1、BRITISH STANDARD BS ISO 15214:1998 Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of mesophilic lactic acid bacteria Colony-count technique at 30 C ICS 07.100.30BSISO15214:1998 This British Standard, having been prepared under the direction of the Consumer Produ
2、cts and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 October 1998 BSI 05-1999 ISBN 0 580 30378 0 National foreword This British Standard reproduces verbatim ISO15214:1998 and implements it as the UK national standard. The UK participatio
3、n in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests inf
4、ormed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Additional information This horizontal method may not be appropriate in all food situations, and other meth
5、ods are more productive with some types of food. In applying this standard it is important to avoid excessive exposure to changes in pH during the preparation of dilutions: in the UK, for lower pH food material 0.1% peptone water without pH adjustment is preferred instead of buffered peptone water a
6、t pH 7.0 as described. To adjust commercially available MRS agar to pH 5.7 use acetic acid. In the UK, microaerobic incubation conditions (510 % oxygen and 5 % carbon dioxide) are commonly applied or the use of a candle jar may be considered. However, if aerobic incubation is the only option then ei
7、ther use the pour plate method or overlay spread plates with 5 ml of MRS agar. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Co
8、rrespondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does
9、 not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theISO title page, pages ii to iv, pages 1 to 6 and a back cover. This standard has been updated (see copyright date) and may have had amendments inco
10、rporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date CommentsBSISO15214:1998 BSI 05-1999 i Contents Page National foreword Inside front cover Foreword iii Text of ISO 15214 1ii blankBSISO15214:1998 ii BSI 05-1999 Content
11、s Page Foreword iii Introduction 1 1 Scope 1 2 Normative references 1 3 Definitions 1 4 Principle 1 5 Diluent and culture medium 2 6 Apparatus and glassware 3 7 Sampling 3 8 Preparation of test sample 3 9 Procedure 3 10 Expression of results 4 11 Confidence limits 4 12 Test report 5 Annex A (informa
12、tive) Bibliography 6 Descriptors: Agricultural products, food, food products, animal feeding products, biological tests, microbiological analysis, counting, bacteria, lactic acid bacteria, bacteria count methods.BSISO15214:1998 BSI 05-1999 iii Foreword ISO (the International Organization for Standar
13、dization) is a worldwide federation of national standards 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
14、 be represented on that committee. International organizations, 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. Draft Internationa
15、l 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 ISO15214 was prepared by Technical Committee ISO/TC 34, Agricultural
16、 food products, Subcommittee SC 9, Microbiology. Annex A of this International Standard is for information only.iv blankBSISO15214:1998 BSI 05-1999 1 Introduction Because of the large variety of food and feed products, this horizontal method may not be appropriate in every detail for certain product
17、s. In this case, different methods which are specific to these products may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt should be made to apply this horizontal method as far as possible. When this International Standard is next reviewed, account will
18、be taken of all information then available regarding to extent to which this horizontal method has been followed and the reasons for deviations from this method in the case of particular products. The harmonization of test methods cannot be immediate, and for certain groups of products International
19、 Standards and/or national standards may already exist that do not comply with this horizontal method. It is hoped that when such standards are reviewed they will be changed to comply with this International Standard so that eventually the only remaining departures from this horizontal method will b
20、e those necessary for well-established technical reasons. 1 Scope This International Standard specifies a horizontal method for the enumeration of viable mesophilic lactic acid bacteria by counting the colonies growing in a solid medium after incubation at 30 C for 3 days. NOTEIn some food products,
21、 there exist psychotrophic or thermophilic lactic acid bacteria necessitating culture temperatures different from 30 C. Moreover, not all lactic acid bacteria grow on MRS agar at pH 5,7 and some grow only weakly. Subject to the limitations discussed in the introduction and in the note above, this In
22、ternational Standard is applicable to products intended for human consumption or animal foodstuffs. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of the publication, the editi
23、ons indicated were valid. All standards are subject to revision, and parties to agreement based on the International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards inidicated below. Members of IEC and ISO maintain registers of currently v
24、alid International Standards. 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 intial suspension and decimal dilutions 1) . ISO 7218:1996,
25、 Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Definitions For the purposes of this International Standard, the following definition applies. 3.1 mesophilic lactic acid bacteria bacteria which form colonies at 30 C in a solid selective medium (MRS a
26、t pH 5,7) under the test conditions specified in this International Standard 4 Principle 4.1 Preparation of two dishes using MRS agar at pH5,7 contained in Petri dishes. Poured-plate or possibly surface 2)inoculation of the dishes with a specified quantity of the test sample if the initial product i
27、s liquid, or with a specified quantity of the initial suspension in the case of other products. 4.2 Inoculation of other pairs of dishes, under the same conditions, using decimal dilutions of the test sample or of the initial suspension. Incubation of the dishes at 30 C for 72 h. 1) To be published.
28、 (Revision of ISO6887:1983) 2) See note 1 in 9.2.BSISO15214:1998 2 BSI 05-1999 4.3 Calculation of the number of mesophilic lactic acid bacteria (3.1) per gram or per millilitre of test sample from the number of colonies obtained in 4.2 in the dishes selected, and possibly confirmed 3) . 5 Diluent an
29、d culture medium 5.1 General For current laboratory practice, see ISO7218. 5.2 Diluent See ISO 6887-1. NOTEThe buffered peptone water does not always allow satisfactory resuscitation of lactic acid bacteria (see Annex A, references 1, 2, 3). 5.3 Culture medium: MRS medium (de Man, Rogosa and Sharpe)
30、 at pH 5,7 (see reference 4) NOTEThe use of commercially available ready-to-use media is acceptable. However, attention is drawn to the fact that variations in composition and pH may occur between products from different manufacturers and could, therefore give results different from the ones obtaine
31、d with the medium as specified in this International Standard. 5.3.1 Composition 5.3.2 Preparation 5.3.2.1 Dissolve the components or the dehydrated complete medium in the water by boiling. Using the pH-meter (6.7), adjust the pH so that after sterilization it is 5,7 0,1 4)at 25 C. Transfer the medi
32、um to bottles of appropriate capacity. Sterilize for 15 min in the autoclave (6.1) set at 121 C. If the medium is to be used immediately, cool it before use to appoximately 47 C in the water bath (6.5), or by any other technique giving equivalent results (see ISO7218). If not, in order to avoid any
33、delay when pouring the medium before beginning the microbiological examination, completely melt the medium, in a boiling water bath (6.6), then cool it to approximately 47 C in the water bath (6.5). 5.3.2.2 If there is a risk of extensive yeast contamination (e.g. in dried sausage), add sorbic acid
34、to the MRS medium as follows. Dissolve 1,4 g of sorbic acid in about 10 ml of a 1 mol/l solution of sodium hydroxide. Sterilize by filtration. Add this solution to 1 000 ml of sterilized MRS agar, previously cooled to approximately 47 C. The final pH of the medium shall be 5,7 0,1 at 25 C. 3) See no
35、te 2 in 9.3. Enzymatic digest of casein 10,0 g Meat extract 10,0 g Yeast extract 4,0 g Triammonium citrate (NH 4 ) 3 C 6 H 5 O 7 2,0 g Sodium acetate (CH 3 COONa) 5,0 g Magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 0,2 g Manganese sulfate tetrahydrate (MnSO 4 4H 2 O) 0,05 g Dipotassium hydrogen pho
36、sphate (K 2 HPO 4 ) 2,0 g Glucose (C 6 H 12 O 6 ) 20,0 g Polyoxyethylenesorbitan monooleate (Tween 80) 1,08 g Agar 12 g to 18 g a Water 1 000 ml a Depending on the gel strength of the agar 4) In order that the pH-value does not fall below 5,6, the tolerance here is 0,1 instead of 0,2 as usual.BSISO1
37、5214:1998 BSI 05-1999 3 6 Apparatus and glassware Usual microbiological laboratory apparatus (see ISO7218) and, in particular, the following. 6.1 Apparatus for dry sterilization (oven) or wet sterilization(autoclave) See ISO7218. 6.2 Incubator, capable of operating at 30 C 1 C. 6.3 Petri dishes, mad
38、e of glass or plastic, of diameter 90 mm to 100 mm. 6.4 Total-delivery graduated pipettes, of nominal capacity 10 ml and 1 ml, graduated respectively in 0,5 ml and 0,1 ml divisions. 6.5 Water bath, or similar apparatus, capable of operating at 47 C 2 C. 6.6 Boiling water bath. 6.7 pH-meter, capable
39、of being read to the nearest 0,01 pH unit at 25 C, enabling measurements to be made which are accurate to 0,1 pH unit. 7 Sampling Sampling is not part of the method specified in this International Standard. If there is no specific International Standard dealing with sampling of the product concerned
40、, it is recommended that the parties concerned come to an agreement on this subject. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage (see ISO7218). 8 Preparation of test sample Prepare the test sample
41、in accordance with the specific International Standard appropriate to the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject. 9 Procedure 9.1 Test portion, initial suspension and dilutions Prepare the i
42、nitial suspension and dilutions in accordance with ISO6887-1. 9.2 Inoculation and incubation NOTE 1Surface plating in combination with incubation under anaerobic or microaerobic conditions can be applied instead of the pour-plating procedure described. Candle jars may be used to obtain appropriate c
43、onditions. NOTE 2It is also possible to use a double-layer MRS medium. 9.2.1 Take two sterile Petri dishes (6.3). Using a sterile pipette (6.4), transfer to each dish 1 ml of the test sample if the product is liquid, or 1 ml of the initial suspension in the case of other products. Take two other ste
44、rile Petri dishes. Using a fresh sterile pipette, transfer to each dish 1 ml of the first decimal dilution of the test sample if the product is liquid, or 1 ml of the first decimal dilution of the initial suspension in the case of other products. Repeat the procedure described with the further dilut
45、ions, using a fresh sterile pipette for each decimal dilution. NOTEIf high numbers of lactic acid bacteria are expected, it is possible to inoculate only those dilutions necessary to be able to enumerate according to the general case (see 10.1). 9.2.2 Pour into each Petri dish approximately 15 ml of
46、 the MRS medium (5.3) which has been prepared then cooled to approximately 47 C in the water bath (6.5). Carefully mix the inoculum with the medium and allow the mixture to solidify. 9.2.3 Invert the prepared dishes and incubate them in the incubator (6.2) set at 30 C for 72 h 3 h. Avoid desiccation
47、 of the agar during incubation so that the medium does not become too inhibitory. 9.3 Counting of colonies After the specified period of time (see 9.2.3), count the colonies in each dish (see notes 1 and 2). Retain dishes containing fewer than 300 colonies at two successive dilutions, and more than
48、15 colonies on at least one dish.BSISO15214:1998 4 BSI 05-1999 NOTE 1Some Leuconostoc spp. may form large slimy colonies which may hinder the development of other colonies, thus causing an underestimation of the number of lactic acid bacteria. NOTE 2Due to the possible development of microorganisms
49、other than lactic acid bacteria on MRS medium, as described in 9.2, it may be necessary in some cases and for some products to confirm the colonies obtained in 9.2 by simple techniques (such as Gram staining, or the test for catalase). Such a procedure, if conducted, should be mentioned in the test report. 10 Expression of results 10.1 General case Calculate the number N of mesophilic lactic acid bacteria present in the test sample, as the weighted mean from two successive dilutions, using the equation: where Roun
