1、BRITISH STANDARDBS EN ISO 16266:2008Water quality Detection and enumeration of Pseudomonas aeruginosa Method by membrane filtrationICS 13.060.70g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g3
2、7g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 16266:2008This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 March 2008 BSI 2008ISBN 978 0 580 59736 7National forewordThis British Standard is the UK implementation of EN ISO 16266:2008.
3、It supersedes BS EN 12780:2002 which is withdrawn.The UK participation in its preparation was entrusted by Technical Committee EH/3, Water quality, to Subcommittee EH/3/4, Microbiological methods.A list of organizations represented on this committee can be obtained on request to its secretary.This p
4、ublication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.Amendments/corrigenda issued since publicationDate CommentsEUROPEAN STANDARDNORME EURO
5、PENNEEUROPISCHE NORMEN ISO 16266February 2008ICS 13.060.70 Supersedes EN 12780:2002 English VersionWater quality - Detection and enumeration of Pseudomonasaeruginosa - Method by membrane filtration (ISO 16266:2006)Qualit de leau - Dtection et dnombrement dePseudomonas aeruginosa - Mthode par filtrat
6、ion surmembrane (ISO 16266:2006)Wasserbeschaffenheit - Nachweis und Zhlung vonPseudomonas aeruginosa - Membranfiltrationsverfahren(ISO 16266:2006)This European Standard was approved by CEN on 14 January 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate th
7、e conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in
8、three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of
9、Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COM
10、MITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 16266:2008: EForeword The text of I
11、SO 16266:2006 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 16266:2008 by Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN. This European Standar
12、d shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2008, and conflicting national standards shall be withdrawn at the latest by August 2008. Attention is drawn to the possibility that some of the elements of this
13、document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 12780:2002. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are
14、 bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Swede
15、n, Switzerland and the United Kingdom. Endorsement notice The text of ISO 16266:2006 has been approved by CEN as a EN ISO 16266:2008 without any modification. BS EN ISO 16266:2008iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 2 4 Principle
16、. 2 5 Diluents, culture media and reagents. 2 6 Apparatus and glassware 5 7 Sampling 5 8 Procedure 6 9 Expression of results . 7 10 Test report . 8 11 Performance data 8 12 Interferences . 9 13 Quality assurance. 9 Annex A (informative) Further information about Pseudomonas aeruginosa. 10 Annex B (i
17、nformative) Alternative media . 11 Bibliography . 12 BS EN ISO 16266:2008iv 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
18、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 in the work. ISO collaborates clo
19、sely 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 prepare International Standards. Dra
20、ft 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 elements of this docu
21、ment may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 16266 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4, Microbiological methods. This International Standard is the equivalent of EN 12780:20
22、02. BS EN ISO 16266:2008vIntroduction Pseudomonas aeruginosa is an opportunistic pathogen of man that is capable of growth in water at very low nutrient concentrations. At source and during marketing, a natural mineral water or a spring water is to be free from Pseudomonas aeruginosa in any 250 ml s
23、ample examined (see, e.g. Council Directive 80/777/EEC1and Council Directive 96/70/EC2). Other bottled waters offered for sale are also to be free of Pseudomonas aeruginosa in any 250 ml sample (see, e.g. Council Directive 98/83/EC3). Other waters, including pool waters and water for human consumpti
24、on, may sometimes be tested for Pseudomonas aeruginosa for reasons of public health. In these cases, it is typical to examine 100 ml volumes. BS EN ISO 16266:2008blank1Water quality Detection and enumeration of Pseudomonas aeruginosa Method by membrane filtration WARNING Persons using this Internati
25、onal Standard should be familiar with normal laboratory practice. This standard 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 nationa
26、l regulatory conditions. IMPORTANT It is absolutely essential that tests conducted according to this International Standard be carried out by suitably trained staff. 1 Scope This International Standard specifies a method for the isolation and enumeration of Pseudomonas aeruginosa in samples of bottl
27、ed water by a membrane filtration technique. This method can also be applied to other types of water with a low background flora, for example, pool waters and waters intended for human consumption. 2 Normative references The following referenced documents are indispensable for the application of thi
28、s document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696, Water for analytical laboratory use Specification and test methods ISO 5667-1, Water quality Sampling Part 1: Guidance
29、 on the design of sampling programmes and sampling techniques ISO 5667-21), Water quality Sampling Part 2: Guidance on sampling techniques ISO 5667-3, Water quality Sampling Part 3: Guidance on the preservation and handling of water samples ISO 6887-1, Microbiology of food and feeding stuffs Prepara
30、tion of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 7704, Water quality Evaluation of membrane filters used for microbiological analyses ISO 8199, Water quality Ge
31、neral guidance on the enumeration of micro-organisms by culture ISO 194582), Water quality Sampling for microbiological analysis 1) ISO 5667-1 and ISO 5667-2 are currently undergoing joint revision, which will be published as ISO 5667-1. 2) To be published. BS EN ISO 16266:20082 3 Terms and definiti
32、ons For the purposes of this document, the following terms and definitions apply. 3.1 Pseudomonas aeruginosa micro-organisms that grow on selective media containing cetrimide and produce pyocyanin, or micro-organisms that grow on selective media containing cetrimide, are oxidase positive, fluoresce
33、under UV radiation (360 20) nm, and are able to produce ammonia from acetamide 4 Principle 4.1 Filtration A measured volume of the water sample, or a dilution of the sample, is filtered through a membrane filter of 0,45 m. The membrane filter is placed on the selective medium and incubated under the
34、 conditions specified for the medium. 4.2 Enumeration The numbers of presumptive Pseudomonas aeruginosa are obtained by counting the number of characteristic colonies on the membrane filter after incubation. Pyocyanin-producing colonies are considered as confirmed Pseudomonas aeruginosa but other fl
35、uorescing or reddish brown colonies require confirmation. 4.3 Confirmation Subcultures of colonies requiring confirmation are made from the membrane filter onto plates of nutrient agar (but see Annex B). After incubation, cultures that were not initially fluorescent are tested for the oxidase reacti
36、on, and oxidase-positive cultures are tested for the production of fluorescein and the ability to produce ammonia from acetamide. Cultures that were fluorescent initially are tested for the ability to produce ammonia from acetamide. 5 Diluents, culture media and reagents Use reagents of analytical r
37、eagent quality in the preparation of culture media and diluents, unless otherwise specified. Prepare the medium as follows and add the selective agents as supplements at the given concentrations or use commercially available media and reagents prepared according to the manufacturers instructions. Pr
38、epare media and reagents using water grade 3 as specified in ISO 3696, or water of equivalent purity and free from substances which might inhibit growth under the conditions of the test. 5.1 Culture medium Use the following medium for the determination of Pseudomonas aeruginosa. 5.1.1 Pseudomonas ag
39、ar base/CN-agar 5.1.1.1 Composition Gelatin peptone 16,0 g Casein hydrolysate 10,0 g Potassium sulfate (anhydrous) (K2SO4) 10,0 g BS EN ISO 16266:20083Magnesium chloride (anhydrous) (MgCl2) 1,4 g Glycerol 10 ml Agar 11,0 g to 18,0 g Water (distilled or equivalent) 1 000 ml NOTE The amount of agar re
40、quired depends on the gel strength. Follow the manufacturers instructions for the agar used. CN supplement Hexadecyltrimethyl ammonium bromide (cetrimide) 0,2 g Nalidixic acid 0,015 g 5.1.1.2 Preparation Suspend the peptone, casein hydrolysate, potassium sulfate, magnesium chloride and agar in 1 000
41、 ml of distilled water (or equivalent). Add 10 ml of glycerol. Heat to boiling in order to dissolve completely and sterilize by autoclaving at (121 3) C for 15 min. Allow the medium to cool to (45 to 50) C. Add the CN supplement rehydrated in 2 ml of sterile distilled water, mix well and add to the
42、sterile molten basal medium. Mix well and pour into sterile Petri dishes to give a depth of at least 5 mm of agar. The final pH of the solidified medium should correspond to 7,1 0,2 at 25 C. Store prepared plates in the dark protected from desiccation at (5 3) C and use within 1 month. Do not keep t
43、he agar molten for more than 4 h. Do not remelt the medium. 5.2 Confirmatory media and reagents 5.2.1 Kings B medium 5.2.1.1 Composition Peptone 20,0 g Glycerol 10 ml Di-potassium hydrogen phosphate (K2HPO4) 1,5 g Magnesium sulfate heptahydrate (MgSO47H2O) 1,5 g Agar 15,0 Water (distilled or equival
44、ent) 1 000 ml 5.2.1.2 Preparation Dissolve the ingredients in the water by heating. Cool down to (45 to 50) C and adjust the pH corresponding to 7,2 0,2 at 25 C, using either hydrochloric acid or sodium hydroxide. Dispense the medium in 5 ml aliquots into culture tubes which are capped and autoclave
45、d at (121 3) C for 15 min. Allow the tubes to cool and solidify in slants. Store in the dark at (5 3) C and use within 3 months. BS EN ISO 16266:20084 5.2.2 Acetamide broth 5.2.2.1 Composition Solution A Potassium di-hydrogenphosphate (KH2PO4) 1,0 g Magnesium sulfate (anhydrous) (MgSO4) 0,2 g Acetam
46、ide 2,0 Sodium chloride (NaCl) 0,2 g Water (distilled or equivalent, ammonia free) 900 ml Dissolve the ingredients in water and then adjust the pH to correspond to 7,0 0,5 at 25 C with either hydrochloric acid or sodium hydroxide. CAUTION Acetamide is carcinogenic and irritant appropriate precaution
47、s shall be taken when weighing out, preparing and discarding the medium. Solution B Sodium molybdate (Na2MoO42H2O) 0,5 g Iron sulfate heptahydrate (FeSO47H2O) 0,05 g Water 100 ml 5.2.2.2 Preparation To prepare the acetamide broth, add 1 ml of solution B to 900 ml of a freshly prepared solution A (5.
48、2.2.1). Add water with constant stirring to a total volume of 1 l. Dispense this mixture in 5 ml aliquots to culture tubes which are then capped and sterilized in an autoclave at (121 3) C for 15 min. Store in the dark at (5 3) C and use within 3 months. 5.2.3 Nutrient agar 5.2.3.1 Composition Pepto
49、ne 5,0 g Meat extract 1,0 g Yeast extract 2,0 g Sodium chloride (NaCl) 5,0 g Agar 15,0 g Water 1 000 ml 5.2.3.2 Preparation Dissolve the ingredients in the water by heating. Sterilize by autoclaving at (121 3) C for 15 min. The pH of the solidified prepared medium should correspond to 7,4 0,2 at 25 C. Dry the plates to remove excess surface moisture before use. Store prepared plates in the dark protected from desiccation at (5 3) C and use within 1 month. BS EN ISO 16266:200855.2.4
