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BS EN 14675:2015 Chemical disinfectants and antiseptics. Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area. Test method and requirements (Phase 2, step 1), 2015
- 1 Scope
- 2 Normative references
- 3 Terms and definitions
- 4 Requirements
- 5 Test method [Go to Page]
- 5.1 Principle
- 5.2 Materials and reagents [Go to Page]
- 5.2.1 Test virus
- 5.2.2 Culture media and reagents [Go to Page]
- 5.2.2.1 General
- 5.2.2.2 Water
- 5.2.2.3 Hard water for dilution of products
- 5.2.2.4 Interfering substance
- 5.2.2.5 Antibiotic suspension
- 5.2.2.6 Antibiotics-Trypsin-Versene® (ATV) 10 × Concentrate
- 5.2.2.7 Dulbecco’s Phosphate Buffered Saline pH 7,2 – 7,4 (PBS)
- 5.2.2.8 Earle’s balanced salt solution (BSS), 10 × concentrated
- 5.2.2.9 Phenol red, 1 %
- 5.2.2.10 Eagle’s minimum essential medium (MEM)
- 5.2.2.11 Preparation of the final mixture of 10x Eagle MEM
- 5.2.2.12 Preparation of 1X Eagle MEM
- 5.2.2.13 Foetal calf serum (FCS)
- 5.2.2.14 Crystal violet solution
- 5.3 Apparatus and glassware [Go to Page]
- 5.3.1 General
- 5.3.2 Usual microbiological laboratory equipment) and, in particular, the following: [Go to Page]
- 5.3.2.1 Apparatus for sterilization
- 5.3.2.2 Water baths, capable of being controlled at 4 °C ( 1 °C and 10 °C ( 1 °C and at additional temperatures ( 1 °C (5.5.1).
- 5.3.2.3 CO2 incubator, (95 % air, 5 % CO2), capable of being controlled at either 36 °C ( 1 °C or 37 °C ( 1 °C.
- 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.
- 5.3.2.5 Inverted microscope, for reading cell cultures microscopically.
- 5.3.2.6 Stopwatch.
- 5.3.2.7 Shakers
- 5.3.2.8 96-well microtitre plates, for cell culture use.
- 5.3.2.9 Containers: sterile test tubes, bottles or flasks of suitable capacity.
- 5.3.2.10 Magnetic stirrer, for keeping cells in suspension before seeding.
- 5.3.2.11 Petri dishes (plates), of size 30 mm to 100 mm.
- 5.3.2.12 Graduated sterile pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml or calibrated automatic pipette.
- 5.3.2.13 Volumetric flasks.
- 5.3.2.14 Biological safety cabinet, class II.
- 5.3.2.15 Centrifuge.
- 5.3.2.16 Refrigerator, capable of being controlled at 2 °C to 8 °C.
- 5.3.2.17 Freezer, for storage at temperature below or equal to -20 °C and -70 °C.
- 5.3.2.18 Six well plates for cell culture use.
- 5.4 Product test solutions
- 5.5 Procedure for assessing the virucidal activity of the product [Go to Page]
- 5.5.1 Experimental conditions (obligatory and additional)
- 5.5.2 Preparation of the test virus suspension
- 5.5.3 Preparation of cell line
- 5.6 Infectivity assay [Go to Page]
- 5.6.1 Predilution of viral suspension
- 5.6.2 Virus titration of cells in suspension on microtitre plates
- 5.6.3 Virus titration on monolayers of cells on microtiter plates
- 5.6.4 Plaque assay
- 5.7 Virucidal test – preparation [Go to Page]
- 5.7.1 General
- 5.7.2 Test method
- 5.8 Control of efficiency for suppression of disinfectant activity [Go to Page]
- 5.8.1 Dilution in ice-cold medium
- 5.8.2 Filtration technique
- 6 Calculation and expression of results [Go to Page]
- 6.1 Protocol of the CPE result
- 6.2 Calculation of infectivity titre (TCID50)
- 6.3 Calculation of PFU
- 6.4 Verification of the methodology
- 6.5 Calculation of the virucidal activity of products
- 6.6 Expression of results
- 7 Conclusion [Go to Page]
- 7.1 General
- 7.2 Test report [Go to Page]
