Surface sterilization of explants

• To prevent bacterial and fungal growth, tissue explants are surface sterilized before they are used to establish axenic/in vitro cultures. The most common disinfectants are listed below with the concentration and exposure time (adapted from SIGMA Plant Cell Culture Manual). There is no universal working procedure for disinfecting plant tissue explants, the procedure varies from tissue to tissue and species to species. It is recommended that the experimenter standardizes his/ her own protocol based on the following guidelines:

  • Wash the tissue explants with mild detergent (Tween-20) before treatment with the disinfectant solution.

   

  •  Rinse the explants thoroughly under running tap water for 10-30min.

   

  •  Submerge the explants into the disinfectant solution, seal the bottle and gently agitate.

   

  •  Under sterile conditions, decant the disinfectant solution and rinse the explants several times with sterile distilled water.

    Commonly used disinfectants for plant tissue culture

    S. No.	Name of the Disinfectants	Concentration (%)	Exposure time (min)
    1.	Calcium hypochlorite	9-10	5-30
    2.	Sodium hypochlorite	0.5-5.0	5-30
    3.	Hydrogen peroxide	3-12	5-15
    4	Ethyl alcohol	70-95	1-5.0
    5.	Silver nitrate	1.0	5-30
    6.	Mercuric chloride	0.1-1.0	2-10
    7.	Benzalkonium chloride	0.01-0.1	5-20

   

  • Commercial bleach contains about 5% sodium hypochlorite and thus may be used at a concentration of 10-20% which is equivalent to 0.5-1.0% sodium hypochlorite