EN 12780:2002
(Main)Water quality - Detection and enumeration of Pseudomonas aeruginosa by membrane filtration
Water quality - Detection and enumeration of Pseudomonas aeruginosa by membrane filtration
This European Standard presents a method for the isolation and enumeration of Pseudomonas aeruginosa in bottled water samples 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.
Wasserbeschaffenheit - Nachweis und Zählung von Pseudomonas aeruginosa durch Membranfiltration
Diese Europäische Norm beschreibt ein Verfahren zum Nachweis und zur Zählung von Pseudomonas aeruginosa in Proben von abgefülltem Wasser nach einem Membranfiltrationsverfahren. Dieses Verfahren ist auch geeignet für andere Wasserarten mit geringer Begleitflora, wie z. B. Schwimm- und Badebeckenwasser sowie Wasser für den menschlichen Gebrauch.
Qualité de l'eau - Détection et dénombrement de Pseudomonas aeruginosa par filtration sur membrane
Domaine d'application
La présente Norme européenne prescrit une méthode permettant d'isoler et de dénombrer Pseudomonas aeruginosa dans des échantillons d'eau embouteillée par filtration sur membrane. Cette méthode peut également être appliquée à d'autres types d'eau présentant une faible flore interférente, par exemple les eaux de piscine et les eaux destinées à la consommation humaine.
Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa z membransko filtracijo
General Information
Relations
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Kakovost vode - Ugotavljanje prisotnosti in števila Pseudomonas aeruginosa z membransko filtracijoWasserbeschaffenheit - Nachweis und Zählung von Pseudomonas aeruginosa durch MembranfiltrationQualité de l'eau - Détection et dénombrement de Pseudomonas aeruginosa par filtration sur membraneWater quality - Detection and enumeration of Pseudomonas aeruginosa by membrane filtration13.060.70Preiskava bioloških lastnosti vodeExamination of biological properties of waterICS:Ta slovenski standard je istoveten z:EN 12780:2002SIST EN 12780:2003en01-januar-2003SIST EN 12780:2003SLOVENSKI
STANDARD
SIST EN 12780:2003
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 12780May 2002ICS 07.100.20English versionWater quality - Detection and enumeration of Pseudomonasaeruginosa by membrane filtrationQualité de l'eau - Détection et dénombrement dePseudomonas aeruginosa par filtration sur membraneWasserbeschaffenheit - Nachweis und Zählung vonPseudomonas aeruginosa durch MembranfiltrationThis European Standard was approved by CEN on 17 February 2002.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the 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 Management Centre or to any CEN member.This European Standard exists in 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 Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2002 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 12780:2002 ESIST EN 12780:2003
EN 12780:2002 (E)2ContentspageForeword.3Introduction.41Scope.42Normative references.43Terms and definitions.54Principle.55Diluents, culture media and reagents.56Apparatus and glassware.97Sampling.98Procedure.99Expression of results.1110Test Report.1211Performance data.1212Interferences.1213Quality assurance.12Annex A (informative)
Further information about Pseudomonas aeruginosa.13Annex B (informative)
Alternative media.14Bibliography.15SIST EN 12780:2003
EN 12780:2002 (E)3ForewordThis document EN 12780:2002 has been prepared by Technical Committee CEN/TC 230, "Water analysis", thesecretariat of which is held by DIN.This European Standard shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by November 2002, and conflicting national standards shall be withdrawn at the latestby November 2002.Annexes A and B are informative.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain,Sweden, Switzerland and the United Kingdom.SIST EN 12780:2003
EN 12780:2002 (E)4IntroductionPseudomonas aeruginosa is an opportunistic pathogen of man that is capable of growth in water at very lowconcentrations of nutrients. At source and during marketing, a natural mineral water or a spring water should be freefrom Pseudomonas aeruginosa in any 250 ml sample examined (Council Directives 80/777/EEC and 96/70/EC).Other bottled waters offered for sale must also be free from Pseudomonas aeruginosa in any 250 ml sample (CouncilDirective 98/83/EC). Other waters including pool waters and water for human consumption can sometimes be testedfor Pseudomonas aeruginosa for reasons of public health when it is usual to examine 100 ml volumes.WARNING — Persons using this standard should be familiar with normal laboratory practice. This standarddoes not purport to address all of the safety problems, if any, associated with its use. It is theresponsibility of the user to establish appropriate safety and health practices and to ensure compliancewith any national regulatory conditions.1 ScopeThis European Standard specifies a method for the isolation and enumeration of Pseudomonas aeruginosa in bottledwater samples by a membrane filtration technique. This method can also be applied to other types of water with a lowbackground flora, for example pool waters and waters intended for human consumption.2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text, and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN 25667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes (ISO 5667-1:1980).EN 25667-2, Water quality — Sampling — Part 2: Guidance on sampling techniques (ISO 5667-2:1991).EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987).prEN ISO 5667-3 Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples (ISO5667-3:1994).ISO 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension anddecimal dilutions for microbiological
examination — Part 1: General rules for the
preparation of the initial suspensionand decimal dilutions.ISO 8199, Water quality — General guide to the enumeration of micro-organisms by culture.SIST EN 12780:2003
EN 12780:2002 (E)53 Terms and definitionsFor the purposes of this European Standard, the following term and definition apply.3.1Pseudomonas aeruginosamicro-organisms that grow on selective media containing cetrimide and produce pyocyanin, or micro-organisms thatgrow on selective media containing cetrimide, are oxidase positive, fluoresce under UV light (360 ± 20) nm, and areable to produce ammonia from acetamide4 Principle4.1 FiltrationA measured volume of the water sample, or a dilution of the sample, is filtered through a membrane filter that hasfiltration characteristics equivalent to a rated pore diameter of 0,45 mm. The membrane filter is placed on the selectivemedium and incubated under the conditions specified for the medium.4.2 EnumerationThe numbers of presumptive Pseudomonas aeruginosa are obtained by counting the number of characteristiccolonies on the membrane filter after incubation. Pyocyanin-producing colonies are considered as confirmedPseudomonas aeruginosa but other fluorescing or reddish brown colonies require confirmation.4.3 ConfirmationSubcultures of colonies requiring confirmation are made from the membrane filter onto plates of nutrient agar (seeannex B). After incubation, cultures that were not fluorescent initially are tested for the oxidase reaction andoxidase-positive cultures are tested for the production of fluorescein and the ability to produce ammonia fromacetamide. Cultures that were fluorescent initially are tested for the ability to produce ammonia from acetamide.5 Diluents, culture media and reagents5.1 GeneralUse reagents of analytical reagent 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 usecommercially available media and reagents prepared according to the manufacturer’s instructions. Prepare media andreagents using glass distilled water or water of equivalent purity, in accordance with EN ISO 3696 Grade 3 and freefrom substances which might inhibit growth under the conditions of the test.5.2 Culture mediumUse the following medium for the determination of Pseudomonas aeruginosa.SIST EN 12780:2003
EN 12780:2002 (E)65.2.1 Pseudomonas Agar Base/CN-agar5.2.1.1 CompositionGelatin peptone16,0 gCasein hydrolysate10,0 gPotassium sulfate (anhydrous) (K2SO4)10,0 gMagnesium chloride (anhydrous) (MgCl2)1,4 gGlycerol10 mlAgar11,0 g to 18,0 gWater (distilled or equivalent)1 000 mlThe amount of agar required is dependent on the gel strength. Follow the manufacturer’s instructions for the agarused.CN SupplementHexadecyltrimethyl ammonium bromide (cetrimide)0,2 gNalidixic Acid0,015 g5.2.1.2 PreparationSuspend the peptone, casein hydrolysate, potassium sulfate, magnesium chloride and agar in 1 000 ml of distilledwater (or equivalent). Add 10 ml of glycerol. Bring to the boil 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 ofsterile distilled water, mix well a
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