1、BRITISH STANDARD BS EN ISO 20645:2004 Textile fabrics Determination of antibacterial activity Agar diffusion plate test The European Standard EN ISO 20645:2004 has the status of a British Standard ICS 59.080.30 BS EN ISO 20645:2004 This British Standard was published under the authority of the Stand
2、ards Policy and Strategy Committee on 20 December 2004 BSI 20 December 2004 ISBN 0 580 45126 7 National foreword This British Standard is the official English language version of EN ISO 20645:2004. It is identical with ISO 20645:2004. The UK participation in its preparation was entrusted to Technica
3、l Committee TCI/80, Textiles Chemical testing, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this documen
4、t may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Us
5、ers are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for chang
6、e, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, pages 2 to 13 and a back cover. The BSI copyright notice displayed in th
7、is document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM ENISO20645 December2004 ICS59.080.30 Englishversion TextilefabricsDeterminationofantibacterialactivityAgar diffusionplatetest(ISO20645:200
8、4) EtoffesContrledelactivitantibactrienneEssaide diffusionsurplaquesdeglose(ISO20645:2004) TextileFlchengebildePrfungderantibakteriellen WirkungAgarplattendiffusionstest(ISO20645:2004) ThisEuropeanStandardwasapprovedbyCENon2September2004. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulatio
9、nswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(
10、English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,Fra
11、nce, Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStass
12、art,36B1050Brussels 2004CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.ENISO20645:2004:EEN ISO 20645:2004 (E) 2 Contents Page Foreword3 Introduction .4 1 Scope 5 2 Terms and definitions .5 3 Safety precautions.5 4 Principle5 5 Apparatus, reagents and
13、 culture media 5 5.1 Apparatus .5 5.2 Reagents and culture media.6 6 Test bacteria.7 7 Preparation of the bacteria cultures 7 7.1 General7 7.2 Culturing with lyophilized bacteria 7 7.3 Culturing from the agar.7 8 Preparation of test specimens .8 8.1 General8 8.2 Preparation of the test material8 8.3
14、 Material for blind tests 8 8.4 Conditioning of specimens.8 9 Test procedure.8 10 Assessment of the tests .9 11 Evaluation.10 12 Test report 10 Annex A (informative) Special specimens and tests .11 Annex B (informative) European Suppliers of ATCC.12 Bibliography 13 EN ISO 20645:2004 (E) 3 Foreword T
15、his document (EN ISO 20645:2004) has been prepared by Technical Committee CEN/TC 248 “Textiles and textile products”, the secretariat of which is held by BSI, in collaboration with Technical Committee ISO/TC 38 “Textiles”. This European Standard shall be given the status of a national standard, eith
16、er by publication of an identical text or by endorsement, at the latest by June 2005, and conflicting national standards shall be withdrawn at the latest by June 2005. This document includes a Bibliography. CAUTION This method involves the use of processes that could lead to a hazardous situation. A
17、ttention is drawn to the safety precautions in Clause 3. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germ
18、any, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN ISO 20645:2004 (E) 4 Introduction The application of an antimicrobial finish to a textile can prevent bact
19、erial growth and might reduce the effects of microbial pathway products, biodeterioration and microbiogenous odours. This method determines the activity of such treatments qualitatively when different products are compared. Semi-quantitative information on the effect of treatments can be obtained wh
20、en different concentrations of the same product are compared. EN ISO 20645:2004 (E) 5 1 Scope This document specifies a method for the determination of the effect of antibacterial treatments applied to woven, knitted and other flat textiles. This method is applicable to testing hygienic finishes of
21、hydrophilic, air-permeable materials or antibacterial products incorporated in the fibre. A minimum diffusion of the antibacterial treatment into the test agar is necessary with this procedure. NOTE Other materials may be tested using this method, provided that it is adapted accordingly. This method
22、 is not suitable for testing textiles treated with antibacterial treatments that react with the agar. 2 Terms and definitions For the purposes of this document, the following term and definition applies. antibacterial effect inhibition of bacterial growth in favourable growing conditions 3 Safety pr
23、ecautions This method requires the use of bacteria and conditions that promote bacterial growth. Since the bacteria might be pathogenic the tests should be carried out by trained personnel. Appropriate safety precautions should be observed. 4 Principle Specimens of the material to be tested are plac
24、ed on two-layer agar plates. The lower layer consists of a culture medium free from bacteria and the upper layer is inoculated with the selected bacteria. The textiles are tested on both sides. The level of antibacterial activity is assessed by examining the extent of bacterial growth in the contact
25、 zone between the agar and the specimen and, if present, the extent of the inhibition zone around the specimen. 5 Apparatus, reagents and culture media 5.1 Apparatus 5.1.1 Incubator , capable of maintaining a temperature of (37 1)C. 5.1.2 Autoclave, capable of operating at 121C and 205 kPa (2,05 bar
26、). 5.1.3 Water bath, capable of maintaining a temperature of (45 2)C. 5.1.4 Shaker, for test tubes 5.1.5 Microscope, 20 magnification, lighting from beneath (lens 20x, stereomicroscope 20x). 5.1.6 Petri dishes, of glass or polystyrene construction and 9 cm inner diameter EN ISO 20645:2004 (E) 6 5.2
27、Reagents and culture media 5.2.1 Reagents Use only reagents of recognized analytical grade and distilled water or water of equivalent purity. 5.2.2 Culture media 5.2.2.1 Dry agar, available commercially with the following composition 1 . Should commercially available agars be unsuitable for the bact
28、eria to be tested, the culture media shall be adapted or replaced accordingly. Such changes shall be mentioned in the test report 5.2.2.2 Composition of the nutrient broth for test strains: trypton peptone 17 g phyton peptone 3 g sodium chloride 5 g di-potassium hydrogen phosphate 2,5 g dextrose 2,5
29、 g distilled water 1 000 ml Preparation Prepare the nutrient broth by heating the above solids in water until they are completely dissolved. Sterilize the broth at 121 C (205 kPa) in the autoclave for 15 min. After sterilization, the pH of the broth shall be 7,3 0,1 at 20 C. 5.2.2.3 Composition of t
30、he medium for tests: trypton peptone 15 g phyton peptone 5 g sodium chloride 5 g agar-agar 15 g distilled water 1 000 ml 1)Trypticase Soy Agar / Broth (BBL); Tryptic Soy Agar / Broth (Difco); CASO Agar / Broth (Merck); Trypton Soya Agar / Broth (Oxoid) are examples of suitable products available com
31、mercially This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN or ISO of these products. EN ISO 20645:2004 (E) 7 6 Test bacteria The following gram positive strain and one of the two gram negative strains shall be used. Staphylococcus
32、aureus gram positive ATCC 2)6538 or NCCB 3)46064 Escherichia coli gram negative ATCC 11229 or NCCB 1500 Klebsiella pneumoniae gram negative ATCC 4352 or NCCB 89160 In order to guarantee comparable, reproducible results, only strains supplied by a recognised culture collection shall be used. NOTE Dep
33、ending on the range of application and composition of the textile under test, the spectrum of test bacteria may be enlarged. Should bacteria other than those specified be used, the method of culture, the culture media and the incubation temperature may need to be adjusted. Such changes should be ind
34、icated in the report. 7 Preparation of the bacteria cultures 7.1 General The procedure described refers to the culturing of stock and test strains of bacteria specifically for the tests. Laboratories should apply the procedure EN 12353 for the preservation of microbial strains. 7.2 Culturing with ly
35、ophilized bacteria Suspend the lyophilized bacteria in an adequate quantity of nutrient broth. Prepare liquid sub-cultures from the suspension and prepare an agar plate culture. Check the purity of the culture by streak plates, and confirm the identity by microscopic examination and gram stain. Effe
36、ct “liquid to liquid“ transfers for up to three days to avoid the opportunity for contamination. Incubate for 24 h in each case at (37 1) C. Verify the purity of the colonies again by spreading on agars. Should the series of three to four “liquid to liquid“ transfers be interrupted by a weekend, a 1
37、6 h to 24 h old culture shall be placed in the refrigerator (3 C to 4 C) on Friday and reinoculated at the latest on Tuesday for a “liquid“ transfer over a minimum of 24 h. 7.3 Culturing from the agar Prepare a first liquid sub-culture from the agar and prepare an agar plate culture. The culture sho
38、uld be no older than 4 weeks. Check the purity of the culture by streak plates and confirm the identity by microscopic examination and gram stain. Effect “liquid to liquid“ transfers for up to three days to avoid the opportunity for contamination. Incubate for 24 h in each case at (37 1) C. Verify t
39、he purity of the colonies again by spreading on agar. 2)American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209,USA,. Tel: +703.365.27003)The Netherlands Culture Collection of Bacteria, Utrecht Univ., Uppsalalaan 8, P.O. Box 85167, 3508 AD Utrecht, The Netherlands
40、,Tel: +31 (30) 2122634 EN ISO 20645:2004 (E) 8 Should the series of three to four “liquid to liquid“ transfers be interrupted by a weekend, a 16 h to 24 h old culture shall be placed in the refrigerator (3 C to 4 C) on Friday and reinoculated on Tuesday at the latest for a “liquid“ transfer over a m
41、inimum of 24 h. At the end of the week, destroy all the working cultures and replace with new ones prepared from stock strains cultivated on agar or taken from lyophilized bacteria. After a maximum of 6 months testing, all new cultures shall be based on lyophilized bacteria. 8 Preparation of test sp
42、ecimens 8.1 General The test specimens shall be circular, with a diameter of (25 5) mm. Tests on four specimens are necessary and shall be carried out on both sides of the specimens. NOTE In special cases, the specimens should be prepared in accordance with Annex A. 8.2 Preparation of the test mater
43、ial Unsterilized specimens only shall be used. Any deviations shall be included in the test report. NOTE The test materials or their antibacterial treatment might be altered if the specimens are sterilized. 8.3 Material for blind tests Wherever possible, prepare blind test specimens of identical qua
44、lity and finish but without antibacterial treatment. If such material is not available, use a cotton fabric without antibacterial treatment for the bacterial growth control. 8.4 Conditioning of specimens Store the specimens between 12 h and 24 h in sterilized petri dishes at room temperature. 9 Test
45、 procedure 9.1 Prepare, for the lower layer free from bacteria, the required agar volume. Pour (10 0,1) ml into each of the sterilized petri dishes and let the agar congeal. 9.2 For the upper layer, prepare the required agar quantity and cool to (45 1) C in the water bath (5.1.3). Inoculate 150 ml a
46、gar with (1 0,1) ml of bacterial working culture (1-5 x10 8 cfu/ml). Shake the vessel vigorously to distribute the bacteria evenly. Pour (5 0,1) ml into each petri dish and let the agar congeal. Use the inoculated agar plates within 1 h. 9.3 Since agar plates tend to dry on the surface after casting
47、 and varying degrees of dryness from one zone to the other may lead to an irregular growth of the bacteria, not more than 50 plates, i.e. 500 ml of agar (250 ml per layer) shall be used at a time. 9.4 Press the test specimens taken from woven, knitted and other flat materials with a pair of steriliz
48、ed tweezers or with a bent glass rod evenly on the nutrient medium, until the texture of the fabric is uniformly imprinted and there is good contact between specimen and agar. If necessary, place a sterilized glass ring or inox ring on the specimens to guarantee this contact. NOTE Other materials may need special tests (see Annex A). EN ISO 20645:2004 (E) 9 9.5 Incubate the plates for 18 h to 24 h at (37 1) C immediately after placing the test specimens on the agar and then check for bacterial growth. Ensure that there is con
