2. Sometime a brief alcohol rinse or swabbing with alcohol wetted cheesecloth is appropriate especially with surfaces that are hairy or coated with thick wax.
3. Immerse the explant in the chlorine bleach solution, which should always be made up fresh. Always add 1-2 drops of Tween-20, detergent, or other wetting agent per 100 ml of bleach solution .A 10% bleach to a graduated cylinder and diluting with water too 100ml. When disinfesting in culture tubes, pour the bleach solution into the culture tube cap and then into the culture tube. This helps to disinfest the cap. Place your hand over the tube and mix. Cap and agitate occasionally for 5-30 min for disinfestation. Chlorine bleach can age and lose its effectiveness. For this reason small containers of commercial bleach are preferable to large ones.
4. Decant the bleach solution and rinse the explant in sterile water three to five times. This step is carried out in a transfer hood.
Some tissues are more difficult than others for establishing clean cultures. The concentration of chlorine bleach and the length of time the tissue is in the bleach can vary. Delicate, succulent tissue may be clean after 10 min in a 10% solution. Fern spores may only require a 3-5% bleach for 3-4% min. Some seeds may require 30-50% bleach for 20-60 min. Any cut explant such as a stem or leaf that is surface sterilization. These damaged pieces should be removed before culture.