What is sterilization and Types of sterilization

Sterilization in Plant Tissue Culture

Sterilization is a critical prerequisite in plant tissue culture to prevent microbial contamination that can ruin cultures. All culture materials—vessels, instruments, media, and explants—must be aseptic. The choice of sterilization technique depends on the type of material, heat sensitivity, and culture requirements.

1. Dry Heat Sterilization

Principle: Microorganisms are killed by oxidation of cellular components, including proteins and nucleic acids, at high temperatures.

Applications: Heat-tolerant materials like glassware, Teflon plasticware, and some metal instruments.

Procedure:

• Place items in a hot air oven at 160–180°C for 2–3 hours.
• Glass bead sterilizers (~300°C) are used for instruments like forceps and scalpels.

Advantages:

• No moisture required; useful for materials that cannot be autoclaved.

Limitations:

• Time-consuming; unsuitable for heat-sensitive materials.

2. Flame Sterilization

Principle: Direct heat destroys microbial contaminants by protein denaturation and oxidation.

Applications: Instruments such as forceps, scalpels, and needles, as well as the mouth of culture vessels before inoculation.

Procedure:

• Dip the instrument in 95% ethanol.
• Pass through a Bunsen burner flame until alcohol burns off.

Advantages:

• Rapid; can be repeated multiple times during culture work.

Limitations:

• Only suitable for metal instruments; care needed to avoid burns.

3. Autoclaving

Principle: Moist heat under pressure denatures microbial proteins and nucleic acids, killing all forms of microbial life, including spores.

Applications: Culture media, glassware, plasticware (heat-tolerant), and some instruments.

Procedure:

• Autoclave at 121°C, 15 psi, for 15–40 minutes (longer for large volumes).
• Ensure vessels are properly sealed and only opened after pressure reaches zero.

Advantages:

• Reliable sterilization of large volumes; widely used.

Limitations:

• Heat-sensitive materials cannot be autoclaved.

4. Filter Sterilization

Principle: Microbes are physically removed using membrane filters.

Applications: Heat-sensitive compounds, including growth regulators (GA3, zeatin, ABA), vitamins, urea, and enzymes.

Procedure:

• Pass solutions through a 0.45 μm or smaller membrane filter.
• The filter assembly is pre-sterilized by autoclaving.
• Add compounds to cooled autoclaved medium (~40°C) under laminar flow.

Advantages:

• Preserves activity of heat-labile substances; allows aseptic handling.

Limitations:

• Only applicable to liquids; small volumes; does not sterilize surfaces.

5. Wiping with 70% Ethanol

Principle: Ethanol denatures proteins and disrupts cell membranes, killing microbes.

Applications: Surfaces, laboratory platforms, laminar flow cabinet interiors, and operator hands.

Procedure:

• Wipe the surface thoroughly with 70% ethanol.
• Allow to air dry before starting work.

Advantages:

• Simple, rapid, and effective for routine disinfection.

Limitations:

• Limited to surface sterilization; does not penetrate porous materials.

6. Surface Sterilization of Explants

Principle: Chemicals inactivate microbes present on the surface of plant tissues, allowing clean culture initiation.

Applications: All plant tissues used as explant material.

Procedure:

• Treat explants with sterilants such as:
• Calcium hypochlorite (9–10%)
• Sodium hypochlorite (2%)
• Mercuric chloride (0.1–1%)
• Silver nitrate (1%)
• Hydrogen peroxide (10–12%)
• Bromine water (1–2%)
• Antibiotics (4–50 mg/L)
• Duration: 15–30 minutes, followed by thorough rinsing to remove residues.

Advantages:

• Essential for eliminating microbial contamination on explants.

Limitations:

• Chemical toxicity may damage tissues; concentration and duration must be carefully optimized.

Summary Table

Method	Principle	Applications	Advantages	Limitations
Dry Heat	Oxidation of microbial components	Glassware, Teflon, metal instruments	No moisture; simple	Time-consuming; heat-sensitive materials cannot be used
Flame Sterilization	Direct heat	Forceps, scalpels, needles, culture vessel mouths	Rapid; repeated use	Only for metal instruments; safety concerns
Autoclaving	Moist heat under pressure	Media, vessels, heat-tolerant instruments	Reliable; kills spores	Heat-sensitive items cannot be used
Filter Sterilization	Physical removal of microbes	Heat-sensitive solutions, growth regulators, enzymes	Preserves heat-labile compounds	Limited to liquids; small volumes
70% Ethanol Wiping	Protein denaturation, membrane disruption	Surfaces, laminar flow cabinet, operator hands	Quick, simple, effective	Only surface sterilization; no penetration
Surface Sterilization	Chemical inactivation of microbes	Plant explants	Eliminates surface contamination	May damage tissue; requires optimization