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1、INTERNATIONAL STANDARD IS0 13681 First edition 1995-l 2-l 5 grNsr Interrmat DW s43c Meat and meat products - Enumeration of yeasts and moulds - Colony-count technique Viandes et produits 2 base de viande - Dknombrement des levures et moisissures - Technique par comptage des colonies - - T Egg5 x =x.
2、! - Reference number IS0 13681 :I 995(E) Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/25/2007 01:31:37 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 13
3、681:1995(E) Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 technical committees. Each member body interested in a subject f
4、or 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 the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on
5、 all matters of electrotechnical standardization. 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. International Standard
6、IS0 13681 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Subcommittee SC 6, Meat and meat products. Annex A forms an integral part of this International Standard. Annex B is for information only. 0 IS0 1995 All rights reserved. Unless otherwise specified, no part of this
7、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 the publisher. International Organization for Standardization Case Postale 56 l CH-1211 Geneve 20 l Switzerland Printed in Switzer
8、land ii Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/25/2007 01:31:37 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD 0 IS0 IS0 13681:
9、1995(E) Meat and meat products - Enumeration of yeasts and moulds - Colony-count technique 1 Scope This International Standard specifies a method for the enumeration of yeasts and moulds in all kinds of meat and meat products, including poultry, by means of a colony-count technique at between 20 “C
10、and 25 “C. IMPORTANT - IS0 7954Cll recommends chloram- phenicol or oxytetracycline as antibiotic. These rec- ommended antibiotics do not, however, sufficiently inhibit Gram-negative microorganisms occurring in meat, especially raw meat. To obtain sufficient inhibition in cases of heavy bacterial con
11、tamination, the addition of gentamicin is necessary. Since a com- bination of chloramphenicol and gentamicin inhibits certain types of yeasts, the alternatively specified antibiotic oxytetracycline is the antibiotic of choice. 2 Normative references The following standards contain provisions which,
12、through reference in this text, constitute provisions of this International Standard. At the time of publi- cation, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility o
13、f applying the most re- cent editions of the standards indicated below. Members of IEC and IS0 maintain registers of cur- rently valid International Standards. IS0 3100-2:1988, Meat and meat products - Samp- ling and preparation of test samples - Part 2: Prep- aration of test samples for microbiolog
14、ical examination. IS0 6887: 1983, Microbiology - Genera/ guidance for the preparation of dilutions for microbiological exam- ination. IS0 7218:-l , Microbiology of food and animal feed- ing stuffs - General rules for microbiological exam- inations. 3 Definition For the purposes of this International
15、 Standard, the following definition applies. 3.1 yeasts and moulds: Microorganisms that form colonies within 5 days at between 20 “C and 25 “C under the conditions specified in this International Standard. 4 Principle 4.1 Deep inoculation of poured plates, using a specified selective culture medium
16、contained in Petri dishes, with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of the initial suspension in the case of other products. Inoculation of other plates, under the same con- ditions, using decimal dilutions of the initial suspen- sio
17、n. NOTE 1 To distinguish, if necessary, between yeasts and moulds, the use of a surface plate is advisable. Surface plates are also recommended when heat-sensitive yeasts or moulds are expected. 4.2 Aerobic incubation of the plates at between 20 “C and 25 “C for 3, 4 or 5 days. 4.3 Calculation of th
18、e number of yeasts and moulds per gram or per millilitre of sample from the number of colonies obtained on plates chosen at dilution lev- els so as to give a significant result. I) To be published. (Revision of IS0 7218:1985) 1 Copyright International Organization for Standardization Provided by IHS
19、 under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/25/2007 01:31:37 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 13681:1995(E) 5 Dilution fluid, culture medium and reagents 5.1 General For current laboratory practice, see IS0 72
20、18. 5.2 Dilution fluid See IS0 6887. 5.3 Agar medium of yeast extract, glucose and oxytetracycline/gentamicin 5.3.1 Base medium 5.3.1.1 Composition Yeast extract 59 Glucose (CaH,O,) 20 g Agar 8gto18g ) Water 1 000 ml 1) Depending on the gel strength of the agar. 5.3.1.2 Preparation Dissolve the comp
21、onents in the water by boiling. If necessary, adjust the pH so that after sterilization it is 6.6 at 25 “C. Transfer the medium to suitable containers (6.6). Sterilize for 10 min in the autoclave (6.1) set at 115C. 5.3.2 Oxytetracycline solution 5.3.2.1 Composition 5.3.3 Gentamicin solution 5.3.3.1
22、Composition 25 mg 1) 25 ml 1) According to the manufacturer s specification of the real gentamicin content of the powder. 5.3.3.2 Preparation Dissolve the gentamicin in the water and sterilize the solution by filtration. 5.3.4 Complete medium Add 5 ml of the oxytetracycline solution (5.3.2) and 5 ml
23、 of the gentamicin solution (5.3.3) to each portion of 100 ml of sterile base medium (5.3.1), melted and kept in a water bath (6.4) set at 47 “C. 6 Apparatus and glassware NOTE 2 Disposable apparatus is an acceptable alterna- tive to reusable glassware if it has suitable specifications. Usual microb
24、iological laboratory equipment and, in particular, the following. 6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave) The autoclave can either operate separately or be part of a general apparatus for preparing and distributing media. See IS0 7218. 6.2 Mixer, working on the pr
25、inciple of a centric ro- tation of the contents of the tube (e.g. Vortex mixer), and capable of mixing 1 ml or 2 ml of sample in a tube of adequate dimensions with 9 ml or 18 ml of dilution fluid to obtain a homogeneous suspension. Oxytetracycline (C,H,N,O,) Water 50 mg 25 ml 6.3 Incubator, capable
26、of operating at between 20 “C f 1 “C and 25 “C f 1 “C. 5.3.2.2 Preparation Dissolve the oxytetracycline in the water and sterilize the solution by filtration. 6.4 Water bath, capable of operating at 47 “C f 2 “C. 6.5 pH-meter, accurate to within + 0,l pH unit at 25 “C. 2 Copyright International Orga
27、nization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/25/2007 01:31:37 MDTNo reproduction or networking permitted without license from IHS -,-,- 63 IS0 IS0 13681:1995(E) 6.6 Culture bottles or flasks Bottles or flasks wi
28、th non-toxic metallic screw-caps may be used. 6.7 Graduated pipettes, calibrated for bacterio- logical use only, of nominal capacities 10 ml and 1 ml, graduated respectively in 0,5 ml and 0,l ml div- isions and with an outflow opening of nominal diam- eter 2 mm to 3 mm. the test sample and the momen
29、t when the medium is poured into the dishes shall not exceed 15 min. Carefully mix the inoculum with the medium and al- low the mixture to solidify by leaving the Petri dishes to stand on a cool horizontal surface. 9.2.4 invert the plates and place them in the incubator (6.3) set at between 20 “C an
30、d 25 “C. 9.3 Counting and selection of colonies 6.8 Petri dishes, of diameter 90 mm to 100 mm. 7 Sampling It is important that the laboratory receive a sample which is truly representative and has not been dam- aged or changed during transport or storage. Sampling is not part of the method specified
31、 in this International Standard. A recommended sampling method is given in IS0 3100-I *I. 8 Preparation of test sample Prepare the test sample in accordance with IS0 3100-2. Start the examination of the pretreated sample as soon as possible. It may be stored, if necessary, at a temperature between 0
32、 “C and + 2 “C, but for no longer than 24 h. 9 Procedure 9.1 Test portion, initial suspension and dilutions Prepare the initial suspension and dilutions in accord- ance with IS0 6887. 9.2 Inoculation and incubation 9.2.1 Take two sterile Petri dishes (6.8). Transfer, by means of a sterile pipette (6
33、.71, 1 ml of the test sam- ple to each dish if liquid, or 1 ml of the initial suspen- sion in the case of other products. 9.2.2 Repeat the procedure described in 9.2.1 with the other dilutions. 9.2.3 Pour, from a culture bottle (6.61, about 15 ml of the agar medium of yeast extract, glucose and oxyt
34、etracycline/gentamicin (5.31, melted and kept at 47 “C in the water bath (6.41, into each Petri dish. The time elapsing between the end of the preparation of Count the colonies on each plate after 3, 4 and 5 days of incubation. After 5 days, retain those plates con- taining fewer than 150 colonies.
35、If parts of the plates are overgrown with moulds, or if it is difficult to count well-isolated colonies, record the counts obtained af- ter 4 (or even 3) days of incubation. In this event, re- cord the incubation period, 3 or 4 days, in the test report. If necessary, proceed to a microscopic examina
36、tion in order to distinguish the colonies of yeasts and moulds from size. 10 10.1 colonies of bacteria which Expression of results General case are mostly of small Use dishes containing fewer than 150 colonies (see 9.3). Calculate the number, N, of microorganisms per gram or per millilitre of produc
37、t, using the following equation: N= CC (q + 0,1 n is the number of dishes retained at the first dilution; n2 is the number of dishes retained at the second dilution; d is the dilution factor corresponding to the first dilution retained (i.e. that with the higher concentration of test sample). Round
38、off the results calculated to two significant figures. For this, if the last figure is below 5, the pre- ceding figure is not modified; if the last figure is 5 or more, the preceding figure is increased by one unit. 3 Copyright International Organization for Standardization Provided by IHS under lic
39、ense with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/25/2007 01:31:37 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 13681:1995(E) 0 IS0 Proceed stepwise until two significant figures are ob- tained. Take as the result the number of microorga
40、nisms per millilitre (liquid product) or per gram (other products), expressed as a number between I,0 and 9,9 multi- plied by the appropriate power of 10. EXAMPLE A direct count of yeasts and mould gave the following results (two Petri dishes per dilution were incubated): - at the first dilution (lo
41、- ): 83 and 97 colonies; - at the second dilution (1 OV”): 33 and 28 colonies. Therefore Nz = 83 + 97 + 33 + 28 (a + O,lf$)d = 2 + (0,l x 2) x 1o-2 241 =-= 10 954 0,022 Rounding the result as specified above gives 11 000 or 1 ,I x IO4 yeasts and moulds per millilitre or per gram of product. 10.2 Est
42、imation of small numbers If the two dishes, at the level of the test sample (liq- uid products) or the initial suspension (other pro- ducts), contain less than 15 colonies, calculate the arithmetic mean y of the colonies counted on both dishes. Express the result as follows: - for liquid products: e
43、stimated and moulds per millilitre NE = y - for other products: estimated and moulds per gram NE = y/d number of yeasts number of yeasts where d is the dilution factor of the initial suspen- sion. 10.3 No colonies on plates If there were no colonies on plates from the test sample (liquid products),
44、the number of yeast and moulds per millilitre of product should be reported as fewer than 1. If there were no colonies on plates from the initial suspension (solid products), the number of yeasts and moulds per gram of product should be reported as fewer than 10. 11 Precision For statistical reasons
45、 alone, in 95 % of cases the confidence limits of the colony-count technique vary from + 16 % to f 52 % (see reference 3); for col- ony counts of less than 15 per plate, the confidence limits are given in annex A. In practice, even greater variation may be found, especially among results ob- tained
46、by different workers. 12 Test report The test report shall specify - the method in accordance with which sampling was carried out, if known; - the method used, including the number of days of incubation: - the test result(s) obtained; and - if the repeatability has been checked, the final quoted res
47、ult obtained. It shall also mention all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the test result(s). The test report shall include all information necessary for the complete identificatio
48、n of the sample. 4 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/25/2007 01:31:37 MDTNo reproduction or networking permitted without license from IHS -,-,- 0 IS0 IS0 13681:1995(E) Anne
49、x A (normative) Confidence limits for the estimation of small numbers of colonies The confidence limits at the 95 % level for the estimation of small numbers, when the number of colonies retained is less than 15, are as follows. Number of Number of coloniesl) microorganisms 1 1 2 1 3 2 4 2 5 2 Confidence limit at 95 % level Lower Upper I 3 I 4 I 4