1、BRITISH STANDARD BS ISO 10705-3:2003 BS 6068-4.16:2003 Water quality Detection and enumeration of bacteriophages Part 3: Validation of methods for concentration of bacteriophages from water ICS 07.100.20 BS ISO 10705-3:2003 This British Standard was published under the authority of the Standards Pol
2、icy and Strategy Committee on 30 October 2003 BSI 30 October 2003 ISBN 0 580 42830 3 National foreword This British Standard reproduces verbatim ISO 10705-3:2003 and implements it as the UK national standard. The UK participation in its preparation was entrusted by Technical Committee EH/3, Water qu
3、ality, to 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 documen
4、t may be 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. Us
5、ers are 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 chang
6、e, and keep 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 13 and a back cover. The BSI copyright notice di
7、splayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments Reference number ISO 10705-3:2003(E)INTERNATIONAL STANDARD ISO 10705-3 First edition 2003-10-01 Water quality Detection and enumeration of bacteriophages Part 3: Validatio
8、n of methods for concentration of bacteriophages from water Qualit de leau Dtection et dnombrement des bactriophages Partie 3: Validation des mthodes de concentration des bactriophages dans leau BSISO107053:2003IS-50701 O3:(3002E) DPlcsid Fremia ihTs PDF file mya ctnoian emdebt dedyfepcaes. In ccacn
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12、fice saCe tsopale 65 eneG 1121-HC 02 av leT. 4 + 10 947 22 1 11 xaF0 947 22 14 + 9 74 E-mial coirypthgis.o gro We bwww.is.o groii BSISO107053:2003 iiiContents Page Foreword iv 1 Scope 1 2 Normative references. 1 3 Terms and definitions. 2 4 Principle. 2 5 Reagents 2 6 Apparatus and glassware. 3 7 Sa
13、mpling 3 8 Preparation of sewage samples for spiking. 3 9 Procedure. 4 10 Calculation. 5 11 Analytical quality control . 6 12 Test report 7 Annex A (informative) Recommended methods for concentration of bacteriophages from water depending on the volume, turbidity and particle content. 8 Annex B (inf
14、ormative) Example of a validation process of a concentration method 10 Bibliography . 13 BSISO107053:2003iv 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 n
15、ormally 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, governmental and non-governmental, in liaison with ISO, also take part i
16、n 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 Directives, Part 2. The main task of technical committees is to prep
17、are International Standards. 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 s
18、ome of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 10705-3 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. ISO 10705 consists of the
19、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: Enumeration of bacte
20、riophages infecting Bacteroides fragilis BSISO107053:2003INTENRATIONAL TSANDADR IS-50701 O3:(3002E)1Water quality Detection and enumeration of bacteriophages Part 3: Validation of methods for concentration of bacteriophages from water WARNING Persons using this part of ISO 10705 should be familiar w
21、ith normal laboratory practice. This part of ISO 10705 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.
22、IMPORTANT It is imperative that personnel involved in validation of methods for concentration of bacteriophages from water have relevant experience with the methods of enumeration of bacteriophages (see ISO/TR 13843 1 ). 1 Scope This part of ISO 10705 specifies the general principles for assessing t
23、he performance of methods for the concentration of bacteriophages from water. Concentration is recommended for those water samples expected to contain 3 pfp (plaque-forming particles) per millilitre. Concentration methods can be applied to all kinds of water provided that the amount and nature of su
24、spended solids and/or dissolved matter do not interfere with the concentration procedure. This part of ISO 10705 does not give specific details of concentration methods, but outlines the fundamental principles for evaluating the suitability of a particular method for a given type and volume of water
25、. Annex A gives examples of methods that have been found satisfactory and their fields of application. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the l
26、atest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbio
27、logical 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 ISO 10705-1, Water quality Detection and enumeration of bacteriophages Part 1: Enumeration of F-specifi
28、c RNA bacteriophages ISO 10705-2, Water quality Detection and enumeration of bacteriophages Part 2: Enumeration of somatic coliphages BSISO107053:20032 ISO 10705-4, Water quality Detection and enumeration of bacteriophages Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis ISO/IEC
29、Guide 2, Standardization and related activities General vocabulary 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO/IEC Guide 2 and the following apply: 3.1 bacteriophages bacterial viruses which are capable of infecting selected host strains NOTE Bac
30、teriophages produce visible plaques (clearance zones) in a confluent lawn of the host strain grown under appropriate culture conditions. 4 Principle The sample is treated according to a method of choice, by which the bacteriophages are concentrated from a relatively large volume of sample (100 ml up
31、 to several litres) to a smaller volume (typically from a few to 20 ml). The concentrated sample is then analysed for bacteriophages according to an International Standard method or other suitable protocol. The concentration method to be evaluated should be carefully described in a protocol, followi
32、ng ISO standard layout as much as possible. The description should include the target group(s) of bacteriophages and their detection method(s), the types of water and ranges of volumes to be analysed, as well as exceptions to the field of application, e.g. turbidity. The method is validated accordin
33、g to principles laid down in this part of ISO 10705. The validation procedure consists of determining the recovery of bacteriophages from a series of samples, seeded with naturally polluted water (raw or treated sewage). The recovery is studied in a range of volumes, and particular attention is paid
34、 to its reproducibility. 5 Reagents Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media. For information on storage see ISO 8199, except where indicated in this part of ISO 10705. Alternatively, use dehydrated complete media and follow strictly t
35、he manufacturers instructions. Other grades of chemicals may be used provided they can be shown to lead to the same results. 5.1 Water, for the preparation of media, glass-distilled water or de-ionized water free from substances that might inhibit bacterial growth under the conditions of the test, a
36、nd at least Grade 3 as specified in ISO 3696. 5.2 Diluent, for making dilutions, peptone-saline solution or another suitable diluent in accordance with ISO 6887-1 or ISO 8199. 5.3 Culture media and reference cultures, as specified in the corresponding standard method of ISO 10705-1, ISO 10705-2 and
37、ISO 10705-4 for the phage assay. 5.4 Glycerol ( = 870 g/l), autoclaved at (121 3) C for 15 min and stored in the dark at room temperature for a period no longer than 1 year. BSISO107053:2003 36 Apparatus and glassware SAFETY PRECAUTIONS Field apparatus should be disinfected before use. Apply safety
38、precautions appropriate to the disinfectant solution used. Some stages of the concentration process may involve the application of hydrostatic or pneumatic pressure. Observe relevant safety precautions. Use usual microbiological laboratory equipment as specified in the method for the phage assay (Cl
39、ause 8), and the protocol for the concentration method. 7 Sampling Samples up to 10 l can conveniently be transported to the laboratory. Take the samples and deliver them to the laboratory as specified in ISO 8199 (see also ISO 19458 2 ). For larger samples, it is advisable to perform the first step
40、 of the concentration procedure in the field. This process may take up to several hours. If parallel examination for indicator bacteria or other micro-organisms is carried out, take a time-proportional sample for these analyses, preferably by filling a sample bottle with a side flow from the concent
41、ration apparatus. Filters, precipitates or other products from the first concentration step may be further treated in the field, or may be transported to the laboratory. Include the transport and storage conditions of intermediate stages of the process in the validation procedure. 8 Preparation of s
42、ewage samples for spiking Obtain a sample of primary or secondary (biologically treated) sewage and centrifuge at 1 000 g for 20 min or filter through an 8 m to 12 m membrane filter. Store supernatant or filtrate on melting ice. Enumerate the target bacteriophages in 1 ml volumes according to the ch
43、osen method. If necessary, dilute the sample to obtain a concentration of 60 pfp to 200 pfp (plaque-forming particles) per millilitre. Add glycerol to obtain a final volume fraction of 5 %; mix well. Distribute 10-ml aliquots into glass or plastic bottles (or tubes, or vials) and freeze at (20 5) C
44、or (70 10) C. Thaw two bottles at room temperature. From each bottle, examine two 0,5-ml aliquots for the target bacteriophages. The average counts should be within the limits as specified above (i.e. 30 pfp to 100 pfp per plate). Analyse the counts for within and between bottle homogeneity as follo
45、ws: 2 1 11 IJ ii ij ij zz Tz JJ + = = where T 1is Cochrans dispersion test statistic to determine the variation in pfp within one vial of reference material; z i+is the total count of plaques of the duplicates of one vial. 1 J ii j j z z + = = I is the number of vials (in this case 2); J is the numb
46、er of duplicates (in this case 2); BSISO107053:20034 The number of degrees of freedom for T 1is equal to I(J1) and 2 2 1 J i j zz Tz II + + + = = where T 2is Cochrans dispersion test statistic to determine the variation in pfp within different vials of one batch of reference material; z +is the tota
47、l count of plaques for all vials and duplicates () 1 I ij i z z + = = . The number of degrees of freedom for T 2is equal to I1. If the phages are randomly distributed within and between the vials, T 1and T 2follow approximately a 2 distribution with respectively 2 and 1 degrees of freedom. Accept th
48、e samples if 0,01 T 1 5,99 and T 2 3,84. NOTE Somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis naturally occurring in raw sewage partially purified as indicated above, do not suffer significant inactivation when frozen with a volume fraction of 5 %
49、glycerol and they can be preserved frozen below (20 5) C and preferably at (70 10) C without significant decrease in numbers for at least one year. 9 Procedure 9.1 Preparation of spiked samples 9.1.1 Batch methods Obtain samples from all types of water mentioned in the scope of the concentration procedure. Obtain th
