1、Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate testBS EN 16936:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.in
2、dd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16936 May 2017 ICS 65.120 English Version Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compo
3、und feed by a microbiological plate test Aliments pour animaux : Mthodes dchantillonnage et danalyse - Dpistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine des niveaux sous-additifs dans les aliments composs par essai sur plaque microbiologique Futtermitte
4、l - Probenahme- und Untersuchungsverfahren - Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels mikrobiologischem Plattentest This European Standard was approved by CEN on 6 Febr
5、uary 2017. 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 references concerning such national standards may be ob
6、tained on application to the CEN-CENELEC 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 member into its own language and notified to the
7、CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Irela
8、nd, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Managem
9、ent Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16936:2017 ENational forewordThis British Standard is the UK implementation of EN 16936:2017.The UK participation in its preparation
10、 was entrusted to Technical Committee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct applicat
11、ion. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 91796 7ICS 65.120Compliance with a British Standard cannot confer immunity from legal obligations. This British Standard was published under the authority of the Standards Policy and Strategy Committee
12、on 30 June 2017.Amendments/corrigenda issued since publicationDate Text affected BRITISH STANDARDBS EN 16936:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16936 May 2017 ICS 65.120 English Version Animal feeding stuffs: Methods of sampling and analysis - Screening on the antibiotics tylos
13、in, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in compound feed by a microbiological plate test Aliments pour animaux : Mthodes dchantillonnage et danalyse - Dpistage des antibiotiques tylosine, virginiamycine, spiramycine, bacitracine-zinc et avoparcine des nive
14、aux sous-additifs dans les aliments composs par essai sur plaque microbiologique Futtermittel - Probenahme- und Untersuchungsverfahren - Screening auf die Antibiotika Tylosin, Virginiamycin, Spiramycin, Bacitracin-Zink und Avoparcin in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermittel
15、n mittels mikrobiologischem Plattentest This European Standard was approved by CEN on 6 February 2017. 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.
16、Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 tra
17、nslation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finl
18、and, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STAND
19、ARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16936:2017 EBS EN 16936:2017EN 16936:201
20、7 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Normative references 4 3 Principle . 4 4 Reagents and materials . 4 5 Apparatus . 8 6 Sample preparation 8 6.1 Preparation of the test plates . 8 6.2 Sample extraction . 9 7 Measurements 9 7.1 Procedure. 9 7.2 Interpretation . 9 7.3 Identificat
21、ion 9 8 Determination of concentrations . 10 9 Precision 10 9.1 Interlaboratory study . 10 10 Test report 11 Annex A (informative) Preparation of bacterial suspensions 12 A.1 General . 12 A.2 Storage . 12 Annex B (informative) Procedure for the additional identification of interfering antibiotics .
22、13 B.1 General . 13 B.2 Tetracyclines . 13 B.3 Quinolones/colistin . 13 B.4 Aminoglycosides . 13 B.5 Avilamycin 14 B.6 Macrolides / -lactams 14 B.7 Mixture of antibiotics . 14 Bibliography . 16 BS EN 16936:2017EN 16936:2017 (E) 3 European foreword This document (EN 16936:2017) has been prepared by T
23、echnical Committee CEN/TC 327 “Animal feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2017, and
24、 conflicting national standards shall be withdrawn at the latest by November 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. This document has
25、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 organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
26、 Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
27、 Turkey and the United Kingdom. BS EN 16936:2017EN 16936:2017 (E) 4 1 Scope This European Standard presents a method describing the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a
28、 microbiological 3-plate test. The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin, and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method. Furthermore, high concentrations of metals (Cu, Zn) may
29、interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC; for confirmatory purposes LC-MS is required 1. A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house val
30、idation first. 2 Normative references The following documents, in whole or in part, are normatively 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 (incl
31、uding any amendments) applies. EN ISO 13969, Milk and milk products - Guidelines for a standardized description of microbial inhibitor tests (ISO 13969) 3 Principle The feed sample is extracted with a mixture of acetone, hydrochloric acid (HCl) and water. Neutralized extract is dispensed into wells
32、in three different test plates. Each of these test plates holds a different composition with respect to culture medium, indicator bacterium and/or pH. After a 16-18 h incubation period, the presence of antibiotic residues is indicated by the appearance of a zone of growth inhibition around the sampl
33、e. Comparison of the inhibition pattern with a reference set (Table 1) may yield a presumptive identification of the antibiotic. 4 Reagents and materials WARNING The use of this protocol involves hazardous materials, operations and equipment, This protocol does not purport to address all the safety
34、problems associated with its use. It is the responsibility of the user of this protocol to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 4.1 Test organisms: Kocuria rhizophila ATCC 9341 (formerly: Micrococcus luteus) Microco
35、ccus luteus ATCC 10240 Bacillus megaterium ATCC 10778 See Annex A for the preparation of the bacterial suspensions. 4.2 Culture media: In order to improve the reproducibility of the method, it is recommended to use dehydrated basic components or dehydrated complete media for the preparation of cultu
36、re media. Follow the manufacturers instructions. BS EN 16936:2017EN 16936:2017 (E) 5 4.2.1 Culture medium for Kocuria rhizophila ATCC 9341. Meat peptone 6 g Tryptone 4 g Yeast extract 3 g Meat extract 1,5 g Glucose 1 g Agar 15 g Dipotassium hydrogen phosphate (K2HPO4) 20 g (to be added after sterili
37、zation) Demineralized water 1000 ml The basic dehydrated medium is commercially available as Antibiotic medium No. 1. Dissolve the components in the water by heating. Autoclave the medium at 121 C 1 C for 15 min. Adjust the pH to 8,0 0,1. 4.2.2 Culture medium for Micrococcus luteus ATCC 10240. Meat
38、peptone 6 g Tryptone 4 g Yeast extract 3 g Meat extract 1,5 g Glucose 1 g Agar 15 g Demineralized water 1000 ml The basic dehydrated medium is commercially available as Antibiotic medium No. 1. Dissolve the components in the water by heating. Autoclave the medium at 121 C 1 C for 15 min. Adjust the
39、pH to 6,5 0,1. 4.2.3 Culture medium for Bacillus megaterium ATCC 10778. Pancreatic digest of casein 5 g Yeast extract 2,5 g Glucose 1 g Agar 15 g Demineralized water 1000 ml The basic dehydrated medium is commercially available as Plate Count Agar. Dissolve the components in the water by heating. Au
40、toclave the medium at 121 C 1 C for 15 min. Adjust the pH to 6,0 0,1. 4.3 Water, demineralized or deionized or at least equivalent. 4.4 Sodium chloride (NaCl). BS EN 16936:2017EN 16936:2017 (E) 6 4.5 Acetone, analytical grade. 4.6 Hydrochloric acid (HCI). 4.7 Dipotassium hydrogen phosphate (K2HPO4).
41、 4.8 Potassium dihydrogen phosphate (KH2PO4). 4.9 Sodium hydroxide (NaOH). 4.10 Methanol, analytical grade. 4.11 Extraction solvent: acetone/ hydrochloric acid/water mixture (475/25/500 v/v/v): Transfer 500 ml of water (4.3) into a 1000 ml volumetric flask, ad 25 ml of hydrochloric acid (4.6) and mi
42、x. Make up to 1000 ml with acetone (4.5). 4.12 Phosphate buffer pH 6,5: Dissolve 18,6 g potassium dihydrogen phosphate (4.8) and 14,8 g dipotassium hydrogen phosphate (4.7) in water (4.3). Adjust the pH to 6,5 0,1 and fill up to 1000 ml with water (4.3). 4.13 Hydrochloric acid solution: 0,1 M: Trans
43、fer 500 ml of water (4.3) into a 1000 ml volumetric flask. Slowly add 8,212 ml hydrochloric acid (4.6). Make up to a final volume of 1000 ml with water (4.3). 4.14 Standard solvent: Transfer 400 ml of 0,1 M hydrochloric acid (4.13) into a 1000 ml volumetric flask, ad 600 ml of acetone (4.5) and mix.
44、 4.15 Standard solutions and control samples: Correct all weighing for purity and salt contents in accordance with EN ISO 13969. 4.15.1 Neomycin standard stock solution. Dissolve 50,0 mg 0,1 mg of neomycin in 100 ml water (4.3) and mix. The thus-prepared neomycin standard stock solution contains 500
45、 g/ml of neomycin. The neomycin standard stock solution may be kept for one month if stored at 0 C to + 5 C. 4.15.2 Tylosin solutions and control samples: 4.15.2.1 Tylosin standard stock solution: Dissolve 50,0 mg 0,1 mg of tylosin in 100 ml water (4.3) and mix. The thus-prepared tylosin standard st
46、ock solution contains 500 g/ml of tylosin. The tylosin standard stock solution may be kept for one month if stored at 0 C to + 5 C. 4.15.2.2 Tylosin working solution: Dilute 2 ml of the tylosin standard stock solution (4.15.2.1) with water (4.3) to 100 ml and mix. The thus-prepared tylosin working s
47、olution contains 1 g/ml of tylosin. BS EN 16936:2017EN 16936:2017 (E) 7 4.15.2.3 Tylosin control sample: Weigh 10,0 g of feed sample and spike with 0,2 ml of the tylosin standard stock solution (4.15.2.1). Mix and leave at room temperature for 30 min, before starting the analysis. This positive cont
48、rol sample contains 1 mg/kg of tylosin. 4.15.3 Avoparcin standard stock solution and control samples: 4.15.3.1 Avoparcin standard stock solution: Dissolve 5,0 mg 0,1 mg of avoparcin in standard solvent (4.14), and fill up to 100 ml. The thus-prepared avoparcin standard stock solution contains 50 g/m
49、l of avoparcin. The avoparcin standard stock solution may be kept for one month if stored at 0 C to + 5 C. 4.15.3.2 Avoparcin control sample: Weigh 10,0 g of feed sample and spike with 0,2 ml of the avoparcin standard stock solution (4.15.3.1). Mix and leave at room temperature for 30 min, before starting the analysis. This positive control sample contains 1 mg/kg of avoparcin. 4.15.4 Spiramycin standard stock solution and control samples: 4.15.4.1 Spiramycin standard stock solution: Dissolve 5,0 mg 0,1 mg of spiram
