1、BS EN 15786:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feedingstuffs Isolationand enumeration ofPediococcus spp.This British Standardwas published underthe authority of theStandards Policy andStrategy Committee on 30September 2009 BSI 2
2、009ISBN 978 0 580 61802 4Amendments/corrigenda issued since publicationDate CommentsBS EN 15786:2009National forewordThis British Standard is the UK implementation of EN 15786:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of orga
3、nizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations
4、.BS EN 15786:2009EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15786 September 2009 ICS 65.120 English Version Animal feeding stuffs - Isolation and enumeration of Pediococcus spp. Aliments des animaux - Isolement et dnombrement du Pediococcus spp. Futtermittel - Keimzhlung von Pediococcus sp
5、p. 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 alteration. Up-to-date lists and bibliographical re
6、ferences 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 translation under the responsibility of a CEN memb
7、er 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, Germany, Greece, Hungary, Iceland, Ireland, Italy,
8、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 NORMUNG Management Centre: Avenue Marnix 17, B-1000 Br
9、ussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15786:2009: EBS EN 15786:2009EN 15786:2009 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle 6 5 Dil
10、uent, selective media and phenotypic characterisation .6 6 Apparatus and glassware .8 7 Sampling 10 8 Preparation of test sample . 10 9 Procedure 10 10 Expression of results . 12 11 Precision 12 12 Test report . 13 Annex A (informative) Notes on procedure 14 Annex B (informative) Results of the inte
11、rlaboratory study . 15 Bibliography . 17 BS EN 15786:2009EN 15786:2009 (E) 3 Foreword This document (EN 15786:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN. This European Standard shall be given the status of a national sta
12、ndard, 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 this document may be the subject of patent rights. C
13、EN 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/CENELEC Internal Regulations, the national standards or
14、ganizations 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, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
15、 Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15786:2009EN 15786:2009 (E) 4 Introduction This methodology has been developed to enumerate probiotic pediococci to enable the European Commission to control proper labelling of animal feeding products (EU project
16、 SMT4-CT98-2235 “Methods for the official control of probiotics (microorganisms) used as feed additives”) 1. The described methodology was validated in an interlaboratory study 2. BS EN 15786:2009EN 15786:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration of probioti
17、c pediococci in feed samples (additives, premixtures and feeding stuffs) that contain pediococci as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable for mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals
18、and containing at least 40% crude ash (Council Directive 79/373/EEC) 3. There are different categories of feed samples: a) Additives containing about 1010(colony forming units) CFU/g b) Premixtures containing about 108CFU/g c) Feeds, meal or pellets, which contain about 106CFU/g and include complete
19、 feeding stuffs, and milk replacers. The detection limit is as defined in EN ISO 7218. 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 latest edition of
20、 the referenced document (including any amendments) applies. EN ISO 6887-1, Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 1: General rules for the preparation of the initial suspension and
21、 decimal dilutions (ISO 6887-1:1999) EN ISO 7218, Microbiology of food and animal feeding stuffs - General requirements 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,
22、 the following terms and definitions apply. 3.1 pediococci (described by their characteristics as used for this standard) pediococci are characterised as bacteria which form colonies fitting the description of these species on the specified media after incubation of 24 h to 48 h for MRSA, AMRSA and
23、MRSA+TTC and 24 h for Pediococcus selective medium at a temperature of 37 C under anaerobic conditions 4. Morphology of colonies on agar plates: a) circular; b) convex; c) entire; BS EN 15786:2009EN 15786:2009 (E) 6 d) white; e) glistening surface; f) opaque. Colony size varies between 1 mm and 3 mm
24、 in diameter. Phase contrast microscopical examination of selected colonies shows that cells are spherical arranged in pairs or tetrads. 4 Principle a) Preparation of sterile and dry poured agar plates. b) Drawing a representative test sample under sterile conditions. c) Preparation of the initial s
25、uspension to obtain a homogeneous distribution of bacterial cells from the test portion. d) Preparation of further decimal dilutions of the initial suspension in order to reduce the number of microorganisms per unit volume, to allow, after incubation, the counting of colonies. e) Inoculation of the
26、prepared plates with an aliquot of the optimum dilutions and dispersion of the inoculum by using a sterile spreader. f) Incubation of inverted plates for 24 h to 48 h at 37 C 1 C, under anaerobic conditions. g) Counting of typical colonies, considering the specific properties of pediococci as listed
27、 above. h) Morphological verification of isolates within the Pediococcus genus through the use of microscope analysis. i) Calculation of the colony count per g or kg of feed sample. 5 Diluent, selective media and phenotypic characterisation 5.1 Diluents 5.1.1 Diluent for initial suspension This dilu
28、ent 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 disodium hydrogen phosphate, 0,2
29、 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, screw cap bottles are re
30、commended. Bring the diluent to room temperature before use. Measure the pH of the diluent to ensure the suitable buffer capacity. BS EN 15786:2009EN 15786:2009 (E) 7 5.1.2 Diluent for serial dilutions This diluent is used to decimally dilute the initial sample suspension and subsequent dilutions. P
31、eptone salt solution: A peptone salt solution is made complying with EN ISO 6887-1. 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. Dissolve the ingredients in water. Ad
32、just 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 temperature before use. 5.2 Media
33、 5.2.1 General Four different media are proposed: a) MRS agar b) MRS agar supplemented with Triphenyl Tetrazolium Chloride (TTC) c) AMRSA: Acidified MRS agar d) Selective media: MRS medium supplemented with cysteine hydrochloride, vancomycin, novobiocin and nystatin. For routine enumeration of pedio
34、cocci the use of MRS agar will be sufficient assuming that the probiotic strain is present in far higher numbers than any other microorganism. The medium is designed to encourage the growth of lactic acid bacteria such as pediococci, enterococci and lactobacilli. Selection can be made by pH adjustme
35、nt, as pediococci and lactobacilli will tolerate a lower pH than enterococci (pH 5,0 to 6,5). When enterococci are expected to be present in similar concentrations as pediococci, acidified MRS agar (AMRSA) should be used. When pediococci in combination with lactobacilli are expected, MRS agar supple
36、mented with TTC allows differentiation of colonies by different colouration after anaerobic incubation. The MRS medium supplemented with three antibiotics is selective for pediococci and should be used when the probiotic lactobacilli colony count exceeds the pediococci colony count by a factor of 50
37、 or more. 5.2.2 Composition 5.2.2.1 MRS agar The composition of the MRS agar per l of distilled water is as follows 5: 20,0 g dextrose, 10,0 g polypeptone, 10,0 g meat extract, 5,0 g yeast extract, 5,0 g sodium acetate 3xH20, 2,0 g sodium phosphate, 2,0 g tri-ammonium citrate, 1,0 g Tween 80, 0,2 g
38、magnesium sulphate 7xH2O, 0,05 g manganese sulphate 4xH2O, agar 15,0 g, pH 6,2 0,2. 5.2.2.2 MRS agar supplemented with TTC Sterilise MRS agar (5.2.2.1) by autoclaving at 121 C 1 C for 15 min. Supplement with 1 ml of a filter sterilised 1 g/100 ml water solution of Triphenyl Tetrazolium Chloride (TTC
39、) per 100 ml MRS agar. BS EN 15786:2009EN 15786:2009 (E) 8 5.2.2.3 AMRSA Acidified MRS agar can be obtained by adjusting the pH of MRS agar (see 5.2.2.1) to 5,4 0,1 with HCl prior to autoclaving. 5.2.2.4 Selective medium MRS agar supplemented with 0,05% cysteine hydrochloride. The medium is suppleme
40、nted with 10 g/ml vancomycin, 0,1 g/ml novobiocin and 50 U/ml nystatin. 5.2.3 Preparation 5.2.3.1 MRS agar Suspend all ingredients in distilled water and sterilise by autoclaving at 121 C 1 C for 15 min. 5.2.3.2 MRS agar supplemented with TTC Prepare 1 g Triphenyl Tetrazolium Chloride (TTC) in 100 m
41、l water and filter sterilise. Add 1 ml per 100 ml MRS agar medium (see 5.2.3.1) which is temperated at 48 C 1 C after autoclaving. NOTE TTC is destroyed by autoclaving. 5.2.3.3 AMRSA Adjust the pH of MRS agar with HCl to 5,4 0,1 prior to autoclaving. Sterilise at 121 C 1 C for 15 min. 5.2.3.4 Select
42、ive medium Prepare MRS medium with cysteine hydrochloride according to the manufacturers directions. Autoclave the medium for 15 min at 121 C 1 C. Melt the medium the day before use and keep at 48 C 1 C. Add the selective agents from filter-sterilised stocks to the medium immediately before pouring.
43、 Pour approximately 15 ml portions of medium into each Petri dish (6.12) under sterile conditions and spread to give a homogeneous layer. Add filter sterilised (0,45 m pore-size) selective agents, vancomycin (10 g/ml), novobiocin (0,1 g/ml), and nystatin (50 U/ml), to sterile molten media held at 48
44、C, prior to plate pouring and drying. Warning Care is needed in preparing the novobiocin stock as levels above 0,1 g/ml may be inhibitory to the probiotic pediococci component. At a concentration lower than 0,1 g/ml the probiotic lactobacilli component may not be inhibited. A stock solution of 10 g/
45、ml should be made through a series of ten-fold dilutions from an initial 1 mg/ml stock. Stack plates when the medium has solidified, and dryd in an incubator at 37 C for approximately 12 h. Check the dried plates for possible loss of sterility. 5.3 Phenotypic characterisation Characteristic appearan
46、ce of the pediococci under phase contrast microscopy is spherical cells arranged in pairs or tetrads. A test for catalase is done with a drop of hydrogen peroxide (H2O2) on a colony. The formation of bubbles is a positive reaction. Only catalase negative colonies are considered as Pediococcus. 6 App
47、aratus and glassware Usual microbiological laboratory equipment and, in particular, the following: BS EN 15786:2009EN 15786:2009 (E) 9 6.1 Equipment for dry sterilisation (oven) and wet sterilization (autoclave) According to EN ISO 7218. 6.2 Incubator Capable of maintaining a temperature of 37 C 1 C
48、. 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 litre bowl which is sterilised in an oven for 1 h at 170 C to 180 C. 6.5 Mechanical stirrer A mechanical
49、 stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent 6.6 Balance Balance capable of weighing to the nearest 0,01 g. 6.7 Microscope Capable of phase-contrast microscopy at 600x to 1 000x magnification. 6.8 Flasks or screw-cap bottles of appropriate capacities 6.9 Test tubes of appropriate capacities 6.10 Total-delivery graduated pipettes or automatic pipettes Of nominal capacity 1 ml and 100 l. With wide bore tips to pipette homogenised feed stuff for dilution. 6.11 pH meter 6.12 S
