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 prEN 4159:2005 2 Contents Page Foreword2 Introduction.3 1 Scope 3
7、 2 Terms, definitions and abbreviations3 3 Principle3 4 Apparatus .4 5 Specimen4 6 Procedure .5 6.1 Test 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.
8、1.5 Microscopic examination5 6.1.6 Time course of germination6 7 Designation 7 8 Test report 7 Bibliography8 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-ST
9、AN defined in ASD-STANs General Process Manual, this standard has received approval for Publication. prEN 4159:2005 3 Introduction Certain fungi are known to be capable of proliferating in fuel systems which can cause corrosion and blockage. Conidiospores are the dispersal form of these fungi. Germi
10、nation 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 performed only by persons qualified in the microbiology of fungi. The standard can be used to asses
11、s 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 germination of conidiospores of certain fungi known to be capable of proliferating in fuel system
12、s 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 structures produced by the mycelial mould form of the fungus. Conidiospores are spherical or nea
13、rly 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 germination, a conidiospore produces a long tube-like outgrowth which then forms dense branchi
14、ng 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 activity. This fungistatic activity may be assessed quantitatively by assessing the success rate
15、 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 possess no fungistatic activity. Laboratories which undertake work to this method should first o
16、btain 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 agarose gel (see below) on the surface of sterile plastic petri dishes rather than on the sur
17、face 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 proportion of these conidia germinate begin growth rapidly unless some material in the coating di
18、ffuses 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 the number of cells examined (germinated + nongerminated). The success rate of germination is
19、 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 conidiospores approximately spherical and the long filamentous outgrowth that is the result of g
20、ermination. 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 mic
21、robiological 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 avail
22、able disposable 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 tip
23、s) 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 4.11.3 National culture collections contain several different strains of each of these fungi. The laboratory should cho
24、ose 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 Chloramphenicol Agar. The Oxoid product Rose Bengal Chloramphenicol Agar is suitable. 4.13 Agarose The product catal
25、ogue No. 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
26、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 are 10 mm 20 mm (length width) are suitable. Panels should be less than 10 mm thick. Panels are stored at room temp
27、erature. 5.2 Three test panels and three control panels are required. prEN 4159:2005 5 6 Procedure 6.1 Test methods 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 clea
28、r 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 Application of gel to coating Ensure that the test specimens are at room temperature. Cut the gel formed
29、in 7.1.1 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 for 22 to 24 hours. 6.1.3 Preparation of spore suspension Ten days prior to test, make up Rose Beng
30、al 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 allow the agar to form a gel. Seed the prepared medium with the fungus under test. Incubate this cu
31、lture (see 5.1) for 10 days. Harvest conidiospores from the culture by passing a sterile microbiological loop gently 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
32、 conidiospores per ml of suspension using a blood cell counting chamber haemocytometer according to the manufacturers directions. A suitable suspension will be made up almost entirely 95 % of conidiospores, the remainder being hyphal fragments readily distinguishible from conidiospores. Remove hypha
33、l 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 centrifuging the suspension and resuspending the conidiospores in a suitable volume of sterile water. Use this suspension within
34、 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 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 sprea
35、d 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 for microscopic examination for time = zero. Mount this on a microscope slide. Place the remainder of the gel + coating + panel in
36、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 fitting plastic bags. Each such bag should also contain a petri dish which contains sterile filter paper saturated with sterile distil
37、led water. 6.1.5 Microscopic examination Examine the slide prepared in 7.1.4 at a magnification of 100. Count all 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,
38、with no evidence of germination i.e. no evidence of hyphal outgrowth from the conidiospore. Discard the sample after examination. prEN 4159:20056 6.1.6 Time course of germination Follow the time course of germination in the preparations placed in the incubator 7.1.4 by taking a sample from time to t
39、ime 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 germination (success rate 50 %) should be evident at 24 hours in the conidiospores exposed to t
40、he 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 coatings under test during this first 48 hours, then further samples may be taken to
41、 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 duration of exposure of the fungi to the test panels shall be agreed with the organizati
42、on 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 coatin
43、g 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 67/103 0/208 0/211 2 Not tested 0/210 0/217 6 0/215 0/210 13 23/217 a0/250 19 12/216 0/2
44、25 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. Coating A did not prevent or delay germination, as seen by the day 1 result. Extensive
45、 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 placed on a plastic surface of a petri dish rather than on a coated metal panel. Simi
46、lar 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 reasonable to conclude from the result
47、s shown that: 1) the conidiospore suspension contained many conidiospores that were capable of rapid germination and that; 2) the coating B previously known to be effective as a fungistatic agent prevented germination for 19 days and; 3) that the new coating also prevented germination for the 19 day
48、 duration of the trials. These trials were carried out in parallel, using portions of the same original conidiospore suspension, the same original agarose preparation, and the same conditions of incubation and examination. The three sets of results are strictly comparable. prEN 4159:2005 7 7 Designa
49、tion EXAMPLE Description block RESISTANCE TO MICROBIAL GROWTH Identity block EN4159 Number of this standard 8 Test report The test report should include the following information: a) Identification of the coatings under test: suppliers code number, batch number, date of manufacture, etc.; b) Reference to this test method; c) The number of specimens tested; d) A list of the fungi used, with reference to their origin in national culture collections; e) The results obtained using the methods
