1、BSI Standards PublicationSludge, treated biowaste and soil Detection and enumeration of Escherichia coliPD CEN/TR 16193:2013National forewordThis Published Document is the UK implementation of CEN/TR 16193:2013.This technical report contains details of three methods evaluated for theirsuitability fo
2、r horizontal application for sludge, treated biowaste and soil.The UK committee believes that these methods are not fit for their intendedpurpose and are not appropriate to samples of all such materials, as the sample matrices are not sufficiently similar.It should be noted that in merging three sep
3、arate drafts to achieve the finaltext, incompatibilities and conflicting interpretation have been introduced: the document does not adhere to a recognised definition of E. coli as target organism (see SCA methods referred to below); the inter laboratory validation undertaken (as found in Tables 4, 9
4、 and 14)is considered by UK technical experts as demonstrating poor reproducibility; soil matrices were not included in the validation.The recommended methods for UK laboratories intending to analyze waste-water sludge can be found in The Microbiology of Sewage Sludge: Part 3(2003) Methods for the i
5、solation and enumeration of Escherichia coli,including verocytotoxigenic Escherichia coli published by the EnvironmentAgency Standing Committee of Analysts (SCA) in the series Methods for theexamination of water and associated materials. SCA methods are also considered suitable for determining E. co
6、li in sometreated biowaste applications such as compost, under PAS 100:2005, anddigestates, under PAS 110:2010. BS ISO 16649-2:2001 provides a methodsuitable for enumeration of E. coli in food and animal feeding stuffs. Thesemethods might also be applicable to soil but this would need to be verified
7、using the approach described in DD ENV ISO/TR 13843:2001, Water Quality Guidance on Validation of Microbiological Methods.The UK participation in its preparation was entrusted to Technical CommitteeH/-/4, Environmental testing programmes.A list of organizations represented on this committee can be o
8、btained onrequest to its secretary.This publication does not purport to include all the necessary provisions of acontract. Users are responsible for its correct application. The British Standards Institution 2013Published by BSI Standards Limited 2013ISBN 978 0 580 70860 2ICS 13.030.01Compliance wit
9、h a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of the Standards Policy and Strategy Committee on 31 July 2013.Amendments issued since publicationAmd. No. Date Text affectedPUBLISHED DOCUMENTPD CEN/TR 16193:2013TECHNICAL REP
10、ORT RAPPORT TECHNIQUE TECHNISCHER BERICHT CEN/TR 16193 May 2013 ICS 13.030.01 English Version Sludge, treated biowaste and soil - Detection and enumeration of Escherichia coli Boue, biodchet trait et sol - Recherche et dnombrement des Escherichia coli Schlamm, behandelter Bioabfall und Boden - Nachw
11、eis und Zhlung von Escherichia coli This Technical Report was approved by CEN on 1 March 2011. It has been drawn up by the Technical Committee CEN/TC 400. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Y
12、ugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EURO
13、PEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TR 16193:2013: EPD CEN/TR 16193:2013CEN/TR 16193:2013 (E) 2 Contents Pag
14、e Foreword . 4 Introduction 5 1 Scope 6 2 Normative references . 6 3 Terms and definitions 6 4 Abbreviations . 7 5 Quality assurance 7 6 Method A Membrane filtration method for quantification 7 6.1 Scope 7 6.2 Principle 8 6.3 Reagents, diluents and culture media . 8 6.4 Apparatus . 9 6.5 Sampling .
15、10 6.5.1 General . 10 6.5.2 Storage . 11 6.5.3 Handling . 11 6.6 Procedure . 11 6.6.1 Sample preparation 11 6.6.2 Sample dilution 12 6.6.3 Membrane filtration 12 6.6.4 Resuscitation and enumeration of colonies on chromogenic agar . 13 6.6.5 Confirmation of colony identity . 13 6.6.6 Determination of
16、 the dry residue content . 13 6.7 Calculation and expression of results 13 6.8 Performance data of the interlaboratory comparison Method A . 14 6.8.1 Material used in the interlaboratory comparison study . 14 6.8.2 First assessment of the precision of the method . 15 6.8.3 Interlaboratory comparison
17、 results . 16 6.9 Pre-filtration and centrifugation Comparison tests 17 7 Method B Miniaturised method (Most Probable Number) by inoculation in liquid medium . 19 7.1 Scope 19 7.2 Principle 19 7.3 Reagents, diluents and culture media . 19 7.4 Apparatus . 21 7.5 Sampling . 22 7.5.1 General . 22 7.5.2
18、 Storage . 22 7.5.3 Handling . 22 7.6 Procedure . 22 7.6.1 Sample preparation 22 7.6.2 Analysis 23 7.6.3 Determination of the dry residue content . 24 7.7 Expression of results . 24 7.7.1 Determination of the characteristic number . 24 7.7.2 Calculation of the MPN and its confidence interval 25 7.8
19、Performance data . 27 7.8.1 MPN Statistical table 27 PD CEN/TR 16193:2013CEN/TR 16193:2013 (E) 3 7.8.2 Performance data of the interlaboratory comparison . 35 7.8.3 First assessment of the precision of the method . 36 7.8.4 Interlaboratory comparison results . 37 7.9 Preparation of synthetic sea sal
20、t . 39 7.9.1 Major ion composition of a convenient ocean synthetic sea salt 39 7.9.2 Example for preparation from defined substances 39 7.10 Quality criteria for the manufacturing of the medium in microtitre plates (E. coli) . 40 8 Method C Macromethod (Most Probable Number) in liquid medium . 41 8.
21、1 Scope 41 8.2 Principle 41 8.3 Reagents, diluents and culture media . 41 8.4 Apparatus . 42 8.5 Sampling . 43 8.5.1 General . 43 8.5.2 Sample storage. 43 8.5.3 Sample handling . 43 8.6 Procedure . 43 8.6.1 Sample preparation 43 8.6.2 Analysis 44 8.6.3 Determination of the dry residue content . 44 8
22、.7 Expression of the results . 44 8.8 Performance data . 46 8.8.1 MPN Statistical table for 3-tubes MPN procedure . 46 8.8.2 Repeatability and reproducibility . 47 8.8.3 First assessment of the precision of the method . 47 8.8.4 Interlaboratory comparison results . 48 9 Test report 50 Bibliography 5
23、1 PD CEN/TR 16193:2013CEN/TR 16193:2013 (E) 4 Foreword This document (CEN/TR 16193:2013) has been prepared by Technical Committee CEN/TC 400 “Project Committee - Horizontal standards in in the fields of sludge, biowaste and soil”, the secretariat of which is held by DIN. Attention is drawn to the po
24、ssibility that some of the elements of this 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 has been prepared under a mandate given to CEN by the European Commission and the European Free Trad
25、e Association. This document is part of a modular horizontal approach in which this document belongs to the analytical step. The preparation of this document by CEN is based on a mandate by the European Commission (Mandate M/330). The mandate considers standards on sampling and analytical methods fo
26、r hygienic and biological parameters as well as inorganic and organic determinants. It was the aim of the mandate to develop standards that are applicable to sludge, treated biowaste and soil and lead to equivalent results as far as this is technically feasible. Until now, test methods determining p
27、roperties of materials within the environmental area were prepared in Technical Committees (TCs) working on specific products/matrices (soil, waste, sludge etc). However, it is recognised that many steps in test procedures can be used in test procedures for other products/matrices. By careful determ
28、ination of these steps and selection of specific questions within these steps, elements of the test procedure can be described in a way that can be used for more matrices and materials with certain specifications. This optimisation is in line with the development among end-users of standards. A majo
29、rity of routine environmental analyses are carried out by institutions and laboratories working under a scope which is not limited to one single environmental matrix but covers a wide variety of matrices. Availability of standards covering more matrices contributes to the optimisation of laboratory
30、procedures and standard maintenance costs, e.g. costs related to accreditation and recognition. A horizontal modular approach was developed in the project Horizontal. Modular means that a test standard developed in this approach concerns a specific step in assessing a property and not the whole “cha
31、in of measurement” (from sampling to analyses). A beneficial feature of this approach is that “modules” can be replaced by better ones without jeopardising the standard “chain”. The results of the desk study as well as the evaluation and validation studies have been subject to discussions with all p
32、arties concerned in the CEN structure during the development by project Horizontal. The results of these consultations with interested parties in the CEN structure have been presented to and discussed in CEN/TC 400. This Technical Report contains the most common detection and enumeration methods for
33、 the determination of E. coli consolidated in one document. The individual methods are specified in the following clauses: Clause 6: Method A - Membrane filtration method for quantification; Clause 7: Method B - Miniaturised method (Most Probable Number) by inoculation in liquid medium; Clause 8: Me
34、thod C - Macromethod (Most Probable Number) in liquid medium. PD CEN/TR 16193:2013CEN/TR 16193:2013 (E) 5 Introduction Escherichia coli is a non-pathogenic, Gram negative bacterium with a faecal origin. Consequently, it can be used as an indicator of faecal contamination. It can also be used to moni
35、tor the effectiveness of pasteurisation or disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the behaviour of all pathogens in these materials. This Technical Report contains three different methods for the detection and enumeration of Escheric
36、hia coli which were included in a validation trial in 2007. The results achieved in this validation trial have been judged differently by experts. Consequently, it was decided by CEN/TC 400 to publish the methods as a Technical Report, aiming for further improvement of the methods and a later public
37、ation as European Standard. Table 1 Matrices for which the methods described in this Technical Report are applicable and tested in a validation trial Matrix Method A Method B Method C Sludge Mesophilic anaerobic digested sewage sludge Mesophilic anaerobic digested sewage sludge Mesophilic anaerobic
38、digested sewage sludge Pelletised air dried sludge Pelletised air dried sludge Pelletised air-dried sludge Digested sewage sludge presscake Digested sewage sludge presscake Digested sewage sludge presscake Composted sewage sludge Composted sewage sludge Composted sewage sludge Biowaste Composted bio
39、waste Composted biowaste Anaerobic treated biowaste Composted green waste Composted green waste Composted green waste Anaerobic treated biowaste Anaerobic treated biowaste Composted biowaste WARNING Persons using this Technical Report should be familiar with normal laboratory practice. This Technica
40、l Report 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. WARNING Samples may contain hazardous and infl
41、ammable substances. They may contain pathogens and be liable to biological action. Consequently, it is recommended that these samples be handled with special care. The gases which can be produced by microbiological activity are potentially inflammable and will pressurise sealed bottles. Exploding bo
42、ttles are likely to result in infectious shrapnel and/or pathogenic aerosols. Glass bottles should be avoided wherever possible. National regulations should be followed with respect to microbiological hazards associated with this method. IMPORTANT It is absolutely essential that tests conducted acco
43、rding to this Technical Report be carried out by suitably trained staff. PD CEN/TR 16193:2013CEN/TR 16193:2013 (E) 6 1 Scope This Technical Report specifies three methods for the detection and enumeration of Escherichia coli in sludge, treated biowaste and soil: Method A - Membrane filtration method
44、 for quantification (see Clause 6); Method B - Miniaturised method (Most Probable Number, MPN) by inoculation in liquid medium (see Clause 7); Method C - Macromethod (Most Probable Number) in liquid medium (see Clause 8). 2 Normative references The following documents, in whole or in part, are norma
45、tively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN 15934, Sludge, treated biowaste, soil and waste Calculation
46、 of dry matter fraction after determination of dry residue or water content EN ISO 9308-3:1998, Water quality Detection and enumeration of Escherichia coli and coliform bacteria in surface and wastewater Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO 9308-3:1
47、998) ISO 8199, Water quality General guidance on the enumeration of micro-organisms by culture 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 Escherichia coli E.coli -D-glucuronidase-positive microorganism growing at an incubation temperatur
48、e of 44 C in the specified liquid medium containing 4-methylumbelliferyl-D-glucuronide (MUG) SOURCE: EN ISO 9308-3:1998 Note 1 to entry: During growth, indole is produced from tryptophan and gas produced from lactose. 3.2 vegetative bacteria bacteria which are capable of normal growth in broth or on
49、 agar media without pre-culture resuscitation 3.3 sub-lethally damaged bacteria bacteria which have been stressed but not killed by storage or subsequent treatment by, e.g., mesophilic anaerobic digestion, lime stabilisation or composting, and therefore may not be recovered 3.4 resuscitation recovery to vegetative growth of sub-lethally damaged bacteria previously incapable of growth on agar media PD CEN/TR 16193:2013CEN/TR 16193:2013 (E) 7 3.5 quantitative resuscitation recovery to vegetative growth of sub-lethally d
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