Sterilization Methods Overview

Sterilization Methods

• Sterilization is categorized into three main types: physical, chemical, and mechanical.
• Physical methods include heat, radiation, and mechanical methods.
• Chemical methods comprise various chemicals to sterilize explants.
• Mechanical methods involve filtration, a technique for heat-sensitive materials.

Physical Sterilization

• Heat:

  • Dry heat: Utilizes an oven or flame; dry heat sterilizers range from 50-300°C. This method involves drying plant and metal tools to sterilize while causing protein coagulation, destroying microorganisms. It's used for glasswares and metal tools (160°C for 2h or 180°C for 1h).

  • Wet heat (moist): Accomplished through autoclaving and employs saturated steam under pressure. Standard autoclave settings are 121°C at 1.0 to 1.5 bar pressure. Time required depends on the volume of media (direct relationship). This is commonly used for media, glassware, plasticware, cotton plugs, and distilled water.

• Radiation:

  • UV: UV sterilizers provide limited penetration in the air, concentrating sterilization in a restricted area around the UV lamp. It's typically safe for small areas like laminar flow hoods. The combination of UV and sodium hypochlorite releases chlorine gas, which is harmful. Combining UV with a closed laminar flow hood can result in ozone buildup, causing explosions.

Chemical Sterilization

• Sterilization for explants: Uses various chemicals:
  • Ethanol 70%: Dehydrates and coagulates microorganisms.
  • H₂O₂: A safe method.
  • Sodium hypochlorite: May cause chlorine gas release, so should be used cautiously.
  • Silver nitrate (AgNO3): Be aware of this chemical's toxicity to embryos and seeds.
  • Bromine water: A potential sterilizing chemical.
  • HgCl₂: Used for underground stems and roots but requires multiple washes due to residual toxicity.
  • Other chemicals mentioned include tap water, sterilized distilled water, and Tween 20 (to remove surface tension for efficient sterilization).

Mechanical Sterilization

• Filtration:
  • Essential for heat-sensitive materials; particularly for ascorbic acid (AA) and growth regulators like IAA.