1、 Reference number ISO 8607:2003(E) ISO 2003INTERNATIONAL STANDARD ISO 8607 First edition 2003-02-01 Artificial insemination of animals Frozen semen of breeding bulls Enumeration of living aerobic microorganisms Insmination artificielle des animaux Semences congeles de taureaux reproducteurs Dnombrem
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5、the Central Secretariat at the address given below. ISO 2003 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from eith
6、er ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2003 All rights reservedISO 8607:2003(E) ISO 20
7、03 All rights reserved iiiContents Page Foreword iv Introduction v 1 Scope 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle . 2 5 Diluent and culture medium. 2 5.1 Diluent 2 5.2 Agar medium . 3 6 Apparatus. 3 7 Sampling 4 8 Preparation of test sample. 4 9 Procedure. 4 9.1 Test por
8、tion, initial suspension and dilutions . 4 9.2 Inoculation and incubation 4 9.3 Control plates 5 9.4 Interpretation of results 5 10 Expression of results 5 10.1 Method of calculation . 5 10.2 Precision 7 11 Test report 7 Annex A (normative) Confidence limits for the estimation of small numbers of co
9、lony-forming units of microorganisms 8 Bibliography . 9 ISO 8607:2003(E) iv ISO 2003 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is norma
10、lly 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 liaison with ISO, also take part in th
11、e 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 technical committees is to prepare
12、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 drawn to the possibility that some
13、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 8607 was prepared by Technical Committee ISO/TC 34, Food products. This first edition of ISO 8607 cancels and replaces ISO/TR 8607:1991, which ha
14、s been technically revised. ISO 8607:2003(E) ISO 2003 All rights reserved vIntroduction The quantitative microbiological control of the hygienic collection and handling of bovine semen is of great importance in order to predict the efficiency of artificial insemination and to fulfil the requirements
15、 of biosecurity (see reference 1). For the same reason, the investigation of bacterial contamination and the possible presence of facultative pathogenic microorganisms in the preserved bovine semen is also very important. There is a need for an international method suitable for the determination of
16、the microbial count in frozen semen, which indicates the hygienic status during collection, handling and storage. The aim of the colony- count method specified in this International Standard is to enumerate the saprophytic microorganisms that are originally present in and/or are transmitted to the b
17、ovine semen from the environment. With this method only the total count of bacteria is detected, mainly the aerophilic and mesophilic saprophytic ones, as well as a few facultative pathogenic microorganisms that are not very sensitive to environmental conditions. Since samples of frozen bovine semen
18、 contain additional antibiotics, the determination of microbiological contamination of this type of sample is slightly different from the commonly used microbiological methods. When examining preserved semen samples in low dilutions, the number of colonies may be lower than expected and do not follo
19、w the usual proportions. Therefore relatively high decimal dilutions should be used to compensate for the inhibition effect of the antibiotics. As a result of the necessary high dilutions, 15 or less colonies can be observed in each Petri dish and this result should be accepted. This differs from th
20、e usual microbiological examinations of food where the sample dilution can be chosen in such a way that the number of colonies is more than 15 in a Petri dish so more precise examination is possible. Microbial cells often occur as clumps or groups in the samples. Whereas shaking samples and dilution
21、s may uniformly distribute the clumps of bacteria, this may not completely disrupt the clumps themselves into single cells. Consequently, each colony that appears on the medium can arise from a clump of cells or from a single cell and therefore it is more precise to express the result as the number
22、of colony-forming units (CFU) of microorganisms than to give the number of microorganisms (see reference 2). This International Standard does not specify a tolerable limit value for the total CFU of bacteria, which may be a consumer requirement in trade. This should be given in commercial contracts.
23、 A list of publications related to this International Standard is given in the Bibliography. INTERNATIONAL STANDARD ISO 8607:2003(E) ISO 2003 All rights reserved 1Artificial insemination of animals Frozen semen of breeding bulls Enumeration of living aerobic microorganisms 1 Scope This International
24、 Standard specifies a method for the enumeration of living aerobic microorganisms present in the frozen semen of breeding bulls. The colonies growing in a solid medium after aerobic incubation at 37 C are counted. The microbiological contamination of the sample is expressed as a number of colony- fo
25、rming units of microorganisms per millilitre of the test sample. 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 the referenced docume
26、nt (including any amendments) applies. 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 decimal dilutions ISO 7218,
27、Microbiology of food and animal feeding stuffs General rules for microbiological examinations 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO 6887-1 and the following apply. 3.1 semen product of the genital organs of a male, intended for the fertiliz
28、ation of a female 3.2 ejaculate quantity of semen obtained as a result of mating the male 3.3 dose quantity of semen which is packaged individually and carries a unique identification, intended for a single artificial insemination 3.4 series of doses group of doses of semen obtained from one bull an
29、d prepared from one or more ejaculates, obtained on the same day and subjected to the same treatment 3.5 living aerobic microorganisms bacteria, yeasts and moulds which grow aerobically at 37 C under the conditions specified in this International Standard ISO 8607:2003(E) 2 ISO 2003 All rights reser
30、ved3.6 colony-forming unit CFU single microbial cell, or clumps or a group of cells, forming one colony on the medium under the conditions specified in this International Standard 4 Principle Two poured plates are prepared using a specified culture medium. These are deep inoculated with a specified
31、quantity of test sample, followed by aerobic incubation at 37 C. The number of CFU of microorganisms per millilitre of the test sample is calculated from the number of colonies obtained. 5 Diluent and culture medium For general guidance, see ISO 7218. Chemical products shall be of recognized analyti
32、cal quality and suitable for microbiological analysis. The water used shall be distilled water or of equivalent quality (see ISO 7218). 5.1 Diluent The diluent is a peptone salt solution as specified in ISO 6887-1. Its composition, preparation and use are given only for the convenience of the users
33、of this International Standard. In order to improve the reproducibility of the results, it is recommended that, for the preparation of the diluent, dehydrated basic components or a dehydrated complete preparation should be used. The manufacturers instructions shall be rigorously followed. 5.1.1 Comp
34、osition Enzymatic digest of casein 1,0 g Sodium chloride (NaCI) 8,5 g Water 1 000 ml 5.1.2 Preparation Dissolve the components in the water, by heating if necessary. Adjust the pH, if necessary, so that after sterilization it is 7,0 0,2 at 25 C. 5.1.3 Distribution and sterilization Dispense the dilu
35、ent in volumes as necessary for the preparation of the initial suspensions into test tubes or flasks (6.3) of appropriate capacity. Dispense the diluent in volumes as necessary for the preparation of the decimal dilutions into test tubes or flasks (6.3) in quantities such that, after sterilization,
36、each tube or flask contains 9,0 ml. The uncertainty of measurement of this final volume, after sterilization, shall not exceed 2 %. ISO 8607:2003(E) ISO 2003 All rights reserved 35.2 Agar medium 5.2.1 Composition Meat extract 10,0 g Anhydrous D-glucose (C 6 H 12 O 6 ) 1,0 g Dehydrated yeast extract
37、2,5 g Peptone 3,0 g Sodium chloride (NaCI) 2,0 g Disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 12H 2 O) 2,0 g Gelatine 10,0 g Agar in powder or flake form 12,0 g to 18,0 g 1)Water 1 000 ml 5.2.2 Preparation Dissolve the components or the dehydrated complete medium in the water, by heating if
38、 necessary. Adjust the pH, if necessary, so that after sterilization it is 7,2 0,2 at 25 C. Dispense the medium into tubes or flasks (6.3), in quantities such that the container is half-full. Sterilize in an autoclave (6.1) at 121 C 1 C for 15 min. If the medium is to be used immediately, cool it to
39、 44 C to 47 C in the water bath (6.8) and then add 10 % (by volume) inactivated and sterilized 2)bovine or sheep serum. Otherwise, before beginning the microbiological examination, completely melt the medium in the boiling water bath (6.9), cool to 44 C to 47 C in another water bath (6.8), and then
40、add 10 % (by volume) inactivated and sterilized 2)bovine or sheep serum. 6 Apparatus NOTE Disposable apparatus is an acceptable alternative to reusable glassware, if it has suitable specifications. Usual microbiological laboratory apparatus and, in particular, the following. 6.1 Sterilizing oven (fo
41、r dry sterilization) or autoclave (for wet sterilization), see ISO 7218. 6.2 Incubator, capable of being maintained at 37 C 1 C. 6.3 Test tubes, of 16 mm diameter and 160 mm length, or flasks, of capacity not greater than 500 ml. 6.4 Petri dishes, made of glass or plastic, of 90 mm to 100 mm diamete
42、r. 6.5 Pipettes, having a nominal capacity of 1 ml, graduated in 0,1 ml divisions. Blow-out pipettes shall not be used. 1) According to the gel strength of the agar. 2) By ultrafiltration (0,2 nm filter). ISO 8607:2003(E) 4 ISO 2003 All rights reserved6.6 pH-meter, electric, accurate to 0,1 pH unit
43、at 25 C. 6.7 Water bath, capable of being maintained at 37 C 1 C. 6.8 Water bath, capable of being maintained at 45 C 1 C. 6.9 Boiling water bath. 6.10 Colony-counting equipment, consisting of an illuminated base with a dark background, fitted with a magnifying lens suitable for use at a magnificati
44、on of 1,5, and a mechanical or electronic digital counter. 7 Sampling Choose at random from a series of doses the necessary number of doses of deep-frozen semen of any type (pellets or minitubes of 0,25 ml or 0,5 ml) so that the volume of sample is 1,0 ml per series of doses. Store the test samples
45、in liquid nitrogen. When required for examination, the test samples may be transferred from the large liquid-nitrogen storage container to a small liquid-nitrogen laboratory container. 8 Preparation of test sample Before use, thaw the test sample in the water bath (6.7) set at 37 C for 3 min. Thawed
46、 test samples may be kept in a refrigerator at 4 C but for no longer than 1 h. 9 Procedure 9.1 Test portion, initial suspension and dilutions Prepare the initial suspension in accordance with ISO 6887-1 in a safety cabinet. The number of further dilutions to be carried out depends on the antibiotics
47、 content of the initial suspension as specified below. a) If the initial suspension contains the usual content of antibiotics i.e. 10 3International Units (IU) 3)of penicillin and 1 mg of streptomycin or another broad-spectrum antibiotic per millilitre, use a 10 5final dilution. b) If the quantity o
48、f antibiotics differs from that mentioned in a), use a final dilution such that the content of penicillin is not more than 0,1 IU/ml and that of the broad-spectrum antibiotic not more than 0,1 g/ml. NOTE A higher antibiotics concentration can inhibit growth of the microorganisms and so produce false
49、 results. 9.2 Inoculation and incubation 9.2.1 Take two sterile Petri dishes (6.4). Transfer, by means of a sterile pipette (6.5), 1 ml of the final dilution of the initial suspension (see 9.1) to each dish. Take two other sterile Petri dishes. Using a new sterile pipette, transfer to each dish 1 ml of the dilution which is diluted by one order of magnitude less than the final one. 3) The IU is the determined quantity of internationally accepted reference material. In the case of penic
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