1、BRITISH STANDARD BS EN ISO 10705-1:2001 BS 6068-4.11: 1996 Incorporating Amendment No. 1 to BS 6068-4.11:1996 (renumbers the BS as BS EN ISO 10705-1:2001) and Incorporating Corrigendum No. 1 Water quality Part 1: Detection and enumeration of bacteriophages Enumeration of F-specific RNA bacteriophage
2、s The European Standard EN ISO 10705-1:2001 has the status of a British Standard ICS 07.100.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN ISO 10705-1:2001 This British Standard, having been prepared under the direction of the Health and Environment Sector Board, was
3、published under the authority of the Standards Board and comes into effect on 15 April 1996 BSI 18 January 2002 The following BSI references relate to the work on this standard: Committee reference EH/3/4 Draft for comment 93/502664 DC ISBN 0 580 25465 8 National foreword This British Standard is th
4、e official English language version of EN ISO 10705-1:2001. It is identical with ISO 10705-1:1995. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/4, Microbiology, which has the responsibility to: aid enquiries to understand the
5、text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this commi
6、ttee and can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or b
7、y using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer imm
8、unity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, pages 1 to 11, the annex ZA page, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when th
9、e document was last issued. Amendments issued since publication Amd. No. Date Comments 13446 10 December 2001 Implementation of the European Standard 13638 Incorp. Corr. No. 1 18 January 2002 Incorporating annex ZAEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 10705-1 August 2001 ICS 07.10
10、0.20 English version Water quality Detection and enumeration of bacteriophages Part 1: Enumeration of F-specific RNA bacteriophages (ISO 10705-1:1995) Qualit de leau Dtection et dnombrement des bactriophages Partie 1: Dnombrement des bactriophages ARN F spcifiques (ISO 10705-1:1995) Wasserbeschaffen
11、heit Nachweis und Zhlung von Bakteriophagen Teil 1: Zhlung von F-spezifischen RNA- Bakteriophagen (ISO 10705-1:1995) This European Standard was approved by CEN on 29 June 2001. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this Eu
12、ropean Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Management Centre or to any CEN member. This European Standard exists in three official versions (English
13、, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic,
14、 Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Management Centre: rue de Stassa
15、rt, 36 B-1050 Brussels 2001 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 10705-1:2001 EEN ISO 10705-1:2001 BSI 18 January 2002 CORRECTED 2001-11-07 Foreword The text of the International Standard from Technical Committee ISO
16、/TC 147, Water quality, of the International Organization for Standardization (ISO) has been taken over as a European Standard 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 b
17、y publication of an identical text or by endorsement, at the latest by February 2002, and conflicting national standards shall be withdrawn at the latest by February 2002. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to
18、implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of the International Standard ISO 10705-1:
19、1995 has been approved by CEN as a European Standard without any modifications. NOTE: Normative references to International Standards are listed in annex ZA (normative).EN ISO 10705-1:2001 BSI 18 January 2002 1 1 Scope This part of ISO 10705 specifies a method for the detection and enumeration of F-
20、specific ribonucleic acid (RNA) bacteriophages by incubating the sample with an appropriate host strain. The method can be applied to all kinds of water, sediments and sludges, where necessary after dilution. In the case of low numbers, a preconcentration step may be necessary for which a separate p
21、art of ISO 10705 will be developed. The method can also be applied to shellfish extracts. Depending on the relative abundance of F-specific RNA bacteriophages to background organisms, additional confirmatory tests may be necessary and are also specified in this part of ISO 10705. The presence of F-s
22、pecific RNA bacteriophages in a water sample generally indicates pollution by wastewater contaminated by human or animal faeces. Their survival in the environment, removal by widely used water treatment processes and concentration or retention by shellfish resembles that of foodborne and waterborne
23、human enteric viruses, for example the enteroviruses, hepatitis A virus and rotaviruses. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this part of ISO 10705. At the time of publication, the editions indicated were v
24、alid. All standards are subject to revision, and parties to agreements based on this part of ISO 10705 are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Stand
25、ards. 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 techniques. ISO 5667-3:1994, Water quality Samp
26、ling 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 by culture. 3 Definition For the purposes
27、 of this part of ISO 10705, the following definition applies. 3.1 F-specific RNA bacteriophages bacterial viruses which are capable of infecting a specified host strain with F-pili or sex-pili to produce visible plaques (clearance zones) on a confluent lawn grown under appropriate culture conditions
28、, whereas the infectious process is inhibited in the presence of a concentration of 40 (occasionally 400) g/ml of RNase in the plating medium 4 Principle 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 nutrient medium. Af
29、ter this, incubation and reading of plates for visible plaques takes place. Where necessary, simultaneous examination of parallel plates with added RNase for confirmation by differential counts is carried out. The results are expressed as the number concentration of plaque-forming particles (C pfp )
30、 per unit of volume. 5 Safety precautions The host strain used is a Salmonella typhimurium mutant of low pathogenicity and should be handled in accordance with the appropriate national or international safety procedures for this bacterial species. F-specific RNA bacteriophages are non-pathogenic for
31、 man and animals, but are very resistant to drying. Appropriate precautions should therefore 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 must be carried out
32、 in a biohazard cabinet or a separate area of the laboratory.EN ISO 10705-1:2001 2 BSI 18 January 2002 6 Diluent, culture media and reagents 6.1 Basic materials Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media and reagents, and follow the inst
33、ructions given in Annex A. For information on storage see ISO 8199, except where indicated in this part of ISO 10705. Alternatively, use dehydrated complete media and follow strictly the manufacturers instructions. For the preparation of media, use glass-distilled water or deionized water free from
34、substances which might inhibit bacterial growth under the conditions of the test, and in accordance with ISO 3696. 6.2 Diluent For making sample dilutions, use peptone saline solution as indicated in A.8. 6.3 Reagents 6.3.1 RNase from bovine pancreas, specific activity approximately 50 units/mg (Kun
35、itz). 6.3.2 Antibiotic discs, for susceptibility testing with nalidixic acid (130 g; 9 mm) and kanamycin (100 g; 9 mm). 6.3.3 Glycerol, 870 g/litre. 6.4 Microbiological reference cultures Salmonella typhimurium strain WG49, phage type 3 Nal r(F 42 lac:Tn5), NCTC 12484. Bacteriophage MS2, NCTC 12487
36、or ATCC 15597-B1. Escherichia coli K-12 Hfr from appropriate culture collection, e.g. NCTC 12486 or ATCC 23631. NOTE 1 The NCTC strains are available from the National Collection of Type Cultures, 61 Colindale Avenue, London NW9 6HT, England. The ATCC strains are available from the American Type Cul
37、ture Collection, 12301 Parklawn Drive, Rockville, Maryland, U.S.A. 7 Apparatus and glassware Usual microbiological laboratory equipment, including 7.1 Hot-air oven for dry-heat sterilization and an autoclave. Apart from apparatus supplied sterile, glassware and other equipment shall be sterilized ac
38、cording to the instructions given in ISO 8199. 7.2 Incubator or water bath, thermostatically controlled at 37 C1 C. 7.3 Incubator or water bath, thermostatically controlled at 37 C1 C and equipped with a rotary platform at 100 min 1 10mi n 1 . 7.4 Water bath, thermostatically controlled at 45 C1 C.
39、7.5 Water bath or equivalent device, for melting agar media. 7.6 pH-meter. 7.7 Counting apparatus, with indirect, oblique light. 7.8 Deep freezer, thermostatically controlled at 20 C5 C. 7.9 Deep freezer, thermostatically controlled at 70 C1 0 C. 7.10 Spectrometer, capable of holding 1 cm cuvettes o
40、r side-arm of nephelometric flasks (7.17) and equipped with a filter in the range 500 nm to 650 nm with a maximum bandwidth of 10 nm. Usual sterile, microbiological laboratory glassware or disposable plastics ware according to ISO 8199 and including the following. 7.11 Petri dishes, of diameter 9 cm
41、 or 15 cm. 7.12 Graduated pipettes, of capacities 1 ml, 5 ml and 10 ml. 7.13 Glass bottles, of suitable volumes.EN ISO 10705-1:2001 BSI 18 January 2002 3 7.14 Culture tubes, with caps. 7.15 Measuring cylinders, of suitable capacity. 7.16 Conical flasks, of capacity 250 ml to 300 ml, with cotton wool
42、 plugs or suitable alternatives. 7.17 Cuvettes, of optical path length 1 cm or nephelometric conical flasks, of capacity 250 ml to 300 ml, with cylindrical side-arms which can be fitted to the spectrometer (7.10) and with cotton wool plugs or suitable alternatives. (See Figure 1.) 7.18 Membrane filt
43、er units, for sterilization, pore size 0,2 m. 7.19 Plastics vials, lidded, of capacity 1,5 ml to 2 ml. 8 Sampling Take samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, ISO 5667-2, and ISO 5667-3. 9 Preparation of test materials 9.1 Culturing and maintenance of host
44、 strains WG49 and E. coli K12 Hfr The culturing and maintenance of host strains involves several stages which are summarized in Figure 2. The figure also indicates the stages where quality control of the host culture is performed. 9.1.1 Preparation of stock cultures Rehydrate the contents of a lyoph
45、ilized ampoule of the reference culture of the host strains in a small volume of TYGB (A.1) using a Pasteur pipette. Transfer the suspension to 50 ml of TYGB in a 300 ml conical flask (7.16). Incubate for 18 h 2h a t3 7 C 1 C while shaking at 100 min 1 1 0m i n 1 . Add 10 ml of glycerol (A.6) and mi
46、x well. Distribute into plastics vials (7.19) in 1,2 ml aliquots and store at 70 C 10 C. NOTE 2 This first culture of the host strain should be stored as a reference standard in the laboratory.EN ISO 10705-1:2001 4 BSI 18 January 2002 9.1.2 Preparation of working cultures Thaw one vial of stock cult
47、ure (9.1.1) at room temperature and inoculate on a plate of McConkey agar (A.7), or another lactose-containing medium, in such a way that single colonies will be obtained. Incubate at 37 C 1 C for 18 h 2 h. Add 50 ml of TYGB to (A.1) a 300 ml conical flask (7.16) and warm to room temperature. Select
48、 three to five lactose-positive colonies from the McConkey agar and inoculate material from each of these colonies in the flask with TYGB. Incubate for 5 h 1h a t3 7C 1 C while shaking at 100 min 1 10 min 1Add 10 ml of glycerol (A.6) and mix well. Distribute into plastics vials (7.19) in 1,2 ml aliq
49、uots and store at 70 C 10 C for a maximum of 2 years. Control the quality of the working culture according to 9.3. NOTE 3 If a great number of tests is anticipated, several conical flasks can be inoculated in parallel. NOTE 4 If quality control fails, prepare new inocula from the stock culture. After repeated failures, or if the stock culture is depleted, obtain a new lyophilized ampoule of the reference culture.
