BS EN ISO 20743:2021 pdf download – Textiles — Determination of antibacterial activity of textile products
Add 10 ml of cryoprotective solution (6.13) to the surface of the TSA plate culture and resuspend the cells in the solution using a sterile glass spreader. Sample the suspended cells from the surface of the agar, dilute them in 100 ml of cryoprotective solution and incubate for 30 min at 20 °C. Using a pipette (5.15), sample 1 ml of the suspension and transfer it to a cryogenic vial (5.16) containing the beads (5.19). Shake the vial in order to spread the suspended cells around the beads.
— Where a cryoprotective solution containing dimethylsulfoxide is used, do not let it stand longer than 1 min at ambient temperature.
— Where a cryoprotective solution containing glycerol is used, let it stand for 30 min at 20 °C.
— Withdraw the excess cryoprotective solution with a sterile pipette. Place the cryogenic vials in a freezer (5.12) set at −70 °C or lower. Prepare 10 −6 and 10 −7 dilutions of the suspension using the serial dilution method. Take a 1,0 ml sample of each dilution and transfer it to separate Petri dishes. Add 12 ml to 15 ml of nutritive solution, cooled down to 45 °C ± 1 °C. Incubate for 18 h to 24 h under the conditions specified for the strain. Enumerate the plate cultures and confirm that the suspension contains less than 5 × 10 8 CFU/ml. Store the cryogenic vials in a freezer at a temperature below −70 °C.
7.2.3 Glycerol suspension method Inoculate a 15 ml culture tube containing 5 ml of appropriate medium with a freshly grown isolated colony. Incubate, usually, for 5 h to overnight at 37 °C until the bacteria culture seems to reach the late logarithm or stationary phase in the growth curve. For each strain to be stored below −70 °C, for the archives, prepare a sterile, labelled cryogenic vial. Place 225 μl of sterile 80 % glycerol in a cryogenic vial. Add 1,0 ml of the bacterial culture (frozen stock shall be 15 % glycerol). Mix well using the vortex mixer (5.4) and store in a tube at −70 °C or lower. For each strain to be stored at −20 °C, as liquid glycerol working stock, pipette equal volumes of 80 % glycerol and bacterial culture into a labelled polypropylene tube. Mix the contents well to avoid formation of ice crystals that will decrease the viability of the cells. Place the tube in a freezer at −20 °C. Check the viability of the cells after 1 week if possible.
To recover a strain from the glycerol stock stored below −70 °C, use a sterile toothpick to scrape pieces of the solid substance, then streak the cells onto the appropriate medium. The frozen stock shall not be thawed because each freeze–thaw cycle will result in a 50 % loss in cell viability. To use the −20 °C working stock, pipette 50 μl to 100 μl as inoculum for a 5 ml overnight culture. 8 Test procedures
8.1 Absorption method (see Annex E)
8.1.1 Incubation of test strain
8.1.1.1 Obtain the strain preserved as stock culture using an inoculating loop, streak onto the plate of EA (6.11) or TSA (6.3) and incubate at 37 °C ± 2 °C for 24 h to 48 h. The plate is kept at 5 °C to 10 °C and should be used within 1 week from the date of preparation. 8.1.1.2 Pour 20 ml of NB (6.5) or TSB (6.2) into a 100 ml Erlenmeyer flask. Apply an inoculating loop to pick one colony up from the incubation as specified in 8.1.1.1 and inoculate it in the broth. Incubate under the following conditions:
Temperature: 37 °C ± 2 °C
Rate of shaking: 110 min −1 and 3 cm width by reciprocal incubation shaker (5.27)
Incubation time: 18 h to 24 h
8.1.1.3 Pour 20 ml of NB (6.5) or TSB (6.2) into a 100 ml Erlenmeyer flask. Add 0,4 ml of the inoculum from the incubation as specified in 8.1.1.2 that contains 1 × 10 8 CFU/ml to 3 × 10 8 CFU/ml in bacteria
concentration or an ATP concentration of 1 × 10 −6 mol/l to 3 × 10 −6 mol/l to the flask and incubate under the following conditions:
Temperature: 37 °C ± 2 °C
Rate of shaking: 110 min −1 and 3 cm width by reciprocal incubation shaker (5.27)
Incubation time: 3 h ± 1 h
Target CFU or ATP concentration after incubation: 10 7 CFU/ml or 10 −7 mol/l.
The prepared inoculum shall be preserved by ice-cooling and used within 8 h.