1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 4159:2011Aerospace series Paints andvarnishes Determination ofresistance to microbial growthBS EN 4159:2011 BRITISH STANDARDNational forewordThis British Standard is the UK
2、 implementation of EN 4159:2011.The UK participation in its preparation was entrusted to TechnicalCommittee ACE/65/-/3, Paints, Surface Finish and ProtectiveTreatments for Aerospace Purposes for Aerospace Purposes.A list of organizations represented on this committee can beobtained on request to its
3、 secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012ISBN 978 0 580 75593 4ICS 49.040Compliance with a British Standard cannot confer immunity fromlegal obligations.T
4、his British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 January 2012.Amendments issued since publicationDate Text affectedBS EN 4159:2011EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 4159 December 2011 ICS 49.040 English Version Aerospace ser
5、ies - Paints and varnishes - Determination of resistance to microbial growth Srie arospatiale - Peintures et vernis - Dtermination de la rsistance laction des microorganismes Luft- und Raumfahrt - Beschichtungsstoffe - Bestimmung der Widerstandsfhigkeit gegen Schimmelwachstum This European Standard
6、was approved by CEN on 27 August 2011. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such
7、national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own l
8、anguage and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
9、Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Br
10、ussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 4159:2011: EBS EN 4159:2011EN 4159:2011 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Terms and definitions .5 3 Principle 5 4 Apparatus .5 5 Specimen 6 6 Pro
11、cedure .6 6.1 Test methods 6 6.1.1 Preparation of agarose + glucose gel 6 6.1.2 Application of gel to coating 6 6.1.3 Preparation of spore suspension .6 6.1.4 Application of suspension to gel and storage at 25 C .7 6.1.5 Microscopic examination 7 6.1.6 Time course of germination 7 7 Designation 8 8
12、Test report 9 Bibliography . 10 BS EN 4159:2011EN 4159:2011 (E) 3 Foreword This document (EN 4159:2011) has been prepared by the Aerospace and Defence Industries Association of Europe - Standardization (ASD-STAN). After enquiries and votes carried out in accordance with the rules of this Association
13、, this Standard has received the approval of the National Associations and the Official Services of the member countries of ASD, prior to its presentation to CEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at
14、 the latest by June 2012, and conflicting national standards shall be withdrawn at the latest by June 2012. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or al
15、l such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hun
16、gary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 4159:2011EN 4159:2011 (E) 4 Introduction Certain fungi are known to be capable of proliferating in fuel sy
17、stems which can cause corrosion and blockage. Conidiospores are the dispersal form of these fungi. Germination of conidia is the first stage in proliferation of the fungus. If the conidiospore cannot germinate, there can be no proliferation and no blockage of fuel lines, ducts etc. This method shoul
18、d be performed only by persons qualified in the microbiology of fungi. The standard can be used to assess the effectiveness of new candidate coating systems in inhibiting microbial (fungal) growth. BS EN 4159:2011EN 4159:2011 (E) 5 1 Scope This European Standard specifies a method to assess the abil
19、ity of biocide-containing coatings to prevent the germination of conidiospores of certain fungi known to be capable of proliferating in fuel systems for aerospace applications. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 Conidiospores sin
20、gle-celled structures produced by the mycelial mould form of the fungus NOTE Conidiospores are spherical or nearly spherical resting cells, i.e. cells which may be dispersed readily but which do not proliferate. However, conidiospores may germinate if they encounter suitable conditions of moisture a
21、nd nutrients. On germination, a conidiospore produces a long tube-like outgrowth which then forms dense branching structures mycelia which may block fuel ducts etc. A suitable coating will prevent germination of conidiospores. A coating which prevents germination of conidiospores is considered to ha
22、ve fungistatic activity. This fungistatic activity may be assessed quantitatively by assessing the success rate of germination of conidiospores under standard conditions (see below) to determine whether the test coating delays or prevents germination of conidiospores when compared with a coating whi
23、ch is known to possess no fungistatic activity. Laboratories which undertake work to this method should first obtain the test fungi (see 5) and perform control experiments to satisfy themselves that they can follow the process of germination of conidiospores. These initial experiments may be perform
24、ed by placing the agarose gel (see below) on the surface of sterile plastic petri dishes rather than on the surface of coated test panels, as is done in the present method. 3 Principle 3.1 Conidiospores are placed on a gel within a few millimetres of the panel/coating under test. Under the test cond
25、itions a high proportion of these conidia germinate begin growth rapidly unless some material in the coating diffuses through the gel and prevents germination. 3.2 The success rate of germination, after any given interval of exposure to the coating, is expressed as the number of cells that have germ
26、inated divided by the number of cells examined (germinated + nongerminated). The success rate of germination is determined from time to time, beginning when the conidiospores are first exposed to the coating under test. Examination is made using a microscope 100. This allows ready distinction betwee
27、n ungerminated conidiospores approximately spherical and the long filamentous outgrowth that is the result of germination. 3.3 The results obtained with conidiospores exposed to test coatings are to be compared with results of conidiospores exposed to coatings that contain no inhibitor. 4 Apparatus
28、4.1 Incubator, capable of maintaining (25 1) C. 4.2 Autoclave suitable for sterilization of microbiological growth media, i.e. capable of heating the media to 121 C for 15 min. 4.3 Water bath, set to (45 1) C. 4.4 Microscope, magnification 100, and glass microscope slides. 4.5 Plastic disposable pet
29、ri dishes - 90 mm to 100 mm diameter. 4.6 Sterile microbiological loops. Commercially available disposable plastic loops (stated to carry 10 l) are suitable. 4.7 Haemocytometer (blood cell counting chamber) Neubauer ruling. BS EN 4159:2011EN 4159:2011 (E) 6 4.8 Funnel, loosely plugged with nonabsorb
30、ent cotton wool. 4.9 Balance, toploading, 0,1 g resolution. 4.10 Micropipetting device (with disposable sterile tips) to deliver 0,010 ml. 4.11 Test fungi, to be obtained from national culture collections: 4.11.1 Amorphotheca resinae (also known as Cladosporium resinae) 4.11.2 Aspergillus niger 4.11
31、.3 National culture collections contain several different strains of each of these fungi. The laboratory should choose a suitable strain (e.g. one isolated from aeronautical fuel tank) by reference to the information supplied by the culture collection. 4.12 Microbiological growth media Rose Bengal C
32、hloramphenicol Agar. The Oxoid product Rose Bengal Chloramphenicol Agar is suitable. 4.13 Agarose The product catalogue No. A0567 of the Sigma Chemical Company is suitable. 4.14 D-glucose The Sigma product catalogue No. G7528 is suitable. 5 Specimen 5.1 Test panels are panels coated on one face only
33、 with a paint suspected of having fungistatic activity. Control panels are similar panels coated on one face only with a similar paint which lacks any fungistatic additive. These coatings must be applied according to the manufacturers instructions. The panel edges may be left uncoated. Panels that a
34、re 10 mm 20 mm (length width) are suitable. Panels should be less than 10 mm thick. Panels are stored at room temperature. 5.2 Three test panels and three control panels are required. 6 Procedure 6.1 Test methods 6.1.1 Preparation of agarose + glucose gel To 100 ml distilled water in a 250 ml conica
35、l flask, add 2 g agarose and 1 g D-glucose. Heat to boiling and swirl thoroughly to obtain a clear solution. Cool the solution in a 45 C water bath. Pour (20 1) g of the cooled solution into a petri dish and allow the solution to set at room temperature to form a clear colourless gel. 6.1.2 Applicat
36、ion of gel to coating Ensure that the test specimens are at room temperature. Cut the gel formed in 6.1.1 so as to fit, approximately, the specimens under test. Place the cut gel on the coated surface of the specimen and trim any excess. Place the gel/specimen in a petri dish and store it at 25 C fo
37、r 22 h to 24 h. 6.1.3 Preparation of spore suspension Ten days prior to test, make up Rose Bengal Chloramphenicol Agar according to the manufacturers directions. Pour (15 1) ml of the sterilized and equilibrated 45 C medium into each of an appropriate number of petri dishes (90 mm diameter) and allo
38、w the agar to form a gel. Seed the prepared medium with the fungus under test. Incubate this culture (see 5.1) for 10 days. BS EN 4159:2011EN 4159:2011 (E) 7 Harvest conidiospores from the culture by passing a sterile microbiological loop gently over the surface of the fungus and then swirling the l
39、oop in a few millilitres of sterile water. Shake the suspension thoroughly to disaggregate conidiospores. Determine the number of conidiospores per ml of suspension using a blood cell counting chamber haemocytometer according to the manufacturers directions. A suitable suspension will be made up alm
40、ost entirely 95 % of conidiospores, the remainder being hyphal fragments readily distinguishible from conidiospores. Remove hyphal fragments by passing the suspension through a funnel loosely plugged with sterile cotton wool. Adjust the suspension to 106conidiospores per ml by either adding water or
41、 centrifuging the suspension and resuspending the conidiospores in a suitable volume of sterile water. Use this suspension within two hours of the time it was formed. 6.1.4 Application of suspension to gel and storage at 25 C After the specimens have been stored for the required time (see 6.1.2), ap
42、ply 0,010 ml of suspension (see 6.1.3) to the gel surface using a micropipettor. Then use a sterile microbiological loop to spread the suspension uniformly on the gel surface. The surface should appear to be dry within a few minutes. Use a razor blade to cut a piece approximately 5 mm 5 mm of gel fo
43、r microscopic examination for time = zero. Mount this on a microscope slide. Place the remainder of the gel + coating + panel in its petri dish and store at 25 C. The required relative humidity during storage at 25 C is attained by placing the specimens under test in petri dishes held in loose fitti
44、ng plastic bags. Each such bag should also contain a petri dish which contains sterile filter paper saturated with sterile distilled water. 6.1.5 Microscopic examination Examine the slide prepared in 6.1.4 at a magnification of 100. Count all cells whether they are conidia or hyphal fragments in sev
45、eral microscopic fields so that 100 cells are examined and counted. At this point all conidia should be approximately spherical, with no evidence of germination i.e. no evidence of hyphal outgrowth from the conidiospore. Discard the sample after examination. 6.1.6 Time course of germination Follow t
46、he time course of germination in the preparations placed in the incubator 6.1.4 by taking a sample from time to time for microscopic examination as described in 6.1.4 and 6.1.5. Take samples at the beginning (T = 0 when the conidiospores are spread on the agarose surface) and at 24 h and 48 h therea
47、fter. Extensive germination (success rate 50 %) should be evident at 24 hours in the conidiospores exposed to the control panel. If this is not observed, then the experiment must be repeated using a conidiospore preparation which gives the expected rapid and abundant germination. If germination is s
48、een to be prevented by the coatings under test during this first 48 hours, then further samples may be taken to ask whether germination may be observed during this extended period. These subsequent samplings may be done at intervals of 4 d to 7 d. See Table 1 as an example. The test intervals and th
49、e overall duration of exposure of the fungi to the test panels shall be agreed with the organization which requested the test work. Discard each sample after examination. Some typical results follow Table 1. The number of germinated conidiospores of Amorphotheca resinae seen among the stated total number of conidiospores examined is shown for a coating known to be ineffective Coating A, a coating known to be effective Coating B, a new coating under development Coating C. The trials ended
