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1、AS 5013.14.220091 AS 5013.14.22009Australian StandardFood microbiologyMethod 14.2: General procedures and techniquesColony countMembrane filtration methodPREFACEThis Standard was prepared by the Standards Australia Committee FT-024, Food Products andSubcommitteeFT-024-01,FoodMicrobiology(Constituted
2、), to supersede AS 1766.1.51991, Food microbiology, Method 1.5: General procedures and techniques Colony countMembrane filtration method.The objective of this revision is to update the references and to transfer the procedure to AS 5013 series. It also incorporates minor technical variations on the
3、apparatus used in the test technique.METHOD1 SCOPEThis Standard sets out a method for estimating the number of colony-forming units (CFUs)in suitable liquids using a membrane filtration technique.The method is applicable only to liquids that can be efficiently filtered without causing a build-up on
4、the filter.NOTE: The membrane filtration method is most suitable when the microorganisms are in low concentration, e.g. in rinse waters collected from cleaned and sanitized pipelines or tanks. The method is suitable for microbiological assessment of water supplies to processing plants.2 REFERENCED D
5、OCUMENTSThe following documents are referred to in this Standard: AS5013Food microbiology5013.14Method 14: Microbiology of food and animal feeding stuffsGeneral rules for microbiological examinations Standards AustraliaAS 5013.14.22009 23 PRINCIPLEThe method involves passing a liquid sample through
6、a membrane of known physical properties. Microorganisms in the sample are retained on the membrane which is then placed on a filter pad saturated with liquid medium, or on solid medium, and incubated. Colonies, corresponding to the viable organisms collected on the filter, are then counted.4 DILUENT
7、S AND CULTURE MEDIAThe diluents and culture media shall be as specified in the relevant methods of AS 5013 according to the product under examination and the microorganisms to be enumerated.Where the medium does not give rise to a good contrast for the colonies developed, a stain comprising 0.01 per
8、cent aqueous solution of malachite green oxalate is required.5 APPARATUSThe following apparatus is required: (a)Membrane filtration apparatus.NOTE: Various types of apparatus suitable for the membrane filtration method are availablecommercially in Australia.(b) Grid-mark membrane filters of 47 mm to
9、 50 mm diameter to fit the apparatus and having a pore size appropriate to the organism or organisms to be assessed. The usual size for water is 0.45 m.NOTE: Sterile membrane filters are commercially available.(c) A filter flask of capacity appropriate to the volume of liquid to be filtered. (d)A so
10、urce of vacuum.(e) Forceps that will not damage the membrane. (f)Petri dishes.(g)Graduated measuring cylinders, where required, suitable for measuring the volume of sample to be filtered.(h) Graduated pipettes. (i)A tally counter.(j) A suitable incubator.(k)Non-toxic absorbent filter pads of diamete
11、r either equal to or slightly greater than the diameter of the membrane filters with which they are to be used.(l) Pasteur pipettes.NOTE: Items (k) and (l) above are only required where the nutrient pad technique is to be used.6 PREPARATION OF APPARATUS AND MATERIALS6.1 SterilizationThe apparatus an
12、d materials shall be sterilized as follows:(a) Wrap the base of the membrane filtration apparatus, with the membrane support in position, in kraft paper or other suitable material. Wrap the filter funnel separately and sterilize the contents of both parcels by autoclaving.NOTE: Some commercial unit
13、can be sterilized fully assembled.(b) Sterilize the remainder of the glassware as described in AS 5013.14. Standards Australiawww.standards.org.au3 AS 5013.14.22009(c) Sterilize the membrane filters according to the suppliers instructions.NOTES:1The filtration characteristics of the membranes can be
14、 adversely affected by overheating.2Sterile membrane filters are available commercially.6.2 Medium6.2.1 Liquid mediumWhere used, a filter pad shall be soaked with liquid medium as follows: (a)Using forceps, place a filter pad in a Petri dish.(b) Add sufficient liquid medium to saturate the pad and t
15、o result in a small excess.NOTE: A 5 cm (in diameter) filter pad will require 2.0 mL to 2.5 mL of medium.6.2.2 Solid mediumWhere a solid medium is used, the plates should be prepared as follows:(a) Pour sufficient volume of molten medium into a Petri dish to give a depth of approximately 5 mm. Allow
16、 to set.(b) As soon as the medium has set, remove excess moisture from the plates by one of the following methods:(i) Incubate the plates open with the internal surface of the base facing downwards and with the base resting on the lid, for the minimum time necessary to obtain plates free from conden
17、sate, e.g. at 37C for about 2 h or at 45C for about 1 h. Do not dry plates at temperatures above 45C.(ii) Incubate at 37C for 16 h in the inverted position with the lids on. If the plates are not then free of condensate, open them and incubate as described in Step (i) above until dry.(iii) Other sui
18、table time/temperature regimes. For example, on bench over night.NOTE: In humid climates either extend drying time or place a tray of desiccant, such as silica gel, in the base of the drying oven.7 FILTRATION PROCEDURE7.1 GeneralThe optimum volume of liquid to be used will depend upon the amount of
19、undissolved solids in the sample and the expected count.If the expected count is high, suitable dilutions of the sample should be made (see Note). Where the expected count is uncertain, it is recommended that two determinations be made using two different volumes. In this way, the probability that a
20、t least one determination will be within the range 20 to 80 will be increased.NOTE: The optimum number of colonies on the filter is about 50 and the volume of sample filtered should be such that the number of colonies to be counted on the membrane is not greater than 80. For example, samples expecte
21、d to contain less than 80 organisms per 100 mL require the filtration of at least 100 mL of sample for each test.7.2 ProcedureThe procedure shall be as follows:NOTE: Where a pre-assembled filtration unit is used, Steps (a) to (c) below are not required.(a) Assemble the funnel base in the filter flas
22、k and connect the flask to the source of vacuum. Apply a slight vacuum. Standards Australiawww.standards.org.auAS 5013.14.22009 4(b) Using sterile forceps, centre a membrane filter on the membrane support with the grid-marked side upwards.(c) Place the funnel in position and having tightened it, tur
23、n off the vacuum.(d) Pour a measured volume of sample into the filter funnel. Gently apply vacuum and gradually increase it to that recommended by the supplier of the membrane. Where no such value is indicated, adjust the vacuum to about 40 kPa.NOTE: When the volume to be filtered is less than 10 mL
24、, add at least 20 mL of sterile diluent to the funnel before addition of the sample to aid uniform dispersion of the bacteria over the entire surface of the membrane during filtration.(e) When the level of the sample has fallen to within about 6 mm of the membrane, reduce the vacuum to approximately
25、 10 kPa. Rinse the sides of the funnel with20 mL to 30 mL of diluent, added from the graduated cylinder used to measure thevolume of the sample.(f) Remove any liquid medium in the Petri dish in excess of that required to saturate the filter pad.NOTE: A sterile Pasteur pipette is convenient for this
26、operation.(g)Immediately filtration has ceased, turn off the vacuum at the control tap, disconnect the funnel and remove the membrane using sterile forceps. Roll the membrane grid- marked side upwards, on the filter pad or on the solid medium, taking care to avoid entrapping air bubbles between the
27、membrane and the substrate. Replace the lid on the Petri dish.NOTE: Where there is no control tap directly under the funnel, it may be necessary to release the vacuum just before the completion of filtration, to avoid excessive drying of the membrane filter.8 INCUBATIONThe dishes shall be incubated
28、as follows:(a) Transfer the Petri dishes to the incubator. Place the plates in either an upright or an inverted position, according to the instructions for the organisms under test.(b) Distribute the dishes in such a manner that overcrowding is avoided and there is no contact with the sides of the i
29、ncubator.(c) Incubate the dishes at the temperature and for the period specified for the organism to be estimated.9 STAINING, COUNTING AND IDENTIFICATION (See Note 1)Where the medium that has been used does not give rise to a good contrast for the colonies developed, staining of the membrane may be
30、needed. If this is the case, stain the membrane by gently flooding the surface with a 0.01 percent aqueous solution of malachite green oxalate, and after 5 s to 6 s contact, pouring off the excess dye.NOTES:1If required, subculturing of colonies should be carried out before any staining operation.2C
31、olonies normally remain unstained, and the filter area not covered by colonies is stained a light green.Using a tally counter, count the presumptively identified colonies and confirm their identification. Where spreaders occur, count each as a single colony provided that the outer edge of each sprea
32、der can be defined.If the count is greater than 80, repeat the test where possible, using either a smaller volume or a dilution designed to produce a count in the range of 20 to 80. Standards Australiawww.standards.org.au5 AS 5013.14.2200910 CALCULATIONFrom the actual count, the number of organisms
33、per unit volume or per unit mass of the sample shall be calculated taking dilution factors into account.Where tests have been carried out on two different volumes or dilutions (see Clause 7.1) and each membrane has a count within the range 20 to 80, the two counts shall be calculated separately and
34、the mean of the two reported as the result (see AS 5013.14).11 REPORTThe report shall contain the following information:(a) Reference to this Australian Standard, i.e. AS 5013.14.2.(b) The number, and identity if confirmed, of colony-forming units (CFUs) per unit volume or per unit mass sample, stat
35、ing that the count was determined by the membrane filtration method.(c) The presence of spreading organisms, if encountered.(d) The membrane filters used and the suppliers specification of pore size. (e)The culture medium used.(f) The conditions of incubation.(g)Details of confirmation used.www.stan
36、dards.org.au Standards AustraliaAS 5013.14.22009 6NOTES7 AS 5013.14.22009NOTESAS 5013.14.22009 8This Australian Standard was prepared by Committee FT-024, Food Products. It was approved on behalf of the Council of StandardsAustralia on 5 June 2009 and published on 14 July 2009.The following are repr
37、esented on Committee FT-024: Australian Chamber of Commerce and IndustryAustralian Food and Grocery CouncilAustralian Institute of Food Science and TechnologyConsumers Federation of AustraliaDepartment of Agriculture, Fisheries and Forestry (Commonwealth)Department of Primary Industries, Vic.Horticu
38、lture AustraliaMeat & Livestock AustraliaNSW Food AuthorityNational Association of Testing Authorities AustraliaNational Measurement InstituteSafe Food QueenslandSubcommittee representatives: ACT HealthAustralian Food and Grocery CouncilAustralian Institute of Food Science and TechnologyAustralian S
39、ociety for MicrobiologyDairy Industry Association of AustraliaDepartment of Agriculture, Fisheries and Forestry (Commonwealth)Department of Primary Industries, Vic.Food Science AustraliaFood Standards Australia New ZealandFood Technology Association of VictoriaInstitute of Clinical Pathology and Med
40、ical ResearchMeat and Livestock AustraliaNational Association of Testing Authorities AustraliaNational Measurement InstituteQueensland Health Scientific ServicesUniversity of MelbourneKeeping Standards up-to-dateStandards are living documents which reflect progress in science, technology and systems
41、. 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 readers assure themselves they are using a current Standard, which should include any amendments wh
42、ich may have been published since the Standard was purchased.Detailed information about Standards can be found by visiting the Standards Web Shop at .au and looking up the relevant Standard in the on-line catalogue.We also welcome suggestions for the improvement in our Standards, and especially enco
43、urage readers to notify us immediately of any apparent inaccuracies or ambiguities. Contact us via email at mailstandards.org.au, or write to the Chief Executive, Standards Australia Limited, GPO Box 476, Sydney, NSW 2001.Originated as part of AS 1095.11971 and AS 1142.11975. Previous edition AS 176
44、6.1.51991.Revised and redesignated as AS 5013.14.22009.This Standard was issued in draft form for comment as DR 07426.COPYRIGHT Standards AustraliaAll 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 LimitedGPO Box 476, Sydney, NSW 2001, AustraliaISBN 0 7337 9185 9 Printed in Australia国内外标准大全豆丁网国内外标准下载地址
