1、 !“ # # ! $ %“ = ( “ ) /5/: Escherichia coli; Proteus vulgaris; Staphylococcus aureus; Pseudomonas aeruginosa; Streptococcus uberis. DIN EN 1656:2010-03 EN 1656:2009 (E) 6 Staphylococcus aureus. Where indicated, additional specific bactericidal activity shall be determined applying other contact tim
2、es, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take into account intended specific use conditions. NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the obligat
3、ory test conditions. 5 Test method 5.1 Principle 5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready-to-use products) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture is maintained at (10 1) C (or (30 1) C for
4、teat disinfectants) for 30 min 10 s (5 min 10 s for teat disinfectants) (obligatory test conditions). At the end of this contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity in this portion is immediately neutralized or suppressed by a validated method. The meth
5、od of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving bacteria in each sample are determined and the reduction is calculated. 5.1.2 For general disinfectant products, the test is performed using Enterococcus hirae, P
6、roteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus as test organisms. For teat disinfectants the test is performed using Escherichia coli, Staphylococcus aureus and Streptococcus uberis as test organisms. 5.1.3 Additional and optional contact times and temperatures are specified. Addi
7、tional test organisms can be used. 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains as test organisms 1) : a) General disinfection products: Enterococcus hirae ATCC 10541 Proteus vulgaris ATCC 13315 Pseudomonas aeruginosa ATCC 1
8、5442 Staphylococcus aureus ATCC 6538 b) Teat disinfectants: Escherichia coli ATCC 10536 Staphylococcus aureus ATCC 6538 Streptococcus uberis ATCC 19436 1) The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (ATCC). This information is given for the
9、 convenience of users of this standard and does not constitute an endorsement by CEN of the product named. DIN EN 1656:2010-03 EN 1656:2009 (E) 7 NOTE See Annex A for strain references in some other culture collections. The required incubation temperature for these test organisms is (36 1) C or (37
10、1) C (5.3.2.3). The same temperature (either 36 C or 37 C) shall be used for all incubations performed during a test and its control and validation. If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test
11、 report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In
12、 addition, they shall be held by the testing laboratory or national culture collection under a reference for five years. 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms may be used as an alternati
13、ve, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. NOTE 1 To improve rep
14、roducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturers instructions relating to the preparation of these products should be rigorously followed. NOTE 2 For each culture medium and reagent, a shelf life should be
15、 fixed (see ISO/IEC 17025). 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralized water. Sterilize in the autoclave (5.3.2.1 a). NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilized. NOTE 2 If dist
16、illed water of adequate quality is not available, water for injections (see 1 in the bibliography) can be used. NOTE 3 See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein 15,0 g Soya peptone, p
17、apaic digest of soybean meal 5,0 g Sodium chloride (NaCl) 5,0 g Agar 15,0 g Water (5.2.2.2) to 1 000,0 ml Sterilize in the autoclave (5.3.2.1 a). After sterilization the pH of the medium shall be equivalent to 7,2 0,2 when measured at (20 1) C. NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used. DIN EN 1656:2010-03
