv ISO 2001 All rights
9、 reserved Foreword 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 committees. Each member body interested in a subject for
10、 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 International Electrotechnical Commission (IEC) on a
11、ll matters of electrotechnical standardization. International Standards 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
12、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 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. Internation
13、al Standard ISO 10705-4 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 10705 consists of the following parts, under the general title Water quality Detection and enumeration of bacteriophages: Part 1: Enumeration of F-specific RNA bacte
14、riophages Part 2: Enumeration of somatic coliphages Part 3: Validation of methods for concentration of bacteriophages from water Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis Annexes A, B, C and D of this part of ISO 10705 are for information only. INTERNATIONAL STANDARDISO 10
15、705-4:2001(E) ISO 2001 All rights reserved 1 Water quality Detection and enumeration of bacteriophages Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis 1Scope This part of ISO 10705 specifies a method for the detection and enumeration of bacteriophages infecting Bacteroides fragi
16、lis by incubating the sample with an appropriate host-strain. The method is applicable to all kinds of water, sediments and 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 be
17、en developed. The method is also applicable to shellfish extracts. NOTEIt is desirable for International Standards to be 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 rea
18、dily available in developing countries. Use of these alternatives will not affect the performance of this method. 2Normative references The following normative documents contain provisions which, through reference in this text, constitute provisions of this part of ISO 10705. For dated references, s
19、ubsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on 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
20、latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid 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 sampli
21、ng programmes ISO 5667-2, Water quality Sampling Part 2: Guidance on sampling techniques ISO 5667-3, Water quality Sampling 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 deci
22、mal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 8199, Water quality General guide to the enumeration of micro-organisms by culture 3Term and definition For the purposes of this part of ISO 10705, the followin
23、g term and definition applies. 3.1 bacteriophage infecting Bacteroides fragilis bacterial virus which is capable of infecting selected Bacteroides fragilis host strains by attachment to the bacterial cell wall as the first step of the infectious process NOTE 1Such bacteriophages produce visible plaq
24、ues (clearance zones) in a confluent lawn of host bacteria grown under appropriate culture conditions. ISO 10705-4:2001(E) 2 ISO 2001 All rights reserved NOTE 2A general description of bacteriophages infecting B. fragilis is given in annex A. 4Safety precautions The host strain used in this part of
25、ISO 10705 is non-pathogenic to man and animals and should be handled in accordance with the normal (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 t
26、o drying. Appropriate precautions shall be taken to prevent cross-contamination of test materials, particularly 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 la
27、boratory. Chloroform is a carcinogenic substance. Observe relevant safety precautions or use an alternative method of equal efficacy. It is recommended that personnel using this method have or acquire some experience in handling anaerobic bacteria. 5Principle The sample is mixed with a small volume
28、of semi-solid nutrient medium. A culture of host-strain is added and plated on a solid nutrient 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
29、/l, etc.). 6Reagents 6.1Basic materials. Use ingredients of uniform quality and chemicals 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 ISO 10705. Alt
30、ernatively, use dehydrated complete media and follow strictly the manufacturers instructions. Other grades of chemicals may be used provided they can be shown to lead to the same results. 6.2Water, for the preparation of media, glass-distilled or deionized, free from substances which might inhibit b
31、acterial growth under the conditions of the test, and complying with ISO 3696. 6.3Diluent, for making sample dilution, such as peptone-saline solution or another diluent complying with ISO 6887-1. 7Apparatus Apart from apparatus supplied sterile, sterilize any glassware and other equipment in accord
32、ance with ISO 8199. Usual sterile, microbiological laboratory equipment and glassware or disposable plastics-ware in accordance with ISO 8199 and including the following: 7.1Hot-air oven, for dry-heat sterilization, and an autoclave. 7.2Incubator or water bath, thermostatically maintained at . 7.3In
33、cubator or water bath, thermostatically maintained at with a shaking device. 7.4Water bath or heating block, thermostatically maintained at . 7.5Water bath or equivalent device, for melting of agar media. 7.6pH meter, and pH paper. (362) C (362) C (451) C ISO 10705-4:2001(E) ISO 2001 All rights rese
34、rved 3 7.7Counting apparatus, with indirect, oblique light. 7.8Deep freezer, thermostatically maintained at . 7.9Deep freezer, thermostatically maintained at or liquid nitrogen storage vessel. 7.10Spectrophotometer, equipped with a filter for the range of to with a maximum bandwidth of , capable of
35、holding cuvettes (7.21) having an optical path length of or Hungate glass tubes (7.20) with butyl rubber stopper and screw cap or screw-capped glass culture tubes. 7.11Anaerobic cabinet, or jars or bags, as well as anaerobiosis generators and anaerobiosis indicators. 7.12Refrigerator, temperature se
36、t at . 7.13Petri dishes, having a diameter of , and vented. 7.14Graduated pipettes, having a capacity of , , and and Pasteur pipettes. 7.15Glass bottles, of suitable volumes. 7.16Screw-capped glass bottles, of suitable volumes. 7.17Culture tubes, with caps or suitable alternatives. 7.18Screw-capped
37、glass culture tubes. 7.19Measuring cylinders, of suitable capacity. 7.20Hungate glass tubes, with butyl rubber stopper and screw cap or screw-capped glass culture tubes which can fit in the spectrophotometer (see Figure 1). 7.21Cuvettes, having an optical path length of . 7.22Membrane filter units,
38、for decontamination, having a pore size of , preferably low protein-binding membranes, as for example, those composed of polyvinylidene difluoride. 7.23Plastics vials, lidded, having a capacity of . 7.24Glass vials, screw-capped, having a capacity of . 7.25Syringes and needles. 7.26Cotton swabs. 8Mi
39、crobiological reference cultures The 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). NOTEThe ATCC strains are available from American Type Cultur
40、e Collection (ATCC), 10801 University 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
41、results. (205) C (7010) C 500 nm650 nm 10 nm1 cm (53) C 9 cm 0,1 ml 1 ml 5 ml10 ml 1 cm 0,2m 3 ml 3 ml ISO 10705-4:2001(E) 4 ISO 2001 All rights reserved 9Sampling Take samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, ISO 5667-2 and ISO 5667-3. 10Preparation of tes
42、t materials 10.1Culturing and maintenance of host strains 10.1.1General 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.
43、 Incubation of cultures in solid media should be carried out in an anaerobic cabinet, or anaerobic jars or bags. When using liquid media it is sufficient to ensure that containers are completely filled and closed with a screw cap. Figure 1 Hungate glass tube with rubber stopper and screw cap ISO 107
44、05-4:2001(E) ISO 2001 All rights reserved 5 10.1.2Preparation of stock cultures Rehydrate the content of a lyophilized ampoule of the reference culture of the host strain in of Bacteroides phage recovery medium broth (BPRMB) (B.1) using a Pasteur pipette (7.14). Inoculate the suspensions in of BPRMB
45、 (B.1) in a screw-capped glass tube (see 10.1.1) and incubate at for . Aseptically soak a sterile cotton swab with the culture and streak it onto a plate of Bacteroides phage recovery medium agar (BPRMA) (B.2). Incubate the culture in an anaerobic cabinet, jar or bag at for . Alternatively, if a cul
46、ture in slant is available, streak it with a sterile cotton swab directly onto a plate of BPRMA (B.2). Incubate the culture in an anaerobic cabinet, jar or bag at for . Inoculate cells (mass inoculation with a sterile cotton swab) from the plate into of BPRMB (B.1) in a screw-capped glass tube (7.18
47、). Be sure that the tube is completely filled (see 10.1.1). If dense growth occurs, inoculate of the growth onto the BPRMA plate; if poor growth occurs, use of the growth onto the plate. Incubate the culture at for . Figure 2 Scheme for the culturing and maintenance of host strains 1 ml 10 ml 10 ml(
48、362) C (213)h (362) C (444)h (362) C (444)h 10 ml10 ml 1/81/2 (362) C (213)h ISO 10705-4:2001(E) 6 ISO 2001 All rights reserved Mix culture and cryoprotector (B.6) in a ratio 1:1 (volume). Mix well avoiding bubble formation. Distribute into screw- capped, preferably glass, vials (7.24) in aliquots o
49、f approximately and store at or in liquid nitrogen for up to five years. This first passage of the host strain should be stored as a reference in the laboratory. Purity of the culture should be checked before storage by Gram staining, by testing absence of growth under aerobic conditions and by testing sensitivity to a reference bacteriophage (i.e. B56-3). 10.1.3Preparation of working cultures Remove a vial of stock culture (10.1.2) from frozen storage, allow the v
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