Sterilization Methods
Sterilization is the complete destruction or removal of all forms of microbial life,
including bacteria, viruses, fungi, and spores, from an object, surface, or medium.
It is an absolute process—an item is either sterile or not sterile.
Spores are dormant, tough, and non-reproductive structures formed by certain bacteria,
fungi, algae, and plants to survive extreme conditions like heat, radiation, desiccation, and
chemicals.
Spores can survive in the environment for long periods, then reactivate when conditions
are right, causing infection.
Sterile vs Disinfected:
Sterile Disinfected
No microorganisms present Some microorganisms may remain
Absolute process Partial reduction of microbes
Achieved by sterilization methods Achieved by disinfectants
Type’s
➡Physical
➡Chemical
➡Gaseous
➡Radiation
➡Mechanical Methods of Sterilization
1. Physical Methods of Sterilization
A. Moist Heat Sterilization (e.g., Autoclave)
Principle:
Moist heat sterilization involves the use of steam under pressure. It kills microorganisms by
denaturing their proteins, disrupting membranes, and coagulating cellular components. Moist
heat is more effective than dry heat due to better heat penetration and faster action.
Procedure:
Items are placed inside an autoclave chamber.
Conditions: 121°C, 15 pounds per square inch (psi) pressure for 15–30 minutes.
Steam must contact all surfaces of the item.
After sterilization, the pressure is released gradually and items are dried.
Merits:
Highly effective against all microorganisms including spores.
Requires less time compared to dry heat.
Suitable for sterilizing liquids, culture media, and heat-stable instruments.
Penetrates through fabrics and porous materials.
Demerits:
Cannot be used for oils, powders, and heat-sensitive materials.
May damage plastics, rubber, and electronic items.
Requires proper maintenance and monitoring.
Applications:
Culture media (nutrient agar, broth), surgical dressings, glassware, instruments,
gowns, cotton swabs, rubber gloves.
B. Dry Heat Sterilization (e.g., Hot Air Oven)
Principle:
Dry heat kills microorganisms by oxidation of cellular components, protein denaturation, and
dehydration. It requires higher temperatures and longer exposure times than moist heat.
Procedure:
Articles are placed in a hot air oven.
Standard conditions: 160°C for 2 hours or 180°C for 30 minutes.
Heat is distributed by natural or forced air circulation.
Merits:
No corrosion of metal instruments.
Suitable for sterilizing anhydrous materials (non-aqueous).
Does not dull sharp instruments.
Demerits:
Time-consuming.
High temperatures may damage sensitive items.
Poor heat penetration compared to steam.
Applications:
Glassware (petri dishes, pipettes), metallic instruments (forceps, scalpels), powders
(zinc oxide), oils and fats.
C. Filtration
Principle:
Filtration removes microorganisms physically from liquids or gases by passing them through
filters with specific pore sizes. No killing occurs; only separation.
Types of Filters:
Membrane filters (cellulose ester): 0.22 µm pore size.
Depth filters (e.g., Seitz filters, sintered glass).
HEPA filters: remove particles from air.
Procedure:
Liquid or air is passed through a filter unit under pressure or vacuum.
The filter retains bacteria and larger organisms.
The filtered liquid is collected in a sterile container.
Merits:
Suitable for heat-sensitive liquids like serum, antibiotics.
Fast and effective.
No chemical residue.
Demerits:
Not effective against viruses and pyrogens (unless ultrafiltration is used).
Filters may clog easily.
Fragile and expensive.
Applications:
Sterilization of vaccines, antibiotic solutions, IV fluids, enzyme preparations, air
filtration in clean rooms and laminar airflow hoods.
2. Radiation Methods of Sterilization
A. Ionizing Radiation (Gamma Rays, X-rays)
Principle:
Ionizing radiation works by producing free radicals and causing DNA strand breakage,
which damages cellular structure and inhibits replication, leading to cell death.
Sources:
Cobalt-60 (commonly used for gamma radiation).
Linear accelerators (for X-rays).
Procedure:
Items are packed and exposed to gamma rays in a radiation chamber.
Dosage depends on the type of material and microbial load (usually 25 kGy).
Performed in a controlled facility.
Merits:
Deep penetration through sealed packaging.
No heat or moisture involved—ideal for heat-sensitive materials.
Can sterilize large volumes.
Demerits:
High cost of installation and maintenance.
Requires shielding and safety protocols.
May affect physical properties of some materials (e.g., plastics).
Applications:
Sterilization of disposable syringes, catheters, surgical implants, IV sets,
pharmaceutical packaging, and tissue grafts.
B. Non-ionizing Radiation (Ultraviolet (UV) Light)
Principle:
UV radiation causes formation of thymine dimers in DNA, which blocks DNA replication
and transcription, ultimately leading to cell death.
Procedure:
UV light at wavelength of 254–260 nm is used.
Exposure time: 15–45 minutes.
Distance: Close range for maximum effectiveness.
Surfaces and air are exposed directly.
Merits:
Simple and inexpensive.
No heat or chemicals required.
No residue formation.
Demerits:
Poor penetration; only effective for surface sterilization.
Prolonged exposure can degrade plastics and damage human skin and eyes.
Ineffective in the presence of dust or organic matter.
Applications:
Sterilization of work surfaces, laminar flow hoods, biosafety cabinets, water
purification, operating rooms, and pharmaceutical packaging areas.
3. Chemical Methods of Sterilization
A. Use of Disinfectants and Antiseptics
Principle:
Chemical agents kill or inhibit the growth of microorganisms by protein denaturation, cell
membrane disruption, or enzyme inactivation.
Examples of Chemicals:
Alcohols: Ethanol, isopropanol (70%).
Phenols: Lysol, cresol.
Halogens: Chlorine (bleach), iodine (tincture).
Aldehydes: Formaldehyde, glutaraldehyde.
Oxidizing agents: Hydrogen peroxide.
Quaternary ammonium compounds: Benzalkonium chloride.
Procedure:
Items are soaked, sprayed, or wiped with chemical disinfectants.
Contact time varies from 5 to 30 minutes depending on the chemical.
Merits:
Rapid action.
Broad-spectrum efficacy.
Some agents (e.g., glutaraldehyde) are sporicidal.
Demerits:
Toxicity, irritation to skin or mucous membranes.
Some agents are corrosive or flammable.
Not suitable for internal body sterilization.
Applications:
Surface disinfection (floors, tables), instrument soaking, hand sanitization, skin
antiseptics before injection.
4. Gaseous Methods of Sterilization
🔸 A. Ethylene Oxide (C₂H₄O) Sterilization
Principle:
C₂H₄O is a potent alkylating agent that reacts with amino, carboxyl, and hydroxyl groups in
proteins and DNA, leading to microbial death.
Procedure:
Materials are exposed to C₂H₄O gas in a sealed chamber.
Conditions: 30–60°C temperature, 40–80% humidity, and exposure time of 2–5
hours.
After sterilization, items are aerated for 8–24 hours to remove gas residues.
Merits:
Ideal for heat- and moisture-sensitive materials.
Penetrates deeply into crevices and porous items.
Can sterilize complex devices without damage.
Demerits:
Toxic, flammable, and carcinogenic—requires strict handling protocols.
Long total processing time (including aeration).
Expensive equipment and gas monitoring required.
Applications:
Plastic syringes, catheters, pacemakers, electronic medical devices, gloves, surgical
kits, endoscopes.
5. Mechanical Methods of Sterilization
A. Scrubbing/Washing
Principle:
Removes microorganisms and organic matter through mechanical action, using brushes,
water, and detergents.
Procedure:
Items are scrubbed manually or mechanically with hot water and detergents.
Often done prior to actual sterilization to reduce microbial load.
Merits:
Simple, low-cost method.
Reduces risk of sterilization failure.
No specialized equipment needed.
Demerits:
Does not kill microorganisms.
Labor-intensive for large-scale operations.
Risk of contamination if improperly handled.
Applications:
Pre-cleaning of surgical instruments, containers, and reusable lab equipment.
B. Sedimentation and Centrifugation
Principle:
Uses gravity (sedimentation) or centrifugal force (centrifugation) to separate microorganisms
from fluids based on size and density.
Procedure:
Liquids are allowed to stand (sedimentation) or spun at high speed in centrifuges.
Microorganisms collect at the bottom and can be removed.
Merits:
Useful for concentrating or removing cells from fluids.
Aids in purification steps.
Demerits:
Not a complete sterilization method.
Ineffective for viruses or dissolved contaminants.
Applications:
Water purification (pre-treatment), separation of microbial cells in biotech processes,
harvesting cell cultures.
