Methodology

At the working bench

1. Young leaves are removed from the plants.
2. The plant tissues are washed properly with running tap water for 10 to 15 minutes.
In the Laminar flow
Preparation of Clorox solution:
1. 100 ml of 20% Clorox in sterile water are prepared using the sterile measuring
cylinder.
2. The Clorox solution is poured into the sterile 250 ml empty beaker.
3. Three drops of Tween 20 are added into the solution and left inside the laminar flow.
Surface sterilization and culturing of the explants:
1. The plant tissues are carefully transferred into the Clorox solution with sterile
forceps. The mixture is swirled for 15 minutes to surface sterilize.
2. The plant tissues are transferred out from the Clorox solution and are washed in
sterile water for 5, 10 and 15 minutes per wash using a sterile forceps.
3. The plant tissues are cut into square form with approximated size of 5 X 5 mm on a
sterile Petri dish.
4. Six pieces of explants are placed on the surface of the medium in both Petri dishes
and the Petri dishes are sealed with Parafilm.
5. The Petri dishes are labelled and placed in the culture room.
6. The cultures are observed for one week. The type, percentage of contamination,
Morphologies of the contaminants (if any), the percentage of survived explants are
recorded and the pictures of the cultures are taken after one week.
7. The contaminated cultures are discarded.