1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 10932:2010Milk and milk products Determination of the minimalinhibitory concentration(MIC) of antibiotics applicableto bifidobacteria and non-enterococcal lactic acid bact
2、eria(LAB)BS ISO 10932:2010 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 10932:2010.The UK participation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this commi
3、ttee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 60097 5ICS 67.100.01Compliance with a British Standard cannot confer immunity fromlegal obl
4、igations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 July 2010Amendments issued since publicationDate Text affectedBS ISO 10932:2010Reference numbersISO 10932:2010(E)IDF 223:2010(E)ISO and IDF 2010INTERNATIONAL STANDARD ISO10932IDF223Fi
5、rst edition2010-06-15Milk and milk products Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB) Lait et produits laitiers Dtermination de la concentration minimale inhibitrice (CMI) dantibiotiques app
6、licable aux bifidobacteria et bactries lactiques non-entrocoques BS ISO 10932:2010ISO 10932:2010(E) IDF 223:2010(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces
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10、ced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Bo
11、ulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2010 All rights reservedBS ISO 10932:2010ISO 10
12、932:2010(E) IDF 223:2010(E) ISO and IDF 2010 All rights reserved iiiContents Page Foreword iv Introductionvi 1 Scope1 2 Normative references1 3 Terms and definitions .2 4 Principle .2 5 Diluents, culture media and reagents .2 5.1 Basic materials 2 5.2 Diluents 2 5.3 Culture media.3 6 Apparatus and g
13、lassware.10 7 Sampling 11 8 Procedure.11 8.1 Propagation11 8.2 Quality control strains and testing12 8.3 Growth conditions for antibiotic susceptibility test 12 8.4 Preparation of microdilution plate.13 9 Expression of results18 10 Precision 18 10.1 Interlaboratory test18 10.2 Repeatability 18 10.3
14、Reproducibility 18 11 Test report18 Annex A (informative) Interlaboratory trial.19 Bibliography30 BS ISO 10932:2010ISO 10932:2010(E) IDF 223:2010(E) iv ISO and IDF 2010 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standard
15、s bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organiz
16、ations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in
17、the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % o
18、f the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 10932IDF 223 was prepared by Technical Committee ISO/TC 34, F
19、ood products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. BS ISO 10932:2010ISO 10932:2010(E) IDF 223:2010(E) ISO and IDF 2010 All rights reserved vForeword IDF (the International Dairy Federation) is a non-pro
20、fit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well as regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing Comm
21、ittees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. The main task of Standing Committees is to prepare International Standards. Draft International Standards adopted by the Standing Committees a
22、re circulated to the National Committees for endorsement prior to publication as an International Standard. Publication as an International Standard requires approval by at least 50 % of IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this d
23、ocument may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 10932IDF 223 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is b
24、eing published jointly by IDF and ISO. All work was carried out by a Joint ISO-IDF Project Group on Minimal inhibitory concentration (MIC) of antibiotics of the Standing Committee on Analytical methods for dairy microorganisms under the aegis of its project leader, Mr. M. Danielsen (DK). BS ISO 1093
25、2:2010ISO 10932:2010(E) IDF 223:2010(E) vi ISO and IDF 2010 All rights reservedIntroduction There are several reports on minimal inhibitory concentration (MIC) determination of lactic acid bacteria according to various methods. However, the MIC value obtained depends on the determination used and th
26、e strain cultivation technique. For example, MIC determined by different quantitative methods are not always equivalent. Also some media components are antagonistic to certain antibiotics. Consequently, a standardized MIC determination which employs a suitable growth medium having little or no antag
27、onistic effects towards the antibiotics studied is necessary. Two EU projects (PROSAFE and ACE-ART) were launched to tackle these issues, and propose appropriate media and method to measure MIC. This International Standard is based on the SOP (standard operating procedure) proposed by ACE-ART. BS IS
28、O 10932:2010INTERNATIONAL STANDARD ISO 10932:2010(E)IDF 223:2010(E) ISO and IDF 2010 All rights reserved 1Milk and milk products Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB) WARNING Antibiotics
29、 are substances that may be hazardous. Necessary precautions should be taken to avoid contact with these substances. In particular, kanamycin may cause harm to the unborn child (risk phrase R61) and chloramphenicol may cause cancer (risk phrase R45). 1 Scope This International Standard specifies a m
30、ethod for determining the minimal inhibitory concentration (MIC) of a series of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB). NOTE Unlike the disk diffusion method, which is semi-quantitative, the frequently used broth microdilution method gives quantitati
31、ve MICs of the test organism in a dilution series of the antibiotics. The lowest concentration of an antibiotic that prevents visible growth of a test organism is considered to be the MIC. This International Standard recommends the broth microdilution method as the standard method. 2 Normative refer
32、ences 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 the referenced document (including any amendments) applies. ISO 6887-5, Microbiology of food and animal
33、 feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 5: Specific rules for the preparation of milk and milk products ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological ex
34、aminations ISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory ISO/TS 11133-2, Microbiology of food and animal feeding stuffs Gu
35、idelines on preparation and production of culture media Part 2: Practical guidelines on performance testing of culture media BS ISO 10932:2010ISO 10932:2010(E) IDF 223:2010(E) 2 ISO and IDF 2010 All rights reserved3 Terms and definitions For the purposes of this document, the following terms and def
36、initions apply. 3.1 minimal inhibitory concentration MIC lowest concentration that, under defined in vitro conditions, prevents visible growth of bacteria within a defined period of time ISO 20776-1:20066, 2.4 NOTE MIC is expressed in micrograms per millilitre. 4 Principle Most individual colonies f
37、rom an agar plate are picked up and suspended in sterile saline. However, Bifidobacterium spp. are suspended in pre-reduced LSM-Cys medium. The bacterial suspension is diluted with recommended medium. The microdilution plate is prepared with a series of twofold dilutions of antibiotic. The diluted b
38、acterial suspension is distributed into the wells of the plate and incubated under recommended conditions. The lowest concentration of an antibiotic that prevents visible growth is considered to be the MIC. 5 Diluents, culture media and reagents 5.1 Basic materials Use only reagents of recognized an
39、alytical grade, unless otherwise specified, and sterile distilled or demineralized water or water of equivalent purity. See ISO 6887-5. 5.2 Diluents See ISO 6887-5. BS ISO 10932:2010ISO 10932:2010(E) IDF 223:2010(E) ISO and IDF 2010 All rights reserved 35.3 Culture media 5.3.1 MRS agar 5.3.1.1 Compo
40、sition Peptone 1 (tryptic digest of casein) 10,0 g Meat extract 10,0 g Yeast extract (dried) 5,0 g Glucose 20,0 g Polysorbate 80 (polyethoxylated sorbitan mono-oleate)a1,0 ml Dipotassium hydrogen phosphate (K2HPO4) 2,0 g Sodium acetate trihydrate (NaCH3CO23H2O) 5,0 Diammonium citrate (NH4)2HC6H5O7 2
41、,0 g Magnesium sulfate heptahydrate (MgSO47H2O) 0,2 Manganese sulfate tetrahydrate (MnSO44H2O) 0,05 g Agar 10 g to 15 gbWater up to 1 000 mlcaTween 80 is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard, an
42、d does not constitute an endorsement by ISO or IDF of this product. bDepending on the gel strength of the agar. cWhen using hand-made microdilution plates (8.4.5.1), the MRS medium should be prepared at twice the higher concentration by only adding water up to 500 ml.5.3.1.2 Preparation Suspend the
43、ingredients in the water. Heat the suspension to boiling with frequent agitation until complete dissolution. If needed, adjust the pH (6.7) to 6,35 0,2 with dilute hydrochloric acid or dilute sodium hydroxide before autoclaving. After autoclaving, the pH range of the MRS agar medium should be 6,2 0,
44、2 at 25 C. Distribute the medium in portions of 100 ml 1 ml into bottles (6.8) of capacity 150 ml or in portions of 200 ml 2 ml into bottles (6.8) of capacity 250 ml. Sterilize in the autoclave (6.5) maintained at 121 C for 15 min. If the medium is to be used immediately, cool it in a water bath (6.
45、6) to between 44 C and 47 C. If not used immediately, melt the MRS agar (5.3.1.1) in a boiling water bath (6.6) and mix carefully to avoid gas bubbles, then cool it in a water bath (6.6) to between 44 C and 47 C. Pour 15 ml to 20 ml of prepared medium into Petri dishes (6.10). Allow the medium to co
46、ol. Solidify by placing the Petri dishes with the lids in place on a cool horizontal surface. Before use, dry the agar surface in accordance with ISO/TS 11133-1. The prepared MRS agar plates may be stored in an airtight plastic bag in the dark and held between 2 C and 8 C for up to 2 weeks. Test aga
47、r plates for microbial contamination in accordance with ISO/TS 11133-2. The complete MRS agar is commercially available, but the results obtained may differ significantly from one supplier to another. If used, therefore, check the commercial MRS agar against the same medium prepared in accordance wi
48、th this International Standard. BS ISO 10932:2010ISO 10932:2010(E) IDF 223:2010(E) 4 ISO and IDF 2010 All rights reserved5.3.2 MRS-cysteine agar (MRS-Cys agar) MRS-Cys agar consists of MRS agar (5.3.1) with addition of 0,3 g of L-cysteine per litre of medium. 5.3.2.1 Basic medium MRS agar See 5.3.1.
49、 5.3.2.2 L-Cysteine stock solution 5.3.2.2.1 Composition L-Cysteine hydrochloride 0,3 g Water up to 10,0 ml 5.3.2.2.2 Preparation Dissolve the L-cysteine hydrochloride in the water. Sterilize through a 0,2 m filter (6.12) into a sterile test tube (6.13). The L-cysteine stock solution may be stored in the dark and held between 2 C and 8 C for up to 1 week. Do not expose the solution to direct sunlight. 5.3.2.3 Complete medium 5.3.2.3.1 Composition Basic medium (5.3.1) 100 ml L-Cysteine
