1、BSI Standards PublicationBS ISO 10718:2015Cork stoppers Characterization of a low-in-germs stopper, throughthe enumeration of colony-forming units of yeasts, mouldsand bacteria, capable of bothbeing extracted and growing inalcoholic mediumBS ISO 10718:2015 BRITISH STANDARDNational forewordThis Briti
2、sh Standard is the UK implementation of ISO 10718:2015.It supersedes BS ISO 10718:2002 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee PRI/81, Cork.A list of organizations represented on this committee can beobtained on request to its secretary.This pub
3、lication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2015. Published by BSI Standards Limited 2015ISBN 978 0 580 87805 3ICS 07.100.30; 55.100; 79.100Compliance with a British Standard canno
4、t confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 October 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 10718:2015 ISO 2015Cork stoppers Characterization of a low
5、-in-germs stopper, through the enumeration of colony-forming units of yeasts, moulds and bacteria, capable of both being extracted and growing in alcoholic mediumBouchons en lige Caractrisation dun bouchon pauvre en germes par dnombrement des units formant colonie de levures, de moisissures et de ba
6、ctries, extraites en milieu alcoolique et capables de sy dvelopperINTERNATIONAL STANDARDISO10718Third edition2015-10-15Reference numberISO 10718:2015(E)BS ISO 10718:2015ISO 10718:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015, Published in SwitzerlandAll rights reserved.
7、 Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at
8、the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 10718:2015ISO 10718:2015(E)Foreword iv1 Scope . 12 Normative references 1
9、3 Low-in-germs stoppers 14 Principle 15 Reagents and cultural media . 16 Apparatus . 37 Sampling 38 Test condition 39 Extraction . 410 Procedures 410.1 General . 410.2 Fast determination using a filtration system and a ready to use sterile culture media . 410.2.1 Preparation . 410.2.2 Seeding on WLD
10、 . 410.2.3 Seeding on M-Green 410.3 Determination using a filtration system to be sterilized and a dehydrated cultural media . 510.3.1 Preparation of media 510.3.2 Preparation of filtration system 510.3.3 Seeding on WLD . 510.3.4 Seeding on M-Green 511 Blank test 512 Incubation . 513 Expression of r
11、esults 513.1 Determination of the cfu number of bacteria per cork stopper . 513.2 Determination of the cfu number of yeasts and moulds per cork stopper . 614 Test report . 6 ISO 2015 All rights reserved iiiContents PageBS ISO 10718:2015ISO 10718:2015(E)ForewordISO (the International Organization for
12、 Standardization) 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
13、right to 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.The procedu
14、res used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial
15、rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights ide
16、ntified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation
17、 on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is
18、 ISO/TC 87, Cork.This third edition cancels and replaces the second edition (ISO 10718:2002), which has been technically revised.iv ISO 2015 All rights reservedBS ISO 10718:2015INTERNATIONAL STANDARD ISO 10718:2015(E)Cork stoppers Characterization of a low-in-germs stopper, through the enumeration o
19、f colony-forming units of yeasts, moulds and bacteria, capable of both being extracted and growing in alcoholic medium1 ScopeThis International Standard specifies a method to enumerate the colony-forming units of yeasts, moulds and bacteria which can exist on cork stoppers and in an alcoholic soluti
20、on, and which, under certain conditions, can be extracted during the 3 months following delivery.This International Standard applies to all types of ready-to-use cork stoppers, submitted to a sanitation process and packaged in properly aseptic and hermetic conditions.This International Standard spec
21、ifies the limit values of the colony-forming units of yeasts, moulds and bacteria which can be found on cork stoppers submitted to the test procedures included in this standard.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are ind
22、ispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiologica
23、l examinationsISO 17727, Cork Cork stoppers for still wine Sampling plan for the quality control of cork stoppers3 Low-in-germs stoppersCork stoppers submitted to test methods specified in this International Standard are designated as low-in-germs stoppers when the following results are obtained: 10
24、 cfu bacteria per stopper (see 13.1) 10 cfu yeast and moulds per stopper (see 13.2)4 PrincipleDirect counting of colonies of living microorganisms (yeasts, moulds and bacteria) by incubation in a specific cultural medium after extraction with an alcoholic solution with added tartaric acid and follow
25、ed by a membrane filtration procedure.5 Reagents and cultural media5.1 Physiological solution (0,85 % NaCl)1)or Ringers solution (1/4 X)1)with the following composition:1) This product is commercially available. ISO 2015 All rights reserved 1BS ISO 10718:2015ISO 10718:2015(E)Sodium chloride 2,25 g/l
26、Potassium chloride 0,105 g/lCalcium chloride 6H20 12 g/lSodium bicarbonate 0,05 g/lFinal pH (obtained from the mixture) 7,0 0,25.2 WLD (for counting bacteria) with the following composition:Yeast extract 4,0 g/lCasein hydrolysate 5,0 g/lDextrose 50,0 g/lPotassium dihydrogen phosphate 0,55 g/lPotassi
27、um chloride 0,425 g/lCalcium chloride 0,125 g/lMagnesium sulfate 0,125 g/lManganese sulfate 0,002 5 g/lFerric chloride 0,002 5 g/lBromocresol green 0,022 g/lCycloheximide (actidione) 0,004 g/lFinal pH (obtained from the mixture) 5,5 0,25.3 M-Green (for counting yeasts and moulds) with the following
28、composition:Yeasts extract 9,0 g/lDextrose (cerelose) 50,0 g/lPeptone 10,0 g/lMagnesium sulfate 2,10 g/lPotassium phosphate 2,0 g/lDiastase 0,05 g/lThiamine 0,05 g/lBromocresol green 0,026 g/lFinal pH (obtained from the mixture) 4,6 0,25.4 Tartaric acid.5.5 Ethanol, 96 %.5.6 Wetting agent.2 ISO 2015
29、 All rights reservedBS ISO 10718:2015ISO 10718:2015(E)5.7 Triptone gel.5.8 Diphenyl.5.9 Demineralized water (or water with a similar purity).Reagents and cultural media should be stored according to the manufacturers instructions.6 ApparatusUsual microbiological laboratory apparatus and, in particul
30、ar, the following.6.1 Membrane filtration system.One of the membrane filtration systems described at 6.1.1 or 6.1.2 may be used.6.1.1 Sterile filtration system, ready-to-use, including a polypropylene funnel with at least a capacity of 100 ml, a sterile membrane (porosity 0,45 m), a sterile dish and
31、 a vacuum pump with three-way cock to turn off the vacuum2).6.1.2 Traditional filtration system, including a funnel with a minimum capacity of 100 ml (of stainless steel, glass or polycarbonate which can be sterilized in an autoclave or an oven), a sterile membrane (porosity 0,45 m), a sterile Petri
32、 dish with blotting pad and a vacuum pump.6.2 Incubator, capable of being controlled at 30 C 2 C.6.3 Refrigerator, capable of being controlled at a temperature between 2 C and 8 C.6.4 Orbital shaker, with a plate or a wrist-action shaker or a rocking-motion shaker that, depending on the model, can b
33、e set at a speed between 140 r/min and 160 r/min or 140 to 160 osc/min or 140 to 160 back-and-forth motions/min.6.5 pH-meter with a temperature compensation, accurate to 0,1 at 25 C.6.6 Glass flasks, with screw caps and appropriate capacity to allow the four stoppers to be immersed in a 100 ml solut
34、ion.7 SamplingSampling shall be carried out aseptically according to ISO 17727.Use sterile containers to preserve the sample at a temperature between 2 C to 8 C up to the time of testing.8 Test conditionThe preparation of the material and the test procedure shall be carried out aseptically and follo
35、wing the rules specified in ISO 7218.2) This system is commercially available. ISO 2015 All rights reserved 3BS ISO 10718:2015ISO 10718:2015(E)9 Extraction9.1 Prepare the physiological solution or Ringers solution (5.1). While stirring, add the wetting agent (5.6) to obtain a 10 g/l concentration an
36、d then add triptone gel (5.7) to obtain a 1 g/l concentration. Afterwards, adjust to a pH value between 3 and 3,5 using tartaric acid (5.4). Dispense about 90 ml of the solution to each flask (6.6) and sterilize.9.2 After cooling, add to each flask, 10 ml of ethanol (5.5) aseptically.9.3 Put four co
37、rk stoppers into each flask, checking that the cork stoppers are completely immersed. Shake the flasks for 1 h at a speed between 140 rpm to 160 rpm, and a temperature between 20 C and 25 C.The number of flasks depends on the sampling plan that has been chosen. Half of the flasks are to be used for
38、seeding on WLD and the remaining for seeding on M-Green.For each cultural media, prepare an additional flask for the blank test.10 Procedures10.1 GeneralFollow procedure 10.2 when using a sterile filtration system and a sterile cultural media that is ready to use.Follow procedure 10.3 when using a f
39、iltration system that has to be sterilized and a dehydrated cultural media.10.2 Fast determination using a filtration system and a ready to use sterile culture media10.2.1 PreparationPrepare the filtration system (6.1.1).10.2.2 Seeding on WLDPlace the complete funnel with the sterile membrane on the
40、 vacuum-pump filtration head. Aseptically filter the extraction solution prepared in accordance with Clause 9. At the end of the filtration, turn off the vacuum from the suction circuit to re-equilibrate the atmospheric pressure.Just before the seeding, add diphenyl (5.8) dissolved in a 10 % ethanol
41、 solution to the WLD media (5.2) in order to obtain a 30 ppm diphenyl concentration. Add the WLD media contained in the ampoule suck it lightly and turn off the vacuum. Remove the filtration set and put the stopper on the base of the filtration set to avoid retro-infection. Take away the cylindrical
42、 part of the funnel. Lift the funnel cap and fit it on the set filter/Petri dish set.Repeat this procedure for each flask.10.2.3 Seeding on M-GreenPlace the complete funnel with the sterile membrane on the vacuum-pump filtration head. Aseptically filter the extraction solution prepared in accordance
43、 with Clause 9. At the end of the filtration, turn off the vacuum from the suction circuit to re-equilibrate the atmospheric pressure.Add the M-Green cultural media (5.3) contained in the ampoule, suck it lightly and turn off the vacuum. Remove the filtration set and put the stopper on the base of t
44、he filtration set to avoid retro-infection. Take away the cylindrical part of the funnel. Lift the funnel cap and fit it on the set filter/Petri dish set.4 ISO 2015 All rights reservedBS ISO 10718:2015ISO 10718:2015(E)Repeat this procedure for each flask.Dehydrated cultural media on the membrane sha
45、ll be rehydrated using sterilized and demineralized water.10.3 Determination using a filtration system to be sterilized and a dehydrated cultural media10.3.1 Preparation of mediaPrepare and sterilize the WLD media (5.2) and the M-Green media (5.3), following the manufacturers instructions.Add diphen
46、yl (5.8), dissolved in a 10 % ethanolic solution, to the WLD media to obtain a 30 ppm concentration in diphenyl.Prepare the Petri dishes.10.3.2 Preparation of filtration systemSterilize and prepare the filtration system (6.1.2).10.3.3 Seeding on WLDAseptically filter the extraction solution prepared
47、 in accordance with Clause 9 using a sterile membrane.Place the membrane on a Petri dish containing WLD.Repeat this procedure for each flask.10.3.4 Seeding on M-GreenAseptically filter the extraction solution prepared in accordance with Clause 9, using a sterile membrane.Place the membrane on a Petr
48、i dish containing M-Green.Repeat this procedure for each flask.11 Blank testPrepare a blank test for each media.12 IncubationInvert the WLD and the M-Green dishes and incubate in an incubator (6.2) at 30 C 2 C for 3 days.Observe and count the colonies on each plate at least every 24 h.13 Expression
49、of results13.1 Determination of the cfu number of bacteria per cork stopperAfter the specified incubation period, count the colonies of bacteria on each WLD dish, always referring to the last valid counting. ISO 2015 All rights reserved 5BS ISO 10718:2015ISO 10718:2015(E)For each dish, the number of colony-forming units (cfu) per cork stopper is given by the following formula:Nb4whereNbis the total number of colonies of bacteria counted;4 is the number of cork stoppers tested.The test result is the arithmetic
