1、 Reference numbers ISO 20128:2006(E) IDF 192:2006(E) ISO and IDF 2006INTERNATIONAL STANDARD ISO 20128 IDF 192 First edition 2006-05-15 Milk products Enumeration of presumptive Lactobacillus acidophilus on a selective medium Colony-count technique at 37 C Produits laitiers Dnombrement de Lactobacillu
2、s acidophilus prsomptifs sur un milieu slectif Technique de comptage des colonies 37 C ISO 20128:2006(E) IDF 192:2006(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 type
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7、0 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 2006 All rights reservedISO 20128:2006(E) IDF 192:2006(E) ISO and IDF 2
8、006 All rights reserved iii Contents Page Foreword iv Foreword. v Introduction . vi 1 Scope 1 2 Normative references 1 3 Terms and definitions .1 4 Principle2 5 Diluents, culture media and reagents2 5.1 Basic materials.2 5.2 Diluent.2 5.3 Culture media.2 6 Apparatus and glassware .4 7 Sampling.5 8 P
9、rocedure .5 8.1 Sample preparation and decimal dilutions .5 8.2 Inoculation and incubation .6 8.3 Enumeration of colonies.6 9 Calculation and expression of results.6 9.1 Calculation6 9.2 Expression of results 6 9.3 Example 7 10 Precision.7 10.1 Interlaboratory test 7 10.2 Repeatability.7 10.3 Reprod
10、ucibility.8 11 Test report 9 Annex A (informative) Interlaboratory test .10 Bibliography 11 ISO 20128:2006(E) IDF 192:2006(E) iv ISO and IDF 2006 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bo
11、dies). 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 organizations, governmental an
12、d 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 the ISO/IEC Directives,
13、 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 % of the member bodies cas
14、ting 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 20128|IDF 192 was prepared by Technical Committee ISO/TC 34, Food products, Subcommi
15、ttee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. ISO 20128:2006(E) IDF 192:2006(E) ISO and IDF 2006 All rights reserved v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with
16、 a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft Internat
17、ional Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. 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 elem
18、ents of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 20128|IDF 192 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk pr
19、oducts. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team on Lactic acid bacteria and starters, of the Standing Committee on Microbiological methods of analysis, under the aegis of its project leader, Mrs D. Ellekaer (DK). ISO 20128:2006(E) IDF 1
20、92:2006(E) vi ISO and IDF 2006 All rights reservedIntroduction Because of the large variety of fermented and non-fermented milks, this method may not be appropriate in every detail for certain products. This could be the case where the number of presumptive Lactobacillus acidophilus is very much low
21、er than the number of other microorganisms such as Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus helveticus and yeasts. INTERNATIONAL STANDARD ISO 20128:2006(E) IDF 192:2006(E) ISO and IDF 2006 All rights reserved 1 Milk products Enumeration of presumptive La
22、ctobacillus acidophilus on a selective medium Colony-count technique at 37 C 1 Scope This International Standard specifies a method for the enumeration of presumptive Lactobacillus acidophilus in milk products on a selective medium by using a colony-count technique at 37 C. The method is applicable
23、to fermented and non-fermented milks, milk powders and infant formulae where presumptive L. acidophilus is present and in combination with other lactic acid bacteria and bifidobacteria. The method is not applicable when the number of presumptive L. acidophilus is less than 10 4CFU/g and the numbers
24、of Lactobacillus rhamnosus, Lactobacillus reuteri and Lactobacillus paracasei subsp. paracasei are greater than 10 6 CFU/g. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For unda
25、ted references, the latest edition of the referenced document (including any amendments) applies. ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 8261|IDF 122, Milk and milk products General guidance for the preparation of test samples, ini
26、tial suspensions and decimal dilutions for microbiological examination 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 presumptive Lactobacillus acidophilus microorganism forming flat, mat, rough, grey to whitish colonies with more or less ir
27、regular edges and a diameter of 1 mm to 3 mm depending on the number of colonies when grown on a solid selective medium under the conditions specified in this International Standard NOTE L. acidophilus is closely related to Lactobacillus johnsonii, Lactobacillus gasseri and Lactobacillus crispatus.
28、The method specified in this International Standard cannot distinguish between these four species and, therefore, only presumptive L. acidophilus is mentioned. ISO 20128:2006(E) IDF 192:2006(E) 2 ISO and IDF 2006 All rights reserved4 Principle 4.1 The antibiotics clindamycin and ciprofloxacin both i
29、nhibit the growth of the most common microorganisms used in fermented milks, non-fermented milks and infant formulae, such as Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, bifidobacteria, lactococci, Lactobacillus casei, Lactobacill
30、us paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri and Leuconostoc species. 4.2 A known amount of sample is homogenized with diluent and decimal dilutions are prepared. 4.3 Appropriate dilutions are spread plated on MRS-agar with the addition of clindamycin and ciprofloxac
31、in. 4.4 The plates are incubated anaerobically at 37 C for 72 h 3 h. 4.5 Typical colonies are counted. 4.6 The number of characteristic microorganisms per gram of sample is calculated from the number of colonies obtained on plates chosen at dilution levels so as to give a significant result. 5 Dilue
32、nts, culture media and reagents 5.1 Basic materials Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. See ISO 7218. 5.2 Diluent See ISO 8261|IDF 122. 5.3 Culture media 5.3.1 MRS/clindamycin/ciprofloxacin
33、agar (MRS/CL/CIP agar) MRS/CL/CIP agar consists of MRS agar (5.3.2) with the addition of 0,1 mg of clindamycin and 10,0 mg of ciprofloxacin per litre of medium (see 5.3.4). ISO 20128:2006(E) IDF 192:2006(E) ISO and IDF 2006 All rights reserved 3 5.3.2 Basic medium: MRS agar 5.3.2.1 Composition Pepto
34、ne 1 (enzymatic digest of casein) 10,0 g Meat extract 10,0 g Yeast extract (dried) 5,0 g Glucose 20,0 g Tween 80 (sorbitan mono-oleate) 1,0 ml Dipotassium hydrogen phosphate (K 2 HPO 4 ) 2,0 g Sodium acetate trihydrate (NaCH 3 CO 2 3H 2 O) 5,0 g Triammonium citrate (NH 4 ) 3 HC 6 H 5 O 7 ) 2,0 g Mag
35、nesium sulfate heptahydrate (MgSO 4 7H 2 O) 0,2 g Manganese sulfate tetrahydrate (MnSO 4 4H 2 O) 0,05 g Agar 12 g to 18 g aWater 1 000 ml aDepending on the gel strength of the agar. 5.3.2.2 Preparation Suspend the ingredients in the water. Heat the suspension to boiling with frequent agitation until
36、 a complete solution is obtained. Distribute the medium in portions of 100 ml 1 ml into bottles (6.9) of 150 ml capacity or in portions of 200 ml 2 ml into bottles (6.9) of 250 ml capacity. If needed, adjust the pH (6.8) so that, after sterilization, it is 6,2 0,2. Sterilize in the autoclave (6.6) s
37、et at 121 C for 15 min. If the medium is to be used immediately, cool it in a water bath (6.7) to between 44 C and 47 C. Do not expose the medium to direct sunlight. The thus-prepared MRS agar may be stored in the dark at 1 C to 5 C for 6 months. NOTE The complete MRS agar is commercially available
38、but the results obtained may differ significantly from one supplier to another (See also ISO/TS 11133-1 and ISO/TS 11133-2.) 5.3.3 Clindamycin stock solution 5.3.3.1 Composition Clindamycin hydrochloride 2,0 mg Water up to 10,0 ml 5.3.3.2 Preparation Dissolve the clindamycin hydrochloride in the wat
39、er. Filter the solution then sterilize through a 0,22 m filter (6.13) into a sterile test tube (6.14). If the solution is not to be used immediately, distribute it in small sterile cryotubes (6.17) and keep the tubes at 20 C. The frozen solution may be stored for 6 weeks. ISO 20128:2006(E) IDF 192:2
40、006(E) 4 ISO and IDF 2006 All rights reserved5.3.4 Ciprofloxacin stock solution 5.3.4.1 Composition Ciprofloxacin hydrochloride 20,0 mg Distilled water up to 10,0 ml 5.3.4.2 Preparation Dissolve the ciprofloxacin hydrochloride in the water and sterilize by filtration through a 0,22 m filter (6.13) i
41、nto a sterile test tube (6.14). If the solution is not to be used immediately then keep it at 20 C. The frozen solution may be stored for 8 weeks. 5.3.5 Complete medium: Preparation of plates 5.3.5.1 Composition MRS agar (5.3.2) 100 ml or MRS agar (5.3.2) 200 ml Clindamycin stock solution (5.3.3) 0,
42、05 ml Clindamycin stock solution (5.3.3) 0,1 ml Ciprofloxacin stock solution (5.3.4) 0,5 ml Ciprofloxacin stock solution (5.3.4) 1,0 ml5.3.5.2 Preparation Immediately before use, melt the MRS agar (5.3.2) in a boiling water bath (6.7). Cool it in a water bath to between 44 C and 47 C. Add 0,05 ml of
43、 the clindamycin stock solution (5.3.3) and 0,5 ml of ciprofloxacin stock solution (5.3.4) to 100 ml of MRS agar (5.3.2), or add 0,1 ml of clindamycin stock solution (5.3.3) and 1,0 ml of ciprofloxacin stock solution (5.3.4) to 200 ml of MRS agar (5.3.2) and mix very carefully. Avoid gas bubbles. Po
44、ur the medium into Petri dishes (6.11) with 12 ml to 15 ml in each. Allow the medium to cool and solidify by placing the Petri dishes with the lids in place on a cool horizontal surface. The prepared MRS/CL/CIP plates may be stored in the dark at 4 C to 7 C for 10 days. Before use, dry the agar surf
45、ace according to ISO 7218. 6 Apparatus and glassware Usual microbiological laboratory equipment required for the preparation of test samples and dilutions as specified in ISO 7218 and, in particular, the following. 6.1 Glassware Sterilize all equipment that will come into contact with the test sampl
46、e, the diluent, the dilutions or the culture medium, as specified in ISO 7218. The glassware shall be resistant to repeated sterilization. 6.2 Incubator, capable of operating at 37 C 1 C. 6.3 Anaerobic incubator, capable of being controlled at 37 C 1 C, or anaerobic culture jars, providing an atmosp
47、here of approx. 9 % to 13 % carbon dioxide. ISO 20128:2006(E) IDF 192:2006(E) ISO and IDF 2006 All rights reserved 5 6.4 Blender, either a peristaltic-type blender (Stomacher) with sterile plastic bags or a rotary blender, capable of operating at a minimum rotational frequency of 20 000 min 1 , with
48、 sterile glass or metal containers of appropriate capacity. 6.5 Colony-counting equipment, consisting of an illuminated base with a dark background, fitted with a magnifying lens to be used at a magnification of 1,5 and a mechanical or electronic digital counter. 6.6 Autoclave, capable of operating
49、at 121 C 1 C. 6.7 Water baths, capable of maintaining a temperature of between 44 C and 47 C and of boiling. 6.8 pH meter, with temperature compensation, accurate to 0,1 pH unit at 25 C. 6.9 Bottles or flasks, of capacity 150 ml or 250 ml, and with suitable sealing caps or stoppers (to hold the culture medium). 6.10 Pipettes, sterile, calibrated for bacteriological use, capable of delivering 0,05 ml 0,002 ml, 0,1 ml 0,02 ml, 1,0 ml 0,02 ml and 10 ml 0,2 ml. 6.11
