1、ASD STANDARD NORME ASD ASD NORM prEN 4159 Edition P1 November 2005 PUBLISHED BY THE AEROSPACE AND DEFENSE INDUSTRIES ASSOCIATION OF EUROPE - STANDARDIZATIONGulledelle 94 - B-1200 Brussels - Tel. + 32 2 775 8126 - Fax. + 32 2 763 3565 - www.asd-stan.orgICS: Descriptors: ENGLISH VERSION Aerospace seri
2、es Paints and varnishes Determination of resistance to microbial growth Srie arospatiale Peintures et vernis Dtermination de la rsistance laction des microorganismes Luft- und Raumfahrt Anstrichstoffe Bestimmung der Widerstandsfhigkeit gegen Schimmelwachstum This “Aerospace Series“ Prestandard has b
3、een drawn up under the responsibility of ASD-STAN (The Aerospace and Defense Industries Association of Europe - Standardization). It is published for the needs of the European Aerospace Industry. It has been technically approved by the experts of the concerned Domain following member comments. Subse
4、quent to the publication of this Prestandard, the technical content shall not be changed to an extent that interchangeability is affected, physically or functionally, without re-identification of the standard. After examination and review by users and formal agreement of ASD-STAN, it will be submitt
5、ed as a draft European Standard (prEN) to CEN (European Committee for Standardization) for formal vote and transformation to full European Standard (EN). The CEN national members have then to implement the EN at national level by giving the EN the status of a national standard and by withdrawing any
6、 national standards conflicting with the EN. Edition approved for publication 30 November 2005 Comments should be sent within six months after the date of publication to ASD-STAN Non-metallic Material Domain Copyright 2005 by ASD-STAN Copyright Association Europeene des Constructeurs de Materiel Aer
7、ospatial Provided by IHS under license with AECMANot for ResaleNo reproduction or networking permitted without license from IHS-,-,-prEN 4159:2005 2 Contents Page Foreword2 Introduction.3 1 Scope 3 2 Terms, definitions and abbreviations3 3 Principle3 4 Apparatus .4 5 Specimen4 6 Procedure .5 6.1 Tes
8、t methods5 6.1.1 Preparation of agarose + glucose gel5 6.1.2 Application of gel to coating 5 6.1.3 Preparation of spore suspension.5 6.1.4 Application of suspension to gel and storage at 25 C .5 6.1.5 Microscopic examination5 6.1.6 Time course of germination6 7 Designation 7 8 Test report 7 Bibliogr
9、aphy8 Foreword This standard was reviewed by the Domain Technical Coordinator of ASD-STANs Non-metallic Material Domain. After inquiries and votes carried out in accordance with the rules of ASD-STAN defined in ASD-STANs General Process Manual, this standard has received approval for Publication. Co
10、pyright Association Europeene des Constructeurs de Materiel Aerospatial Provided by IHS under license with AECMANot for ResaleNo reproduction or networking permitted without license from IHS-,-,-prEN 4159:2005 3 Introduction Certain fungi are known to be capable of proliferating in fuel systems whic
11、h 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 should be perfo
12、rmed 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. 1 Scope This standard specifies a method to assess the ability of biocide-containing coatings to prevent the ger
13、mination of conidiospores of certain fungi known to be capable of proliferating in fuel systems for aerospace applications. 2 Terms, definitions and abbreviations For the purposes of this standard, the following terms, definitions and abbreviations apply. Conidiospores are typically single-celled st
14、ructures produced by the mycelial mould form of the fungus. 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 and nutrients. On
15、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 have fungistatic ac
16、tivity. 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 which is known to po
17、ssess 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 performed by placing the
18、 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 conditions a high pro
19、portion 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 germinated divided by
20、 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 between ungerminated co
21、nidiospores approximately spherical and the long filamentous outgrowth that is the result of germination. Copyright Association Europeene des Constructeurs de Materiel Aerospatial Provided by IHS under license with AECMANot for ResaleNo reproduction or networking permitted without license from IHS-,
22、-,-prEN 4159:20054 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 4.1 Incubator, capable of maintaining (25 1) C. 4.2 Autoclave suitable for sterilization of microbiologi
23、cal growth media, i.e. capable of heating the media to 121 C for 15 minutes. 4.3 Water bath, set to (45 1) C. 4.4 Microscope, magnification 100, and glass microscope slides. 4.5 Plastic disposable petri dishes : 90 mm to 100 mm diameter. 4.6 Sterile microbiological loops. Commercially available disp
24、osable plastic loops (stated to carry 10 microlitres) are suitable. 4.7 Haemocytometer (blood cell counting chamber) Neubauer ruling. 4.8 Funnel, loosely plugged with nonabsorbent cotton wool. 4.9 Balance, toploading, 0,1 g resolution. 4.10 Micropipetting device (with disposable sterile tips) to del
25、iver 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 4.11.3 National culture collections contain several different strains of each of these fungi. The laboratory should choose a sui
26、table 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 Chloramphenicol Agar. The Oxoid product Rose Bengal Chloramphenicol Agar is suitable. 4.13 Agarose The product catalogue No.
27、A-0567 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 with a paint suspected of having fungistatic activity. Control panels are similar panels coated on one face only with a si
28、milar 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 are 10 mm 20 mm (length width) are suitable. Panels should be less than 10 mm thick. Panels are stored at room temperature.
29、5.2 Three test panels and three control panels are required. Copyright Association Europeene des Constructeurs de Materiel Aerospatial Provided by IHS under license with AECMANot for ResaleNo reproduction or networking permitted without license from IHS-,-,-prEN 4159:2005 5 6 Procedure 6.1 Test meth
30、ods 6.1.1 Preparation of agarose + glucose gel To 100 ml distilled water in a 250 ml conical 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 a
31、llow the solution to set at room temperature to form a clear colourless gel. 6.1.2 Application of gel to coating Ensure that the test specimens are at room temperature. Cut the gel formed in 7.1.1 so as to fit, approximately, the specimens under test. Place the cut gel on the coated surface of the s
32、pecimen and trim any excess. Place the gel/specimen in a petri dish and store it at 25 C for 22 to 24 hours. 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 equilibr
33、ated 45 C medium into each of an appropriate number of petri dishes (90 mm diameter) and allow the agar to form a gel. Seed the prepared medium with the fungus under test. Incubate this culture (see 5.1) for 10 days. Harvest conidiospores from the culture by passing a sterile microbiological loop ge
34、ntly over the surface of the fungus and then swirling the loop 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 manufact
35、urers directions. A suitable suspension will be made up almost 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 susp
36、ension to 106conidiospores per ml by either adding water or 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 speci
37、mens have been stored for the required time (see 7.1.2), apply 0,010 ml of suspension (see 7.1.3) 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 r
38、azor blade to cut a piece approximately 5 mm 5 mm of gel for 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 t
39、he specimens under test in petri dishes held in loose fitting 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 7.1.4 at a magnification of 100. Count al
40、l cells whether they are conidia or hyphal fragments in several 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 a
41、fter examination. Copyright Association Europeene des Constructeurs de Materiel Aerospatial Provided by IHS under license with AECMANot for ResaleNo reproduction or networking permitted without license from IHS-,-,-prEN 4159:20056 6.1.6 Time course of germination Follow the time course of germinatio
42、n in the preparations placed in the incubator 7.1.4 by taking a sample from time to time for microscopic examination as described in 7.1.4 and 7.1.5. Take samples at the beginning (T = 0 when the conidiospores are spread on the agarose surface) and at 24 and 48 hours thereafter. Extensive germinatio
43、n (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 seen to be prevented by the
44、 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 four days to seven days. See Table 1 as an example. The test intervals and the overall dur
45、ation 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 coni
46、diospores 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 at day 19. Table 1 Germinated conidia/total conidia examined Day Coating A Coating B Coating C 0 0/208 0/208 0/208 1 67/
47、103 0/208 0/211 2 Not tested 0/210 0/217 6 0/215 0/210 13 23/217 a0/250 19 12/216 0/225 aSome germination was seen at days 13 and 19 among conidiospores exposed to Coating B. Further growth of the germinated conidia did not occur. Germination was not observed in conidiospores exposed to Coating C. C
48、oating A did not prevent or delay germination, as seen by the day 1 result. Extensive fungal growth followed, such that microscopic examination was not practicable at day 2 or later. The results for Coating A are similar to results obtained when the coating is omitted, i.e. when the agarose slab is
49、placed on a plastic surface of a petri dish rather than on a coated metal panel. Similar results not shown were obtained for the fungus Aspergillus niger. However, germination was not observed over the full 19 days of the experiment. The germination (day 13) seen for Amorphotheca resinaeon Coating B was not observed for Aspergillus niger. It is reaso
