1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58Lactobacillus acidophilus on a selective medium Colony-count technique at 37 CICS 07.100.30; 67.100
2、.10Milk products Enumeration of presumptive BRITISH STANDARDBS ISO 20128:2006BS ISO 20128:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 29 June 2007 BSI 2007ISBN 978 0 580 50874 5Amendments issued since publicationAmd. No. Date Comments
3、contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.National forewordThis British Standard was published by BSI. It is the UK implementation of ISO 20128:2006. The UK participation in its preparation was entrust
4、ed to Technical Committee AW/5, Chemical analysis of milk and milk products.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 Reference numbersISO 20128:2006(E)IDF 192:2006(
5、E)ISO and IDF 2006INTERNATIONAL STANDARD ISO20128IDF192First edition2006-05-15Milk products Enumeration of presumptive Lactobacillus acidophilus on a selective medium Colony-count technique at 37 C Produits laitiers Dnombrement de Lactobacillus acidophilus prsomptifs sur un milieu slectif Technique
6、de comptage des colonies 37 C BS ISO 20128:2006ii iiiContents 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 glas
7、sware .4 7 Sampling.5 8 Procedure .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 Re
8、peatability.7 10.3 Reproducibility.8 11 Test report 9 Annex A (informative) Interlaboratory test .10 Bibliography 11 BS ISO 20128:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing
9、 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 and non-governmental, in liais
10、on 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, Part 2. The main task of te
11、chnical 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 casting a vote. Attention is dr
12、awn 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, Subcommittee SC 5, Milk and milk pro
13、ducts, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. BS ISO 20128:2006vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the rig
14、ht 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 International Standards adopted by the Action Teams and Standing Committees are circulated
15、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 elements of this document may be the subject of patent rights. IDF shall not be held re
16、sponsible 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 products. It is being published jointly by IDF and ISO. All work was carried out by t
17、he 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). BS ISO 20128:2006vi Introduction Because of the large variety of fermented and non-fermented milks, this m
18、ethod 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 lower than the number of other microorganisms such as Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillu
19、s helveticus and yeasts. BS ISO 20128:20061Milk products Enumeration of presumptive Lactobacillus 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
20、 selective medium by using a colony-count technique at 37 C. The method is applicable 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
21、 when the number of presumptive L. acidophilus is less than 104CFU/g and the numbers of Lactobacillus rhamnosus, Lactobacillus reuteri and Lactobacillus paracasei subsp. paracasei are greater than 106 CFU/g. 2 Normative references The following referenced documents are indispensable for the applicat
22、ion 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 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations ISO 8261|IDF 1
23、22, Milk and milk products General guidance for the preparation of test samples, initial 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 m
24、icroorganism forming flat, mat, rough, grey to whitish colonies with more or less irregular 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 rel
25、ated to Lactobacillus johnsonii, Lactobacillus gasseri and Lactobacillus crispatus. The method specified in this International Standard cannot distinguish between these four species and, therefore, only presumptive L. acidophilus is mentioned. BS ISO 20128:20062 4 Principle 4.1 The antibiotics clind
26、amycin and ciprofloxacin both inhibit 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, L
27、actobacillus casei, Lactobacillus 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
28、 of clindamycin and ciprofloxacin. 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 giv
29、e a significant result. 5 Diluents, 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
30、 MRS/clindamycin/ciprofloxacin 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). BS ISO 20128:200635.3.2 Basic medium: MRS agar 5.3.2.1 Composition Peptone 1 (enzymatic digest o
31、f 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 (K2HPO4) 2,0 g Sodium acetate trihydrate (NaCH3CO23H2O) 5,0 g Triammonium citrate (NH4)3HC6H5O7) 2,0 g Magnesium sulfate heptahydrate (MgSO47H2O) 0,2
32、g Manganese sulfate tetrahydrate (MnSO44H2O) 0,05 g Agar 12 g to 18 gaWater 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 a complete solution is obtained. Distribute the medi
33、um 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) set at 121 C for 15 min. If the medium is to be used i
34、mmediately, 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 but the results obtained may differ significantly fro
35、m 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 mgWater up to 10,0 ml5.3.3.2 Preparation Dissolve the clindamycin hydrochloride in the water. Filter the solution then sterilize through a 0,22 m
36、 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. BS ISO 20128:20064 5.3.4 Ciprofloxacin stock solution 5.3.4.1 Composition Ciproflo
37、xacin hydrochloride 20,0 mgDistilled water up to 10,0 ml5.3.4.2 Preparation Dissolve the ciprofloxacin hydrochloride in the water and sterilize by filtration through a 0,22 m filter (6.13) into a sterile test tube (6.14). If the solution is not to be used immediately then keep it at 20 C. The frozen
38、 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 mlClindamycin stock solution (5.3.3) 0,05 ml Clindamycin stock solution (5.3.3) 0,1 mlCiprofloxacin stock solution (5.3.4) 0,5 ml Ciprofloxacin stock
39、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 the clindamycin stock solution (5.3.3) and 0,5 ml of ciprofloxacin stock solution (5.3.4) to 100 ml of MRS agar
40、 (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. Pour the medium into Petri dishes (6.11) with 12 ml to 15 ml in each. Allow the medium to cool and solidify by pla
41、cing 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 surface according to ISO 7218. 6 Apparatus and glassware Usual microbiological laboratory equipment required for the
42、 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 sample, the diluent, the dilutions or the culture medium, as specified in ISO 7218. The glassware shall be resistant
43、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 atmosphere of approx. 9 % to 13 % carbon dioxide. BS ISO 20128:200656.4 Blender, either a peristaltic-type blender (St
44、omacher) with sterile plastic bags or a rotary blender, capable of operating at a minimum rotational frequency of 20 000 min1, with 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 magnif
45、ying lens to be used at a magnification of 1,5 and a mechanical or electronic digital counter. 6.6 Autoclave, capable of operating 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
46、 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 Pe
47、tri dishes, sterile, made of clear colourless glass or plastic, of diameter 90 mm, of internal depth 10 mm minimum. The bottom shall have no irregularities that may interfere with counting colonies. 6.12 Spatula, sterile, made of glass or metal. 6.13 Filter, sterile, with cellulose acetate membrane
48、of 0,22 m pore size. 6.14 Test tubes, sterile, of capacity 20 ml, with suitable sealing caps. 6.15 Spreader, sterile, made of glass or metal. 6.16 Drying cabinet, see ISO 7218. 6.17 Cryotubes, sterile, of capacity 2 ml. 7 Sampling A representative sample should have been sent to the laboratory. It s
49、hould not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707|IDF 50. 8 Procedure 8.1 Sample preparation and decimal dilutions Prepare the test samples, test portion, initial suspension and further dilutions in accordance with ISO 8261|IDF 122. The samples shall be plated in a minimum of three serial dilutions in order to verify growth o
