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Sterilization - Complete Notes

Definition

Key distinction: Sterilization kills everything, including spores. Disinfection destroys most but not all microbial forms (spores may survive).

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Key Terminology (Definitions)

• Sherris & Ryan's Medical Microbiology, 8th ed.

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Classification of Sterilization Methods

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A. Physical Methods

1. Heat

a) Moist Heat (Steam Sterilization)

• The gold standard of sterilization.
• Essentially a sophisticated pressure cooker.
• Air is removed either by:
  • Evacuation of the chamber before filling with steam, OR
  • Downward displacement - capitalizes on heaviness of air compared to saturated steam
• Standard cycle: 121°C for 15 minutes at 15 psi pressure
• Spores directly exposed are killed in < 5 minutes; 10-15 minutes accounts for variability in steam penetration
• A drop of just 1.7°C increases the required exposure time by 48%
• "Flash" autoclaves (used in operating rooms) use higher temperatures (~134°C) for shorter times
• Uses: Surgical instruments, dressings, culture media, aqueous solutions
• Cannot be used for: oils, waxes, powders (immiscible in water), heat-sensitive plastics

• High-level disinfection, NOT true sterilization
• Kills most pathogens and some spores
• Vegetative bacteria killed within minutes at 70°C or less
• Some heat-resistant spores may survive prolonged boiling

• Exposure to 55-75°C to remove all vegetative bacteria
• Spores are unaffected
• Activity level: Intermediate
• Uses: milk, beverages, plastic hospital equipment, certain fruit drinks
• Originally proposed by Pasteur for wine-making to prevent microbial spoilage

b) Dry Heat

• Mechanism: Oxidation of cellular components (protein denaturation requires much higher temperatures without moisture)
• Temperature and time: 160°C for 2 hours in a sterilizing oven
• Less efficient than moist heat - carbonization of organic material occurs
• Uses: Metals, glassware, heat-resistant oils and waxes immiscible in water, laboratory glassware preparation
• Not recommended for most instruments due to prolonged exposure times and instrument damage

c) Incineration

• Simplest method - exposure to a naked flame
• Examples: wire loop in microbiology labs, knife blade, needle (emergency sterilization)
• Disposable materials are rapidly decontaminated by incineration
• Destroys all microorganisms including spores

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2. Radiation

a) Ultraviolet (UV) Light

• Absorbed by nucleic acids, causing genetic damage (thymine dimer formation)
• Activity level: Sterilizing (but limited in practice)
• Major limitation: poor penetration ability - cannot penetrate glass, plastic, or most solid objects
• Uses: Irradiation of air in critical hospital sites, decontamination of air in facilities handling hazardous organisms, biosafety cabinets
• Not suitable for materials with irregular surfaces or anything UV cannot reach

b) Ionizing Radiation (Gamma rays, Cathode rays)

• Carries far greater energy than UV
• Mechanism: Direct DNA damage + production of toxic free radicals and hydrogen peroxide from water within microbial cells
• Activity level: Sterilizing - all microorganisms including spores
• Advantage: Can be applied to packaged items (packaging done before exposure to penetrating radiation)
• Uses: Industrial sterilization of disposable surgical supplies (gloves, plastic syringes, specimen containers), some foodstuffs
• Sherris & Ryan's Medical Microbiology, 8th ed.

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3. Filtration

• Both live and dead microorganisms are physically removed from liquids by positive- or negative-pressure filtration
• Membrane filters are available with variable pore sizes: 0.005-1 μm
• For removal of bacteria: pore size of 0.2 μm is effective
• Best used for: sterilization of large volumes of fluid, especially fluids containing heat-labile components (e.g., serum, enzymes, certain drugs)
• Limitation: Not effective for removing viruses (too small to be trapped)
• Used in: pharmaceutical industry, microbiological media preparation, IV solutions

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B. Gas / Vapor Methods

1. Ethylene Oxide (EO)

• An alkylating agent - inactivates microorganisms by replacing labile hydrogen atoms in DNA
• Activity: Sterilizing (all microorganisms)
• Treatment time: generally 4 hours, followed by 12-hour aeration to eliminate toxic gas before use
• Why aeration is required: The gas diffuses into absorbed substances and must be removed before clinical use
• Advantages: Effective for heat-labile devices (artificial heart valves, certain plastics, lensed instruments) damaged by autoclaving
• Disadvantages: Flammable, potentially explosive, carcinogenic to laboratory animals; strict regulations limit use
• Avoided if acceptable alternatives are available

2. Hydrogen Peroxide Vapor

• Mechanism: Oxidizing nature of the gas
• Activity: Sterilizing
• A more advanced variation: Plasma gas sterilization
  • Hydrogen peroxide is vaporized, then reactive free radicals are produced with microwave-frequency or radio-frequency energy
  • Efficient sterilizing method that does not produce toxic by-products
  • Has replaced many applications of ethylene oxide
  • Limitation: Cannot be used with materials that absorb hydrogen peroxide or react with it
• Uses: Sterilization of instruments

3. Formaldehyde Vapor

• An alkylating agent (similar mechanism to ethylene oxide)
• Can be used without pressure to decontaminate larger areas such as rooms
• Less commonly used due to toxicity and irritant properties

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C. Chemical Sterilants (Liquid)

• Medical Microbiology 9e and Sherris & Ryan's Medical Microbiology, 8th ed.

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Levels of Disinfection (vs. Sterilization)

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Summary Table of Sterilization Methods

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Clinical Points to Remember

1. Autoclaving is the method of choice for heat-stable items in clinical settings.
2. Ethylene oxide is reserved for heat-labile equipment (e.g., cardiac valves, plastic instruments) but is being replaced by plasma sterilization due to toxicity concerns.
3. Filtration is used for heat-sensitive liquids but does NOT remove viruses.
4. UV light is used for air and surface disinfection only - its poor penetration makes it unsuitable for solid materials.
5. Ionizing radiation is the preferred method for industrial pre-packaged sterilization.
6. The 3 critical parameters for autoclave sterilization: time, temperature, and moisture (steam must be saturated).

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