1、BRITISH STANDARD BS 4285-3.4.2: 1989 Microbiological examination for dairy purposes Part 3: Methods for detection and/or enumeration of specific groups of microorganisms Section 3.4 Enumeration of lipolytic microorganisms Subsection 3.4.2 Tributyrin agar method NOTEIt is essential that Parts 0 and 1
2、 and Section 2.1 which are published separately, arereadinconjunction with this Subsection. UDC 637.1.055.07:579.8:620.1BS4285-3.4.2:1989 This British Standard, having been prepared under the directionof the Dairying Standards Committee, was published under the authority ofthe Board of BSI and comes
3、 intoeffect on 30June1989 BSI 06-1999 BS 4285 first published March1968 Supplement No. 1 to BS 4285:1968 first published April 1970 First revision, Subsection 3.4.2 June 1989 The Committees responsible for this standard are shown in Part 0 The following BSI references relate to the work on this stan
4、dard: Committee reference DAC/4 Draftfor comment 87/55556 DC ISBN 0 580 17092 6 Foreword This Subsection of BS 4285 has been prepared under the direction of the Dairying Standards Committee. The technique was given neither in BS4285:1968 nor in Supplement No. 1 (1970). The method described in this S
5、ubsection is suitable for routine purposes. The more accurate but more complex Victoria blue method described in Subsection3.4.1 is the reference method. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their co
6、rrect application. Compliance with a British Standard does 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, pages 1 and 2, an inside back cover and a back cover. This standard has been updated (see co
7、pyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover. Amendments issued since publication Amd. No. Date of issue CommentsBS4285-3.4.2:1989 BSI 06-1999 i Contents Page Foreword Inside front cover 1 Scope 1 2 Definition 1 3 Pri
8、nciple 1 4 Diluents and culture medium 1 5 Apparatus 1 6 Sampling 1 7 Preparation of the test sample 1 8 Procedure 1 9 Calculation and expression of results 2 10 Test report 2 Publications referred to Inside back coverii blankBS4285-3.4.2:1989 BSI 06-1999 1 1 Scope This Subsection of BS 4285 describ
9、es a method for the enumeration of lipolytic organisms in milk and milk products and for dairy purposes generally. NOTEThe titles of the publications referred to in this standard are listed on inside back cover. 2 Definition For the purposes of this Subsection of BS 4285 the following definition app
10、lies. lipolytic organisms those organisms which hydrolyse tributyrin (glyceryl tributyrate) and result in the formation of colonies surrounded by a zone of clear medium under the conditions described in this Subsection of BS 4285 3 Principle A test portion or series of decimal dilutions is mixed wit
11、h an opaque peptone yeast extract agar medium of pH 7.5 containing tributyrin and incubated. The tributyrin is hydrolysed by the lipolytic organisms multiplying in the medium, to produce colonies surrounded by a zone of clear medium. The number of colonies is counted. 4 Diluents and culture medium 4
12、.1 Diluents Use diluents described in BS 4285-1.2. 4.2 Tributyrin agar The composition of the tributyrin agar shall be as follows: If the medium is not obtained as a stable prepared homogenate of pH 7.5 0.2 at 25 C (as is normal), prepare it in the laboratory as follows. Dissolve the peptone, yeast
13、extract and agar in the water by boiling. Add the tributyrin and emulsify the medium at about 90 C using a laboratory homogenizer or hand (pump type) emulsifier. NOTE 1A high-speed stirrer is not adequate. NOTE 2If required, the medium may be stored in the dark for up to 2 months at 2 C to 5 C. 5 Ap
14、paratus NOTEFor details of apparatus, including its preparation and sterilization, see BS 4285-1.2. 5.1 Ordinary microbiological laboratory apparatus 5.2 Incubator, capable of being maintained at 30 1 C. 5.3 Water bath, capable of being maintained at45 2 C. 5.4 Colony counting equipment 5.5 Petri di
15、shes 5.6 Total delivery pipettes, of 1 mL nominal quantity. 6 Sampling Take the laboratory sample in accordance with BS4285-1.1. 7 Preparation of the test sample Prepare the test sample from the laboratory sample in accordance with BS 4285-1.1. 8 Procedure 8.1 Preparation of the tributyrin agar medi
16、um Melt the tributyrin agar (see 4.2) and cool to 45 2 C in the water bath (5.3) (see BS 4285-2.1). 8.2 Preparation of the dilutions Prepare serial dilutions from the test sample (seeclause 7), making as many dilutions as are necessary to produce acceptable counts at two successive dilutions. NOTETh
17、e preferable count is between six and60characteristic colonies. 8.3 Inoculation Take two Petri dishes (5.5) for each dilution used. Using a separate 1 mL pipette (5.6) for each dilution and for any undiluted test portion transfer 1 mL into each dish (see BS 4285-2.1). 8.4 Addition of medium 8.4.1 Ad
18、d 10 mL to 12 mL of melted tributyrin agar medium at 45 2 C (see 8.1) to each inoculated dish. 8.4.2 Immediately after pouring, mix by five to and fro movements, followed by five circular clockwise movements, followed by five to and fro movements at right angles to the first set, followed by five ci
19、rcular anticlock-wise movements. Allow the agar to set. peptone 5 g yeast extract 3 g tributyrin 10 g agar 15 g (or according to the manufacturers instructions) water 1000 mLBS4285-3.4.2:1989 2 BSI 06-1999 8.5 Incubation of the plates Invert the plates and incubate in the incubator (5.2), maintained
20、 at 30 1 C for 3 days. If the development of yeasts or moulds is expected continue the incubation for a further 3 days. 8.6 Counting the colonies Take colonies surrounded by a zone of clear medium to be lipolytic. Count the colonies following the procedure given in BS 4285-2.1 (see the note to 8.2).
21、 9 Calculation and expression of results Calculate the number of organisms and express the results as described in BS 4285-2.1 as the total number of lipolytic organisms. 10 Test report The test report shall give the number and date of this standard, i.e. BS 4285-3.4.2:1989, and state the results ob
22、tained. It shall also state any operating conditions not specified in this standard or regarded as optional, as well as any circumstances that may have influenced the result. The report shall include all details required for complete identification of the sample.BS4285-3.4.2:1989 BSI 06-1999 Publica
23、tions referred to BS 4285, Microbiological examination for dairy purposes. BS 4285-0, General introduction. BS 4285-1, Guide to general procedures. BS 4285-1.1, Sampling and preparation of sample. BS 4285-1.2, Diluents, media and apparatus and their preparation and sterilization. BS 4285-1.3, Proced
24、ures for obtaining incubation conditions. BS 4285-2, Methods of general application for enumeration of microorganisms. BS 4285-2.1, Enumeration of microorganisms by poured plate technique for colony count. BS 4285-3, Methods for detection, and/or enumeration of specific groups of microorganisms 1) .
25、 BS 4285-3.4, Enumeration of lipolytic microorganisms. BS 4285-3.4.1, Victoria blue method. 1) Referred to in the foreword only.BS 4285-3.4.2: 1989 BSI 389 Chiswick High Road London W4 4AL BSIBritishStandardsInstitution BSI is the independent national body responsible for preparing BritishStandards.
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