1、December 2009DEUTSCHE NORM English price group 10No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.120!$“1562556www.d
2、in.deDDIN EN 15788Animal feeding stuffs Isolation and enumeration of Enterococcus (E. faecium) spp.English version of DIN EN 15788:2009-12Futtermittel Keimzhlung von Enterococcus spp. (E. faecium)Englische Fassung DIN EN 15788:2009-12www.beuth.deDocument comprises pages16DIN EN 15788:2009-12 Nationa
3、l foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food a
4、nd Agricultural Products Standards Committee), Technical Committee NA 057-03-03 AA Futtermittel. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 6497 DIN EN ISO 6497 National Annex NA (informative) Bibliography DIN EN ISO 6497, Animal f
5、eeding stuffs Sampling 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15788 September 2009 ICS 65.120 English Version Animal feeding stuffs - Isolation and enumeration of Enterococcus (E. faecium) spp. Aliments des animaux - Isolement et dnombrement de lEntrocoque (E. faecium) spp. Futtermit
6、tel - Keimzhlung von Enterococcus spp. (E. faecium) This European Standard was approved by CEN on 1 August 2009. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any al
7、teration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN 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 t
8、ranslation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Ge
9、rmany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMU
10、NG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15788:2009: EEN 15788:2009 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and d
11、efinitions .5 4 Principle 6 5 Diluents, selective medium and test kit for phenotypic characterisation .6 6 Apparatus and glassware .7 7 Sampling .8 8 Preparation of test sample 9 9 Procedure .9 10 Expression of results . 10 11 Precision 11 12 Test report . 11 Annex A (informative) Notes on procedure
12、 12 Annex B (informative) Results of the interlaboratory study . 13 Bibliography . 14 DIN EN 15788:2009-12 EN 15788:2009 (E) 3 Foreword This document (EN 15788:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN. This European St
13、andard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by March 2010, and conflicting national standards shall be withdrawn at the latest by March 2010. Attention is drawn to the possibility that some of the elements of th
14、is 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 Trade Association. According to the CEN/CENEL
15、EC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembo
16、urg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15788:2009-12 EN 15788:2009 (E) 4 Introduction This methodology has been developed to enumerate enterococci (E. faecium) to enable the European Commission to cont
17、rol proper labelling of animal feeding products (EU project SMT4-CT98-2235 - “Methods for the official control of probiotics (microorganisms) used as animal feeds”) 1. The method is based on an extensive screening of 12 pre-selected, commercially available media for the detection and enumeration of
18、enterococci. The described methodology was validated in an interlaboratory study 2. This method is not selective for probiotic enterococci (E. faecium) but can be applied to enumerate enterococci in additives, premixtures and feeding stuffs assuming that the probiotic enterococci (E. faecium) is pre
19、sent in far higher numbers than any other enterococci. DIN EN 15788:2009-12 EN 15788:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration of enterococci in feed samples (additives, premixtures and feeding stuffs) that contain enterococci (E. faecium) as a single microo
20、rganism component or in a mixture with other microorganisms. This standard is not applicable to mineral feeds which are defined as complementary feedingstuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) 3. There are different categories of feed sa
21、mples: a) Additives containing about 1010colony forming units (CFU)/g; b) Premixtures containing 108CFU/g; c) Feeds, meal or pellets which contain about 106CFU/g and include complete feeding stuffs,and milk replacers. The detection limit is as defined in EN ISO 7218. 2 Normative references The follo
22、wing referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 6887-1, Microbiology of food and animal feeding stu
23、ffs - Preparation 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 6887-1:1999) EN ISO 7218, Microbiology of food and animal feeding stuffs - General requirements
24、 and guidance for microbiological examinations (ISO 7218:2007) ISO 6498, Animal feeding stuffs Preparation of test samples 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 enterococcus faecium (described by their characteristics as used for th
25、is standard) enterococcus faecium is charaterised as a bacterium which forms colonies fitting the description of the species on the specified selective medium after incubation of 24 h at a temperature of 37 C under aerobic conditions: a) morphology of colonies on selective medium; b) circular; c) co
26、nvex to dome-shaped; d) entire; DIN EN 15788:2009-12 EN 15788:2009 (E) 6 e) white; f) glistening surface; g) opaque. Colony size varies between 1 mm and 2 mm in diameter The medium surrounding the colonies shows a dark brown to black coloration, due to the hydrolysis of esculin Phase contrast micros
27、copical examination of selected colonies typically shows spherical cells arranged in pairs. 4 Principle An initial suspension of the sample is prepared in a diluent with suitable buffer capacity using a suitable homogeniser. Dilutions of the initial suspension have to be immediately prepared before
28、the suspension settles. Spread plates (Bile Esculin Azide Agar) are inoculated with the chosen dilutions. The plates are incubated aerobically for 24 h 2 h at 37 C 1 C. Presumptive enterococci (E. faecium) colonies are counted and the number of colony forming units per g or kg is calculated. NOTE To
29、 verify the colony count, a phenotypic characterisation and confirmation of a selection of colonies may be done by means of an identification kit. 5 Diluents, selective medium and test kit for phenotypic characterisation 5.1 Diluents 5.1.1 Diluent for initial suspension of premixtures, additives and
30、 feeding stuffs This diluent is used to decimally dilute the sample to prepare an initial decimally diluted sample suspension (10-1) in appropriate containers (e.g. universals, bottles or flasks). Phosphate buffered saline (PBS): Dissolve 8 g sodium chloride, 0,2 g potassium chloride, 1,15 g disodiu
31、m hydrogen phosphate, 0,2 g potassium dihydrogen phosphate, pH 7,3 0,2 in 1 l of distilled water. Aliquote this saline into appropriate containers (e.g. universals, bottles or flasks). Autoclave all capped containers with the initial diluent at 121 C 1 C for 10 min. To avoid loss during autoclaving,
32、 screw cap bottles are recommended. Bring the diluent to room temperature before use. Measure the pH of the diluent to ensure the suitable buffer capacity. 5.1.2 Diluent for serial dilutions This diluent is used to decimally dilute the initial sample suspension and subsequent dilutions. Peptone salt
33、 solution: A peptone salt solution is made complying with EN ISO 6887-1. DIN EN 15788:2009-12 EN 15788:2009 (E) 7 Compose the solution of enzymatic digest of 1 g casein such as pancreatic peptone of casein (or peptone of same quality) and 8,5 g sodium chloride) per liter (l) distilled water. Dissolv
34、e the ingredients in water. Adjust the pH to 7,0 0,2 at 25 C 1 C. For decimal dilutions, prepare test tubes containing 9,0 ml 0,1 ml after sterilisation or use screw cap bottles to avoid weight loss during autoclaving. Sterilise in the autoclave for 15 min at 121 C 1 C. Bring the diluent to room tem
35、perature before use. 5.2 Selective medium Bile Esculin Azide Agar1)is used as a selective medium. Composition in g/l final medium: a) Peptone 1 (pancreatic digest of casein) 17,0 g b) Peptone 2 (peptic digest of meat) 3,0 g c) Yeast extract 5,0 g d) Ox bile (dehydrated) 10,0 g e) Sodium chloride 5,0
36、 g f) Esculin 1,0 g g) Ferric ammonium citrate 0,5 g h) Sodium azide 0,25 g i) Agar 13,5 g Final pH 7,1 0,2. 5.3 Phenotypic characterization and confirmation Check selected colonies microscopically for enterococci-like morphology. NOTE A phenotypic kit may be used for phenotypic characterisation of
37、isolated colonies if required. 6 Apparatus and glassware Usual microbiological laboratory equipment and, in particular, the following: 6.1 Equipment for dry sterilisation (oven) and wet sterilisation (autoclave) According to EN ISO 7218. 6.2 Incubator Capable of maintaining a temperature of 37 C 1 C
38、. 1) the agar is commercially available from various suppliers. DIN EN 15788:2009-12 EN 15788:2009 (E) 8 6.3 Water bath Capable of maintaining a temperature of 48 C 1 C. 6.4 Blending equipment Two-speed or a variable adjustable blender (18 000 rotations per minute (rpm) and 22 000 rpm), with a one l
39、itre bowl which is sterilised in an oven for 1 h at 170 C to 180 C. 6.5 Mechanical stirrer A mechanical stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent 6.6 Balance Capable of weighing to two decimal places. 6.7 Screw-cap bottles Appropriate capacities, 25 ml universals, bottles, test tube
40、s, flasks and 1 000 ml Duran bottles 6.8 Pipettor and sterile tips to dispense 100 l and 1 ml Wide bore tips to pipette homogenised feed stuff for dilution. 6.9 Bacterial Cell spreaders Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders. 6.10 Steril
41、e Petri dishes, triple vent, 90 mm in diameter 6.11 Laminar flow cabinet 6.12 Microscope With phase-contrast-imaging (400x) and for use with oil immersion (1 000x). 7 Sampling Carry out the sampling procedure in accordance with the specific standard appropriate to the product concerned. If such a sp
42、ecific standard is not available, it is recommended that agreement be reached on this subject among the parties concerned. Apply community rules 1 for official control sampling of animal feeds. NOTE Sampling can be done according to ISO 6497 4. Although ISO 6497 is not applicable for microorganisms,
43、 due to the lack of other reference, it seems it is the most suitable protocol to be taken into account WARNING Take precaution to avoid potential cross-contamination of samples with enterococci, particularly after sampling additives and premixtures supplemented with enterococci. DIN EN 15788:2009-1
44、2 EN 15788:2009 (E) 9 8 Preparation of test sample The test sample preparation shall be done in accordance with ISO 6498 and the congruent product standard. If such a specific standard is not available, it is recommended that agreement be reached on this subject among the parties concerned. ISO 6498
45、 gives general guidelines on test sample preparation. 9 Procedure 9.1 Preparation of poured agar plates The selective medium is prepared according to the manufacturers directions. Pour after autoclaving and cooling in a water bath to a temperature of approximately 48 C 1 C, portions of approximately
46、 20 ml into each Petri dish (6.10) under sterile conditions and spread to give a homogeneous layer. WARNING Sodium azide is a heat sensitive ingredient and does not allow repeated liquefaction by heating! When the medium has solidified, pile quantities of four plates reversed on each other and dry a
47、t room temperature or in an incubator at 37 C 1 C for approximately 12 h or over night. Alternatively spread the plates out in a lamina flow cabinet and dry the agar surface with the lids partially removed for about 30 min. Check the dried plates for sterility. Dried plates, if correctly protected f
48、rom dehydration, may be stored for two weeks in a fridge. In this case bring the plates to room temperature about 30 min before use. 9.2 Preparation of the initial suspension and decimal dilutions Weigh 20 g 0,1 g of plain additive or premixture, or 2 g 0,01 g of a microgranule (encapsulated) additi
49、ve or 50 g 0,5 g of feed. Add 180 g 0,1 g of diluent to plain additives or premixtures, 198 g 0,1 ml to microgranule additives or 450 g 1 g to feeding stuffs. Homogenise the mixture with a suitable homogeniser such as a blender. NOTE 1 It is recommended to check the pH of the initial suspension and to correct it to a range of pH 7,3 8,1. The numbers of capsules should be more than 25, to obtain a SD (Standard Deviation) of less than 20% for a good repeat of analysis. Blend additives and prem
