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1、 1 AS 5013.292009 Standards Australia Australian Standard Food microbiology Method 29: Examination for specific organisms Colony count of yeasts and moulds PREFACE This Standard was prepared by the Standards Australia Committee FT-024, Food Products and Subcommittee FT-024-01 Food Microbiology (cons
2、tituted), to supersede AS 1766.2.21997, Food microbiology, Method 2.2: Examination for specific organisms Colony count of yeasts and moulds. This edition confirms the Method without technical change but updates the referenced documents and reflects current editorial style. It is one of a series of m
3、ethods for the microbiological examination of foods for quality control and investigative purposes and, where applicable, for checking that foods comply with regulatory specifications. The laboratory should have a clearly defined quality control system to ensure that the apparatus, culture media, re
4、agents and technique are suitable for the test. The use of positive controls is part of this system. The term normative has been used in this Standard to define the application of the appendix to which it applies. A normative appendix is an integral part of a Standard. METHOD 1 SCOPE This Standard s
5、ets out procedures for determining the colony count of yeasts and moulds in foods. 2 REFERENCED DOCUMENTS AS 5013 Food microbiology 5013.11 Method 11: Microbiology of food and animal feeding stuffsPreparation of test samples, initial suspension and decimal dilutions for microbiological examination A
6、S 5013.292009 AS 5013.292009 2 Standards Australia www.standards.org.au 3 CULTURE MEDIA AND DILUENTS 3.1 General Formulations and instructions for preparing culture media and diluents are given in the Appendix A. 3.2 Diluents NOTE: The diluent SHOULD NOT CONTAIN NaCl, as this is deleterious to yeast
7、s. 3.2.1 Peptone solution 0.1% or specified diluent as detailed in AS 5013 series. 3.2.2 Osmophilic diluent 20% to 30% (w/v) glucose in 0.1% peptone is recommended (see Clause 6, Reference 5). 3.3 Dichloran Rose Bengal Chloramphenicol Agar 3.4 Dichloran 18% Glycerol Agar (DG18) 4 PROCEDURE The proce
8、dure shall be as follows: (a) Prepare dilutions of the sample as described in AS 5013.11, using Peptone Solution 0.1%. Use osmophilic diluent if enumerating yeasts in concentrates, syrups and other low water activity samples (3.2). Several dilutions may be required. (b) Using the procedures describe
9、d for the Surface Spread Method in AS 5013, prepare and inoculate spread plates according to the following: (i) For high water activity foods (aw 0.95), use Dichloran Rose Bengal Chloramphenicol (DRBC) agar. (ii) For reduced water activity foods (aw 0.95) use Dichloran 18% Glycerol (DG18) agar. NOTE
10、: A reference to a Table on water activity of foods is given in Clause 6 (see reference 6). (c) Incubate the plates, upright at 25 1C for 5 d, unless specified otherwise in the AS 5013 series. DG18 plates should be incubated for 7 d. Examine the plates after 68 4 h and then at 24 h intervals, and co
11、unt the colonies on any plates likely to be overgrown before the full incubation period. (d) Confirm presumptive yeast colonies by microscopic examination of each colony type, as some bacteria are capable of growth on DRBC agar. 5 TEST REPORT The following information shall be reported: (a) All deta
12、ils necessary for the complete identification of the sample. (b) Reference to this Australian Standard, i.e. AS 5013.29. (c) Date of testing. (d) Any abnormality observed in the physical condition of the container or product. (e) Number of yeasts per millilitre or gram of material under test. (f) Nu
13、mber of moulds per millilitre or gram of material under test. (g) Any circumstances or conditions that may have influenced the test results. 3 AS 5013.292009 www.standards.org.au Standards Australia 6 BIBLIOGRAPHY Scientific findings published in the following papers were taken into account in the p
14、reparation of this Standard: 1 KING, A.D., HOCKING, A.D. and PITT, J.I. Dichloran-rose bengal medium for enumeration and isolation of moulds from foods. Appl. Environ. Microbiol. 1979., Vol. 37, pp. 959964. 2 HOCKING, A.D. and PITT, J.I. Dichloran-glycerol medium for enumeration of xerophilic fungi
15、from low-moisture foods. Appl. Environ. Microbiol. 1980, Vol. 39, pp. 488492. 3 BEUCHAT, L.R. Media for detecting and enumerating yeasts and moulds. Int. J. Food Microbiol. 1992. Vol. 17, pp. 145158. 4 SAMSON, R.A., HOCKING, A.D., PITT, J.I. and KING, A.D. (eds) 1992, Modern Methods in Food Mycology
16、. Elsevier, Amsterdam. 5 PITT, J.I., HOCKING, A.D., SAMSON, R.A. and KING, A.D. 1992. Recommended methods for mycological examination of food, 1992. In SAMSON, R.A., HOCKING, A.D., PITT, J.I. and KING, A.D. (eds) 1992. Modern Methods in Food Mycology, Elsevier, Amsterdam, pp. 365368. 6 TROLLER, J.A.
17、, CHRISTIAN, J.H.B. Water Activity and Foods Academic Press, New York, San Francisco, London, 1978, p. 214. AS 5013.292009 4 Standards Australia www.standards.org.au APPENDIX A CULTURE MEDIA (Normative) NOTE: For the preparation of diluents, dehydrated basic components or equivalent commercially pre
18、pared reagents may be used. The manufacturers instructions should be rigorously followed. A1 PEPTONE SOLUTION, 0.1% Ref. STRAKA, R.P. and STOKES, J.L. Appl. Microbiol. 1957, Vol. 5, p. 21. Peptone 10 g. Water 10 L. Dissolve peptone and adjust pH to 7.0 0.1. Dispense as required and autoclave at 121C
19、 for 15 min. A2 DICHLORAN ROSE BENGAL CHLORAMPHENICOL AGAR (see Clause 6, reference 1) A2.1 Formulation Glucose.10.0 g. Peptone .5.0 g. KH2PO4.1.0 g. MgSO4.7H2O.0.5 g. Rose Bengal (5% aq. soln, w/v) 0.5 mL. Chloramphenicol .0.1 g. Dichloran (0.2% in ethanol). 1.0 mL. Agar15.0 g. Water. 1.0 L. Dissol
20、ve ingredients and sterilize by autoclaving at 121C for 15 min. Final pH 5.6 0.2. Note that media containing Rose Bengal are sensitive to light. Inhibitory compounds are produced after relatively short exposures (8 h or more) to fluorescent illumination or strong daylight. Prepared plates of DRBC sh
21、ould be stored at 14C, protected from light (e.g. in black plastic bags). Under these conditions, prepared plates may be stored for up to 4 weeks. Inoculated plates should be incubated in the dark. For foods expected to contain large numbers of Gram negative rods, Gentamicin solution, (see Paragraph
22、 A2.2) should be added to prepared medium (50 mL per litre of medium) just before pouring. NOTE: This medium is available commercially in dehydrated form. A2.2 Preparation of Gentamicin solution Gentamicin1.0 g. Water. 1.0 L. Dissolve gentamicin and sterilize by filtration. 5 AS 5013.292009 www.stan
23、dards.org.au Standards Australia A3 DICHLORAN 18% GLYCEROL AGAR (DG18) (see Clause 6, reference 2) A3.1 Formulation Basal medium (see Paragraph A3.2) 1.0 L. Glycerol (analytical reagent grade).220.0 g. After steaming basal medium for 30 min, add 220 g glycerol, mix well, then sterilize by autoclavin
24、g at 121C for 15 min. Final pH 5.6 0.2; aw 0.955. A3.2 Basal medium Glucose.10.0 g. Peptone .5.0 g. KH2PO4.1.0 g. MgSO4.7H2O.0.5 g. Chloramphenicol .0.1 g. Dichloran (0.2% in ethanol). 1.0 mL. Agar15.0 g. Water. 1.0 L. Mix ingredients well, then steam for 30 min or bring to the boil to ensure agar a
25、nd all other ingredients are completely dissolved. NOTE: This basal medium is available commercially in dehydrated form. AS 5013.292009 6 NOTES 7 AS 5013.292009 NOTES AS 5013.292009 8 This Australian Standard was prepared by Committee FT-024, Food Products. It was approved on behalf of the Council o
26、f Standards Australia on 5 June 2009 and published on 14 July 2009. The following are represented on Committee FT-024: Australian Chamber of Commerce and Industry Australian Food and Grocery Council Australian Institute of Food Science and Technology Consumers Federation of Australia Department of A
27、griculture, Fisheries and Forestry (Commonwealth) Department of Primary Industries, Vic. Horticulture Australia Meat & Livestock Australia National Association of Testing Authorities Australia National Measurement Institute Safe Food Queensland Subcommittee representatives: ACT Health Australian Foo
28、d and Grocery Council Australian Institute of Food Science and Technology Australian Society for Microbiology Dairy Industry Association of Australia Department of Agriculture, Fisheries and Forestry (Commonwealth) Department of Primary Industries, Vic. Food Science Australia Food Standards Australi
29、a New Zealand Food Technology Association of Victoria Institute of Clinical Pathology and Medical Research Meat & Livestock Australia National Association of Testing Authorities Australia National Measurement Institute Queensland Health Scientific Services University of Melbourne Keeping Standards u
30、p-to-date Standards are living documents which reflect progress in science, technology and systems. To maintain their currency, all Standards are periodically reviewed, and new editions are published. Between editions, amendments may be issued. Standards may also be withdrawn. It is important that r
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33、476, Sydney, NSW 2001. Originated in part as AS 1095.3.31974. Previous edition AS 1766.2.21994. Revised and redesignated as AS 5013.292009. This Standard was issued in draft form for comment as DR 07428. COPYRIGHT Standards Australia All rights are reserved. No part of this work may be reproduced or copied in any form or by any means, electronic or mechanical, including photocopying, without the written permission of the publisher. Published by Standards Australia Limited GPO Box 476, Sydney, NSW 2001, Australia ISBN 0 7337 9184 0 Printed in Australia