1、August 2013 Translation by DIN-Sprachendienst.English price group 19No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS
2、 71.100.35!%(2165“ (for moulds) or “330“ (for yeasts) for any counts higher than 165 and 330 respectively and determine the Vc values according to 5.6.2.2. c) Calculate the numbers of cfu/ml in the test suspension “N” and in the validation suspension “Nv” using the methods given in 5.6.2.3 and 5.6.2
3、.5. Verify according to 5.7. 5.4.2 Product test solutions The concentration of a product test solution shall be 1,25 times the desired test concentration because it is diluted to 80 % during the test and the method validation (5.5.2 or 5.5.3). Product test solutions shall be prepared in hard water (
4、5.2.2.7) at minimum three different concentrations to include one concentration in the active range and one concentration in the non-active range (5.8.2). The product as received may be used as one of the product test solutions, in this case the highest tested concentration is 80%. Dilutions of read
5、y-to-use products, i.e. products that are not diluted when applied, shall be prepared in water (5.2.2.2). For solid products, dissolve the product as received by weighing at least 1,0 g 10 mg of the product in a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (lower c
6、oncentrations) shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.7). For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.7) on a volume/volume basis using volumetric flasks (5.3.2.12). The product test solutions shall b
7、e prepared freshly and used in the test within 2 h. They shall give a physically homogeneous preparation that is stable during the whole procedure. If during the procedure a visible inhomogeneity appears due to the formation of a precipitate or flocculant (for example, through the addition of the in
8、terfering substance), it shall be recorded in the test report. NOTE Counting micro-organisms embedded in a precipitate or flocculant is difficult and unreliable. The concentration of the product stated in the test report shall be the desired test concentration. Record the test concentration in terms
9、 of mass per volume or volume per volume and details of the product sample as received. DIN EN 1650:2013-08 EN 1650:2008+A1:2013 (E) 18 5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product 5.5.1 General 5.5.1.1 Experimental conditions (obligatory and additional) Besides
10、the obligatory temperature, contact time, interfering substance and test organisms additional experimental conditions (including test organisms) may be selected according to the practical use considered for the product (Clause 4) as follows: a) temperature (in C): the obligatory temperature to be te
11、sted is = 20 C; additional temperatures may be chosen from 4 C, 10 C or 40 C; the allowed deviation for each chosen temperature is 1 C; b) contact time t (in min): the obligatory contact time to be tested is t = 15 min; additional contact times may be chosen from 1 min, 5 min, 30 min or 60 min; the
12、allowed deviation for each chosen contact time is 10 s (except for 1 min: 5 s); c) interfering substance: the obligatory interfering substance to be tested is 0,3 g/l bovine albumin (5.2.2.8.2) for clean conditions or 3 g/l bovine albumin (5.2.2.8.3) for dirty conditions according to practical appli
13、cations; additional interfering substances as given in 5.2.2.8 shall be chosen according to the field of application specified for the product; DIN EN 1650:2013-08 EN 1650:2008+A1:2013 (E) 19 d) test organisms (5.2.1): the obligatory test organisms for testing fungicidal activity are Candida albican
14、s and Aspergillus niger; the obligatory test organism for testing yeasticidal activity is Candida albicans. Additional test organisms may be tested. 5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration) The method of choice is the dilution-neutralization method (5.5.2). To d
15、etermine a suitable neutralizer, carry out the validation of the dilution neutralization method (5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection with 5.5.2.6) using a neutralizer, chosen according to laboratory experience and published data. If this neutralizer is not valid, repeat the validation test us
16、ing an alternative neutralizer taking into account the information given in Annex B. If both neutralizers are found to be invalid, the membrane filtration method (5.5.3) may be used. NOTE In special circumstances it may be necessary to add neutralizer to MEA (5.2.2.3). 5.5.1.3 General instructions f
17、or validation and control procedures The neutralization and/or removal of the fungicidal and/or fungistatic activity of the product shall be controlled and validated - only for the highest product test concentration - for each of the used test organisms and for each experimental condition (interferi
18、ng substance, temperature, contact time). These procedures (experimental condition control, neutralizer or filtration control and method validation) shall be performed at the same time with the test and with the same neutralizer or rinsing liquid used in the test. In the case of ready-to-use-product
19、s use water (5.2.2.2) instead of hard water. If because of problems with neutralization, a neutralizer has been added to MEA (5.5.1.2) used for the validation and control procedures the MEA used for the test shall contain the same amount of this neutralizer as well. 5.5.1.4 Equilibration of temperat
20、ure Prior to testing, equilibrate all reagents (product test solutions (5.4.2), test suspension (5.4.1.4), validation suspension (5.4.1.5), diluent (5.2.2.4), hard water (5.2.2.7) and interfering substance (5.2.2.8) to the test temperature 5.5.1.1a) using the water bath (5.3.2.2) controlled at . Che
21、ck that the temperature of the reagents is stabilized at . The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a temperature of (20 1) C. In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to . 5.5.1.5 Precautio
22、ns for manipulation of test organisms Do not touch the upper part of the test tube sides when adding the test or the validation suspensions (5.4.1). 5.5.2 Dilution-neutralization method5)5)For a graphical representation of this method see C.1. DIN EN 1650:2013-08 EN 1650:2008+A1:2013 (E) 20 5.5.2.1
23、General The test and the control and validation procedures (5.5.2.2 through 5.5.2.5) shall be carried out in parallel and separately for each experimental condition (5.5.1.1). 5.5.2.2 Test “Na“ determination of fungicidal or yeasticidal concentrations The procedure for determining fungicidal or yeas
24、ticidal concentrations is as follows. a) Pipette 1,0 ml of the interfering substance (5.2.2.8) into a tube. Add 1,0 ml of the test suspension (5.4.1.4). Start the stopwatch (5.3.2.5) immediately, mix 5.3.2.6a) and place the tube in a water bath controlled at the chosen test temperature 5.5.1.1a) for
25、 2 min 10 s. At the end of this time, add 8,0 ml of one of the product test solutions (5.4.2). Restart the stopwatch at the beginning of the addition. Mix 5.3.2.6a) and place the tube in a water bath controlled at for the chosen contact time t 5.5.1.1b). Just before the end of t, mix 5.3.2.6a) again
26、. b) At the end of t, take a 1,0 ml sample of the test mixture “Na“ and transfer into a tube containing 8,0 ml neutralizer (5.2.2.5) and 1,0 ml water (5.2.2.2). Mix 5.3.2.6a) and place in a water bath controlled at (20 1)C. After a neutralization time of 5 min 10 s, mix and immediately take a sample
27、 of 1,0 ml of the neutralized test mixture “Na“ (containing neutralizer, product test solution, interfering substance and test suspension) in duplicate and inoculate using the pour plate or spread plate technique. 1) When using the pour plate technique, pipette each 1,0 ml sample into separate Petri
28、 dishes and add 15 ml to 20 ml of melted MEA (5.2.2.3), cooled to (45 1) C. 2) When using the spread plate technique, spread each 1,0 ml sample divided into portions of approximately equal size on an appropriate number (at least two) of surface dried plates containing MEA (5.2.2.3). For incubation a
29、nd counting see 5.5.2.6. c) Perform the procedures a) and b) using the other product test solutions at the same time. d) Perform the procedures a) to c) applying the other obligatory and if appropriate other additional experimental conditions (5.5.1.1). 5.5.2.3 Experimental conditions control “A“ va
30、lidation of the selected experimental conditions and/or verification of the absence of any lethal effect in the test conditions To validate the selected experimental conditions and/or verify the absence of any lethal effect in the test conditions, the procedure is as follows. a) Pipette 1,0 ml of th
31、e interfering substance used in the test (5.5.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch immediately, mix 5.3.2.6a) and place the tube in a water bath controlled at for 2 min 10 s. At the end of this time, add 8,0 ml of hard water (5.2.2.7). In the case
32、of ready-to-use products: water (5.2.2.2) instead of hard water. Restart the stopwatch at the beginning of the addition. Mix 5.3.2.6a) and place the tube in a water bath controlled at for t. Just before the end of t, mix 5.3.2.6a) again. b) At the end of t, take a sample of 1,0 ml of this mixture “A
33、“ in duplicate and inoculate using the pour plate or the spread plate technique 5.5.2.2b). For incubation and counting see 5.5.2.6. DIN EN 1650:2013-08 EN 1650:2008+A1:2013 (E) 21 5.5.2.4 Neutralizer control “B“ verification of the absence of toxicity of the neutralizer To verify the absence of toxi
34、city of the neutralizer, the procedure is as follows. a) Pipette 8,0 ml of the neutralizer used in the test (5.5.2.2) and 1,0 ml of water (5.2.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch at the beginning of the addition, mix 5.3.2.6a), and place the tube
35、in a water bath controlled at (20 1)C for 5 min 10 s. Just before the end of this time, mix 5.3.2.6a). b) At the end of this time, take a sample of 1,0 ml of this mixture “B“ in duplicate and inoculate using the pour plate or the spread plate technique 5.5.2.2b). For incubation and counting see 5.5.
36、2.6. 5.5.2.5 Method validation “C“ dilution-neutralization validation To validate the dilution neutralization method, the procedure is as follows. a) Pipette 1,0 ml of the interfering substance used in the test (5.5.2.2) into a tube. Add 1,0 ml of the diluent (5.2.2.4) and then, starting a stopwatch
37、, add 8,0 ml of the product test solution only of the highest concentration used in the test (5.5.2.2). Mix 5.3.2.6a) and place the tube in a water bath controlled at for t. Just before the end of t, mix 5.3.2.6a) again. b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml
38、 of neutralizer (used in 5.5.2.2). Restart the stopwatch immediately at the beginning of the addition. Mix 5.3.2.6a) and place the tube in a water bath controlled at (20 1)C for 5 min 10 s. Add 1,0 ml of the validation suspension (5.4.1.5). Start a stopwatch at the beginning of the addition and mix
39、5.3.2.6a). Place the tube in a water bath controlled at (20 1)C for 30 min 1 min. Just before the end of this time, mix 5.3.2.6a) again. At the end of this time, take a sample of 1,0 ml of the mixture “C“ in duplicate and inoculate using the pour plate or the spread plate technique 5.5.2.2b). For in
40、cubation and counting see 5.5.2.6. 5.5.2.6 Incubation and counting of the test mixture and the control and validation mixtures For incubation and counting of the test mixture and the control and validation mixtures, the procedure is as follows. a) Incubate (5.3.2.3) the plates for 42 h to 48 h. Disc
41、ard any plates that are not countable for any reason. Count the cfu on the plates to determine the total number of cfu. Only for Aspergillus niger : Incubate the plates for a further 20 h to 24 h and - if the number of colonies has increased - for a third additional period of 20 h to 24 h. Do not re
42、count plates that no longer show well-separated colonies. Recount the remaining plates. If the number has increased, use only the higher number for further evaluation. b) Note for each plate the exact number of colonies, but record “165“ (for moulds) or “330“ (for yeasts) for any counts higher than
43、165 and 330 respectively and determine the Vc values according to 5.6.2.2. c) Calculate the numbers of colony-forming units per millilitre in the test mixture “Na“ and in the validation mixtures “A“, “B“ and “C“ using the method given in 5.6.2.4 and 5.6.2.6. Verify according to 5.7. DIN EN 1650:2013
44、-08 EN 1650:2008+A1:2013 (E) 22 5.5.3 Membrane filtration method6)5.5.3.1 General The test and the control and validation procedures (5.5.3.2 through 5.5.3.5) shall be carried out in parallel and separately for each experimental condition (5.5.1.1). Each membrane filtration apparatus shall be equipp
45、ed with a membrane of 0,45 m pore size and 47 mm to 50 mm diameter (5.3.2.7) and filled with 50 ml of the rinsing liquid (5.2.2.6). The time required for filtering if longer than one minute in exceptional cases shall be recorded in the test report. When transferring the membranes to the surface of a
46、n agar plate, care should be taken to ensure that the test organisms are on the upper side of the membrane when placed on the plate, and to avoid trapping air between the membrane and agar surface. 5.5.3.2 Test “Na“ determination of the fungicidal or yeasticidal concentrations The procedure for dete
47、rmining the fungicidal or yeasticidal concentrations is as follows. a) See 5.5.2.2a). b) At the end of t, take a sample of 0,1 ml of the test mixture “Na“ in duplicate and transfer each 0,1 ml sample into a separate membrane filtration apparatus (5.5.3.1). Filter immediately. Filter through at least
48、 150 ml but no more than 500 ml of rinsing liquid (5.2.2.6). If the rinsing liquid is not water, complete the procedure by filtering 50 ml of water (5.2.2.2). Then transfer each of the membranes to the surface of separate MEA plates. For incubation and counting see 5.5.3.6. c) See 5.5.2.2c). d) See
49、5.5.2.2d). 5.5.3.3 Experimental conditions control “A“ validation of the selected experimental conditions and/or verification of the absence of any lethal effect in the test conditions To validate the selected experimental conditions and/or verify the absence of any lethal effect in the test conditions, the procedure is as follows. a) See 5.5.2.3a). b) At the end of t, take a sample of 1,0 ml of this mixture “A“ in duplicate and tran
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