Summary: Sterilization And Disinfection

Table of Contents
1. Definitions
2. Factors Affecting Effectiveness
3. Classification of Methods
4. Physical Methods - Heat
5. Filtration and Air Filtration
6. Radiation
7. Chemical Methods - Key Agents
8. Prions and Resistant Agents
9. Spore-Killing (Sporicidal) Agents
10. Testing and Controls
11. Clinical Use and Device Classification
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Definitions

• Sterilization: elimination of all living microorganisms including spores (≥10⁶ CFU reduction).
• Disinfection: removal/killing of most pathogenic organisms but not spores (≥10³ CFU reduction).
• Asepsis: use of chemical antiseptics on skin to inhibit/destroy pathogens and some normal skin flora.
• Decontamination (Sanitization): reduction of microbes to a safe level so items can be handled without protective gear (≥1 log CFU reduction; spores excluded).

Factors Affecting Effectiveness

• Organism load: higher numbers require longer treatment.
• Nature of organism: susceptibility varies by organism type.
• Concentration and temperature of agent.
• Nature of sterilant/disinfectant: microbicidal ability, speed, residual effect, and activity in presence of organic matter.
• Duration of exposure: longer increases effect.
• pH: acidic pH can enhance heat lethality.
• Biofilm formation: reduces disinfectant effectiveness.

Classification of Methods

• Physical methods: heat (dry and moist), filtration, radiation (ionizing and non-ionizing), ultrasonic vibration.
• Chemical methods: alcohols, aldehydes, phenolics, halogens, oxidizing agents (H₂O₂, peracetic acid), heavy metal salts, surface-active agents, dyes, gaseous sterilants (e.g., ethylene oxide), plasma sterilization.

Physical Methods - Heat

• Mechanism: dry heat causes charring, oxidative damage and protein denaturation; moist heat denatures and coagulates proteins.
• Dry heat (hot air oven): 160°C for 2 hours.
• Moist heat <100°C: pasteurization (Holder 63°C 30 min; Flash 72°C 20 s then cool), water bath (60°C 1 h), inspissation (80-85°C 30 min on 3 consecutive days).
• Moist heat = 100°C: boiling (15 min kills vegetative forms; spores survive), steaming (100°C, long exposure), tyndallization (20 min steam on 3 days).
• Moist heat >100°C (autoclave): 121°C for 15 minutes at 15 psi; biological indicator: Geobacillus stearothermophilus; controls include thermocouples and autoclave tape.

Filtration and Air Filtration

• Filtration: removes microbes from heat-sensitive fluids; depth filters trap throughout filter depth, membrane filters trap on surface.
• Membrane pore size: typically 0.22 µm for bacterial filtration.
• Depth filters: used for industrial food/drink/chemical filtration (not for bacterial sterility testing).
• Air filters: HEPA removes 99.97% of ≥0.3 µm; ULPA removes 99.999% of ≥0.12 µm.

Radiation

• Ionizing radiation (X-rays, gamma rays): causes DNA breaks (cold sterilization), high penetration, fast action, no temperature rise; destroys spores and vegetative cells; tested with Bacillus pumilus.
• Non-ionizing radiation (UV 250-300 nm for ~30 min): lethal on clean surfaces but does not penetrate glass, dirt, or water; used for clean surface and water treatment.

Chemical Methods - Key Agents

• Alcohols: act on proteins and membranes; effective against bacteria, fungi and some enveloped viruses; do not kill spores; ethyl alcohol commonly used at 70%.
• Aldehydes: inactivate nucleic acids and proteins by crosslinking; sporicidal and used as chemical sterilants; formaldehyde is toxic and corrosive; glutaraldehyde (2%) used for endoscope sterilization (requires activation, active ~14 days); ortho-phthalaldehyde (0.55%) is more stable and needs no activation.
• Phenolic compounds: active in organic matter, toxic/irritating so used as disinfectants; some less irritating phenolics used as antiseptics; chlorhexidine and chloroxylenol are notable phenolics; hexachlorophane limited due to toxicity.
• Halogens: iodine oxidizes and iodinate proteins (tincture, iodophors); chlorine (sodium hypochlorite, bleaching powder, gas) yields hypochlorous acid active vs vegetative bacteria and fungi but not spores; chlorine is inactivated by organic matter, can be corrosive and has other disadvantages.
• Oxidizing agents: hydrogen peroxide releases hydroxyl radicals; ideal 3-6% for general use, 10% for catalase-producers/spores; effective in presence of organic matter and low toxicity; peracetic acid is a strong sterilant often used with H₂O₂ but can corrode metals.
• Plasma sterilization: uses H₂O₂ alone or with peracetic acid to generate UV photons and reactive radicals (O, OH) in a vacuum at low temperature (<50°C); suited for heat-labile instruments; biological controls include Geobacillus stearothermophilus and Bacillus subtilis subsp. niger.
• Heavy metal salts: act by binding cell proteins (sulfhydryl groups) and precipitating proteins; many are bacteriostatic; examples used clinically include silver and copper compounds and historical uses of mercury salts.
• Surface active agents (surfactants): cationic (quaternary ammonium compounds) disrupt membranes and denature proteins but are inactive vs M. tuberculosis and spores; anionic soaps have detergent action with weaker antimicrobial effect; amphoteric agents work across pH ranges but may be affected by organic matter.
• Dyes: aniline and acridine dyes act as skin/wound antiseptics and interfere with cell wall or nucleic acid/protein synthesis; effectiveness differs between gram-positive and gram-negative organisms and may be reduced by organic material.
• Gaseous sterilization - Ethylene oxide (ETO): effective sporicidal gaseous sterilant that alkylates cell proteins; used in 10-20% concentrations mixed with inert gases because ETO is flammable, irritating, explosive and potentially carcinogenic.

Prions and Resistant Agents

• Prions are most resistant; recommended destruction: autoclave 134°C for 3-4 minutes, or 1 N NaOH for 1 hour, or 0.5% sodium hypochlorite for 2 hours.
• Relative resistance of microorganisms (most → least resistant): prions; Cryptosporidium oocysts; coccidian cysts; bacterial spores; mycobacteria; other parasite cysts; small non-enveloped viruses; protozoan trophozoites; gram-negative bacteria; fungi; large non-enveloped viruses; gram-positive bacteria; enveloped viruses.

Spore-Killing (Sporicidal) Agents

• Common sporicidal agents: Ethylene oxide, Formaldehyde, Glutaraldehyde, Hydrogen peroxide (EFGH).
• Other sporicidal options: Peracetic acid, Ortho-phthalaldehyde, Plasma sterilization (3P), plus autoclave and hot air oven.

Testing and Controls

• Disinfectant tests: Phenol coefficient (Rideal-Walker), Modified Rideal-Walker (with organic matter), Chick-Martin, Capacity (Kelsey-Sykes), In-use (Kelsey and Maurer).
• Phenol coefficient: compares disinfectant to phenol against Salmonella Typhi; >1 considered acceptable.
• Biological sterilization indicators: Clostridium tetani (non-toxigenic), Bacillus subtilis subsp. niger, Brevundimonas diminuta, Serratia, Geobacillus stearothermophilus, Bacillus pumilus (for ionizing radiation), Bacillus globigii (ETO control).

Clinical Use and Device Classification

• Sterilization conditions commonly used: hot air oven 160°C for 2 h; autoclave 121°C for 15 min at 15 psi.
• ETO sterilization uses: disposable plastics, syringes, tubing, sutures, catheters and other heat-sensitive devices.
• Spaulding's classification of medical devices is included as a standard reference for disinfection/sterilization selection.