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Sterilization - Important Exam Questions & Answers

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Section 1: Definitions (Short Answer / 1-2 marks)

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• Antisepsis: Use of chemical agents on living skin/tissue to inhibit or eliminate microbes; no sporicidal action implied.
• Disinfection: Use of physical/chemical agents to destroy most microbial forms; spores may survive.
• Sanitization: Reduction of microbial contamination to a level considered safe for public health.
• Pasteurization: Exposure of liquids to 55-75°C to kill vegetative bacteria but not spores (e.g., milk, beverages).
• Decontamination: Removal or neutralization of hazardous agents from a surface/person.

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Section 2: Physical Methods (3-5 marks)

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• Dry heat: Flaming, incineration, hot air oven (160°C/2 hrs)
• Moist heat: Boiling (100°C), autoclave (121°C/15 min), pasteurization, Tyndallization

• Non-ionizing: UV radiation (254 nm)
• Ionizing: Gamma rays, cathode rays (X-rays)

• Membrane filters (0.2 µm pore size)
• HEPA filters (air)

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• Principle: Moist heat causes irreversible denaturation of proteins. Moisture (water molecules) disrupts hydrogen bonds between peptide groups at relatively low temperatures - far more effectively than dry heat.
• Standard cycle: 121°C at 15 psi (1 atm above atmospheric) for 15-20 minutes
• Flash autoclave (operating room): 134°C for 3 minutes
• Critical parameters: Time, temperature, and moisture (all three must be adequate)
• Effect of temperature drop: A drop of just 1.7°C increases required exposure time by 48%

1. Downward displacement (gravity) - air drains out from bottom valve (air is heavier than steam)
2. Pre-vacuum (mechanical evacuation before steam entry)

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• Hot air oven: 160°C for 2 hours
• Mechanism: Protein oxidation/denaturation
• Requires higher temperature and longer time than moist heat because there are no water molecules to assist denaturation

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• Developed by Louis Pasteur
• Exposes liquids to 55-75°C to kill all vegetative bacteria; spores survive
• Used for: Milk, fruit juices, wine, and hospital plastic equipment (inhalation therapy)
• HTST (High Temperature Short Time): 72°C for 15 seconds (most common for milk today)
• LTLT (Low Temperature Long Time / Holder method): 63°C for 30 minutes
• UHT: 138°C for 2 seconds (ultra-high temperature, commercially sterile)
• Kills pathogens like Mycobacterium bovis, Salmonella, Listeria in milk
• NOT sterilization - spores survive

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• Also called intermittent sterilization or fractional sterilization
• Method: Steam at 100°C (flowing steam, NOT under pressure) for 30 minutes on 3 consecutive days
• Principle: First exposure kills vegetative forms. During incubation intervals (at 37°C), surviving spores germinate into vegetative forms. Second and third exposures kill these germinated forms.
• Used for: Materials that cannot withstand autoclave pressure (e.g., sugar-containing media, serum-containing media)

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Section 3: Radiation (3-5 marks)

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• Wavelength: 240-280 nm (optimum 254 nm)
• Mechanism: Causes formation of thymine dimers in DNA, blocking DNA replication and transcription
• Limitation: Very poor penetration - effective only on exposed surfaces; cannot penetrate glass, paper, or most materials
• Uses: Disinfection of air in OT, laminar flow hoods, sterile cabinets; water surface disinfection
• Categorized as sterilizing in activity level (if exposure is long enough) but practically used for surface/air disinfection

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• Types: Gamma rays (from Cobalt-60), cathode rays (electron beams), X-rays
• Mechanism: Direct DNA damage + production of toxic free radicals and H₂O₂ from water inside microbial cells
• Advantage: Excellent penetration - can sterilize items through packaging (can be packaged before exposure)
• Uses: Industrial sterilization of disposable surgical supplies (gloves, syringes, specimen containers), food irradiation
• Gamma radiation is the most widely used form for industrial sterilization

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Section 4: Gas Sterilization (3-5 marks)

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• Used for heat-labile or pressure-sensitive items (plastics, lensed instruments, endoscopes, rubber, electronics)
• Mechanism: Alkylating agent - replaces hydrogen atoms of NH, OH, COOH, SH groups with hydroxyethyl groups, inactivating enzymes and disrupting DNA replication
• Conditions: Generally 4 hours exposure; must be followed by 12 hours of aeration to eliminate toxic gas before use
• Properties:
  • Highly efficient - kills ALL organisms including spores and viruses
  • Flammable and explosive - mixed with CO₂ or freons to reduce risk
  • Carcinogenic to laboratory animals
  • Strict regulations limit its use
• Current status: Being replaced by hydrogen peroxide plasma sterilization due to toxicity concerns
• Monitoring: Bacillus subtilis spore strips are used for biological monitoring

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• A newer alternative to ethylene oxide
• Method: Hydrogen peroxide is vaporized, then exposed to microwave/radio-frequency energy to produce reactive free radicals (plasma)
• Mechanism: Oxidative destruction of cell components (lipids, proteins, DNA)
• Advantages: No toxic by-products; efficient; no aeration needed
• Disadvantages: Cannot be used with materials that absorb or react with H₂O₂ (e.g., cellulose, paper, certain liquids)
• Has replaced many ETO applications

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Section 5: Chemical Agents (5-10 marks)

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High-Level Disinfectants (approach sterilization):

Intermediate-Level Disinfectants:

Low-Level Disinfectants:

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• Types: Ethyl alcohol (ethanol) and isopropyl alcohol
• Optimal concentration: 70-95% aqueous solution
• Mechanism: Protein denaturation - water molecules are essential for this process
• 100% alcohol: LESS effective - dehydrates rapidly but fails to kill because the lethal process requires water
• Spectrum: Kill vegetative bacteria, fungi, some viruses; inactive against bacterial spores
• Use: Skin antisepsis before venipuncture, injections (isopropyl 90-95%)
• Inactivated by: Organic matter (blood, pus)

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• Iodine mechanism: Iodination OR oxidation of essential microbial cell components
• Tincture of iodine: Iodine in alcohol - effective but causes skin staining and dehydration; now largely replaced
• Iodophors: Iodine combined with povidone (polyvinylpyrrolidone) or non-ionic detergents
  • Gradually release small amounts of free iodine
  • Less skin irritation and staining than tinctures
  • Uses: Pre-surgical skin preparation, wound antisepsis
• Inactivated by: Organic matter

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• Exists as hypochlorous acid in aqueous solution
• Mechanism: Oxidation - lethal within seconds to most vegetative bacteria and inactivates most viruses at < 1 ppm
• Uses:
  • Drinking water chlorination
  • Swimming pool disinfection
  • Agent of choice for surfaces contaminated with viruses or bacterial spores
  • Applied as 5% sodium hypochlorite solution for decontamination
• Inactivated by: Organic matter (significantly reduces efficacy)

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• Also called "quats" - e.g., benzalkonium chloride, cetrimide
• Mechanism: Their hydrophobic and lipophilic groups react with the lipid of the bacterial cell membrane - alters membrane surface properties and permeability - leads to loss of essential cell components and death
• Spectrum: Effective against most bacteria and fungi, and lipophilic (enveloped) viruses
• Inactive against: Spores, most non-enveloped (non-lipid) viruses, Mycobacterium tuberculosis
• Inactivated by: Organic matter, anionic detergents (soaps), cotton/gauze (adsorbed)
• Level: Low-level disinfectant

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• High-level disinfectant / sterilant depending on exposure time
• Mechanism: Alkylation of proteins and nucleic acids
• Used for: Endoscopes, surgical instruments that cannot be autoclaved
• Spectrum: Active against all organisms (bacteria, spores, viruses, fungi) with adequate contact time
• Concentration used: 2% alkaline glutaraldehyde (Cidex)
• Safety concern: Toxic, irritant to skin/mucous membranes - requires careful handling
• 2% glutaraldehyde: 10 min = disinfection; 3-10 hours = sterilization

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Section 6: Filtration

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• Used for heat-labile fluids (serum, certain enzymes, antibiotics in solution, IV solutions)
• Membrane filters: Pore size 0.2 µm - removes bacteria effectively
  • Available in pore sizes 0.005 to 1 µm
  • Used for large volumes of fluid
• HEPA filters (High Efficiency Particulate Air): Remove particles from air in laminar flow hoods, OT ventilation, biosafety cabinets
• Limitation: Does NOT remove viruses (too small)
• Works by physical size exclusion - both live and dead organisms are removed

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Section 7: Spaulding's Classification (High-Yield)

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Section 8: Monitoring of Sterilization (High-Yield)

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1. Physical/Mechanical monitoring: Temperature gauges, pressure gauges, time recorders on autoclave
2. Chemical indicators: Bowie-Dick tape, indicator strips (change color with heat/gas) - do NOT confirm sterility
3. Biological indicators (gold standard):
   • Autoclave (steam): Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) spore strips - spores killed at 121°C/15 min
   • Ethylene oxide / dry heat: Bacillus subtilis var. niger (also called B. atrophaeus) spore strips

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Section 9: One-liner / MCQ-type

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