Sterilisation & Disinfection

Sterilisation and disinfection underpin every aspect of safe medical practice, from theatre instruments to vaccine production. For NEET PG this is a guaranteed-yield, low-effort topic: questions revolve around exact temperatures, times, which agent kills spores, and the meaning of named indicators and coefficients. Learn the numbers cold.

Core definitions — get the hierarchy right

These terms are constantly confused in MCQs. The killing power decreases as you move down the list.

Term	Meaning	Spores killed?
Sterilisation	Complete destruction/removal of all microbes including bacterial spores	Yes
Disinfection	Destruction of vegetative pathogens but not necessarily spores; usually for inanimate objects	Usually no
Antisepsis	Destruction/inhibition of microbes on living tissue (skin, wounds)	No
Sanitisation	Reducing microbial load to a safe public-health level (e.g. crockery, food premises)	No
Asepsis	Preventing entry of microbes into a sterile environment	—
Decontamination	Rendering an item safe to handle	Variable

High-yield: A disinfectant is used on inanimate surfaces; an antiseptic is used on living tissue. The same chemical at different concentrations may serve both (e.g. dilute Dettol = antiseptic, concentrated = disinfectant).

Spaulding classification of medical devices dictates the level of processing required:

• Critical items (enter sterile tissue/vascular system — surgical instruments, implants, needles) → must be sterilised.
• Semi-critical items (contact mucous membranes — endoscopes, laryngoscopes, respiratory equipment) → high-level disinfection minimum.
• Non-critical items (contact intact skin — BP cuffs, stethoscopes) → low/intermediate-level disinfection.

Classification of methods

Sterilisation methods broadly → Physical and Chemical.

1. Physical — Heat (dry & moist), Radiation, Filtration.
2. Chemical — Gases (ethylene oxide, formaldehyde, hydrogen peroxide vapour) and Liquids (glutaraldehyde, peracetic acid).

The order of microbial resistance to killing (most to least resistant) is a favourite:

Prions > Bacterial spores > Mycobacteria > Non-enveloped (naked) viruses > Fungi > Vegetative bacteria > Enveloped viruses.

Mnemonic for resistance: "Prions Spoil My Naked Friends Very Easily."

Physical methods — HEAT

Dry heat

Acts by protein denaturation, oxidative damage and toxic effects of elevated electrolyte levels. Less efficient than moist heat (needs higher temperature/longer time because dry air is a poor conductor).

Method	Temperature / Time	Uses
Red heat	Till red hot	Inoculation loops, wires, forceps tips, points of needles
Flaming	Pass through flame	Scalpels, mouths of culture tubes, glass slides
Incineration	Burning to ash	Soiled dressings, animal carcasses, pathological & sharps waste
Hot air oven	160°C for 1 hour (holding period)	Glassware, swabs, all-glass syringes, dry powders, oils, forceps, scalpels, dry instruments

High-yield: Hot air oven = 160°C for 60 minutes. Spore strip used = Clostridium tetani or Bacillus atrophaeus (formerly B. subtilis var. niger / B. globigii). The oven must cool slowly to prevent glassware cracking.

The hot air oven holding time relative to temperature is also asked: 170°C → 18 min, 160°C → 60 min, 150°C → 150 min. The standard answer is 160°C/1 hr.

Moist heat

More efficient than dry heat — acts by coagulation and denaturation of proteins. Subdivided by temperature.

Temperature below 100°C:

• Pasteurisation of milk:
  • Holder (LTLT) method → 63°C for 30 minutes.
  • Flash (HTST) method → 72°C for 15–20 seconds, then rapid cooling.
  • UHT → 140°C for 15 seconds.
  • Kills Mycobacterium bovis, Brucella, Salmonella, Coxiella burnetii (Q fever — the most heat-resistant non-spore-former and the index organism that set pasteurisation standards). Spores survive.
• Inspissation (Koch's/serum coagulator) → 80–85°C for 30 min on 3 successive days — used for Löwenstein–Jensen (LJ) and Loeffler's serum media.
• Vaccine bath → 60°C for 1 hour.

Temperature at 100°C:

• Boiling → 100°C for 10–30 min; kills vegetative forms, not spores. (Disinfection, not sterilisation.)
• Tyndallisation (intermittent steam sterilisation) → free-flowing steam at 100°C for 20 min on 3 successive days. Used for sugar/serum/egg media that would be damaged by autoclaving. Spores germinate between cycles and are killed on subsequent days.

Temperature above 100°C — the AUTOCLAVE (steam under pressure):

This is the single most tested item in the whole topic.

High-yield: Standard autoclave cycle = 121°C at 15 lb/sq inch (psi) ≈ 1.06 kg/cm² pressure for 15 minutes. Other valid combinations: 126°C/10 psi/10 min; 134°C/30 psi/3 min (flash). Moist heat kills by protein denaturation/coagulation, NOT oxidation.

Latent heat of steam is what makes the autoclave so efficient — when steam condenses on a cooler object it releases large latent heat and the resulting reduction in volume draws in fresh steam. Saturated steam is essential; trapped air lowers the temperature and causes failure.

Autoclave uses: culture media, dressings, gloves, gowns, aprons, syringes, surgical instruments, rubber goods. Not suitable for oils, powders, sharp cutting instruments (blunting) or heat-sensitive plastics.

Controls/sterility indicators for autoclave:

Control type	Example
Biological (most reliable)	Spores of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) NCTC 10007 — paper strips; no growth = success
Chemical	Browne's tube (green = sterile), Bowie–Dick tape (tests air removal & steam penetration in pre-vacuum autoclaves)
Physical	Thermocouple, temperature/pressure gauges, automatic process records

High-yield mapping: Autoclave → Geobacillus stearothermophilus. Hot air oven → Bacillus atrophaeus/Clostridium tetani. Ethylene oxide → Bacillus atrophaeus (B. subtilis). Memorise these spore-strip pairings — directly examined.

Physical methods — RADIATION

Type	Nature	Uses
Non-ionising (UV, 240–280 nm; peak ~260 nm)	Low penetration; damages DNA via thymine dimers	Surface/air disinfection — operation theatres, laminar flow hoods, safety cabinets, water
Ionising (gamma rays — Cobalt-60; X-rays; high-energy electrons)	High penetration; "cold sterilisation"	Industrial sterilisation of plastic syringes, catheters, gloves, sutures, bone/tissue grafts, swabs

High-yield: UV light → non-ionising, poor penetration, acts by forming pyrimidine (thymine) dimers; cannot penetrate glass/plastic, hence surface use only. Gamma irradiation → ionising, "cold" sterilisation of disposable single-use medical plastics. Dosage commonly cited: 2.5 megarad (25 kGy).

Physical methods — FILTRATION

Filtration removes rather than kills organisms; the method of choice for heat-labile liquids — sera, antibiotic solutions, sugars, urea, vaccines, and for air.

• Membrane filters (cellulose acetate/nitrate) — most widely used now; pore size 0.22 µm sterilises (removes bacteria), 0.45 µm for routine work. Used for sterility testing, water analysis (colony counts), removing bacteria from fluids.
• HEPA filters (High-Efficiency Particulate Air) — remove particles ≥0.3 µm with ≥99.97% efficiency; used in laminar flow / biosafety cabinets and theatres.
• Older: Seitz (asbestos), sintered glass, candle filters (Berkefeld – diatomaceous earth; Chamberland – porcelain).

High-yield: A standard membrane/bacterial filter (0.22 µm) does NOT retain viruses or mycoplasma, which can pass through. Filtration sterilises by physical exclusion, not killing.

Chemical methods — GASEOUS sterilants

Ethylene oxide (EO/ETO)

• Alkylating agent — alkylates amino, carboxyl, sulphydryl, hydroxyl groups of proteins/nucleic acids.
• Highly penetrating, sporicidal, effective at low temperature → ideal for heat- and moisture-sensitive items: plastic and rubber tubing, catheters, prosthetic heart valves, ventilator/anaesthetic equipment, complex endoscopes, disposable plastics.
• Drawbacks: highly inflammable & explosive (diluted with CO₂ or fluorocarbons), carcinogenic/mutagenic, toxic — requires post-sterilisation aeration to remove residues.
• Indicator spore: Bacillus atrophaeus (B. subtilis var. niger).

Formaldehyde

• Gas used for fumigation of rooms/operation theatres/safety cabinets (formalin + KMnO₄, or formaldehyde vapour). Sporicidal, microbicidal, virucidal.
• Low-temperature steam formaldehyde (LTSF) at 73°C — for heat-sensitive items.
• Irritant; carcinogenic. Used to inactivate toxins → toxoids and to prepare killed vaccines (e.g. Salk polio).

Other gases

• Hydrogen peroxide vapour / plasma (Sterrad) — low-temperature sterilisation of endoscopes; produces free radicals.
• Vapour-phase peracetic acid and chlorine dioxide.
• Beta-propiolactone (BPL) — fumigant, more active than formaldehyde, carcinogenic.

Chemical methods — LIQUID disinfectants/antiseptics

Mechanisms cluster into a few groups — examiners love mechanism-of-action matching.

Agent	Mechanism / Notes	Sporicidal?	Typical use
Glutaraldehyde (2% Cidex)	Alkylation; needs activation (alkaline pH), 10 h contact for sterilisation, 20 min for high-level disinfection	Yes (slow)	Endoscopes, plastic/rubber items, cystoscopes
Alcohols (60–70% ethanol/isopropanol)	Protein denaturation + membrane lipid dissolution; needs water (absolute alcohol is poor)	No	Skin antisepsis, surfaces
Halogens — Chlorine (hypochlorite)	Oxidation; sodium hypochlorite, bleaching powder	Yes (high conc.)	Water, surfaces, blood spills (1% for HIV/HBV)
Iodine / Iodophors (povidone-iodine)	Oxidation/iodination of proteins	Variable	Skin antiseptic, pre-op preparation
Phenolics (phenol, cresol, Lysol, chlorhexidine partly)	Membrane damage + protein precipitation	No	Surfaces, faeces, the reference standard
Chlorhexidine (biguanide)	Membrane disruption	No	Hand scrub, skin, mouthwash
Quaternary ammonium compounds (cetrimide, benzalkonium)	Cationic surfactants — disrupt membranes	No	Skin, instruments (inactivated by soap, organic matter; Pseudomonas can contaminate)
Hydrogen peroxide / peracetic acid	Oxidising free radicals	Yes	Wounds, contact lenses, sterilants
Heavy metals (silver, mercury)	Protein sulphydryl binding	No	Silver nitrate (eyes/burns), merthiolate
Dyes (gentian violet, acriflavine)	—	No	Skin, wounds

High-yield: Glutaraldehyde is the classic chemical agent that is sporicidal and used for sterilising heat-sensitive endoscopes; alcohols and QACs are NOT sporicidal. 70% alcohol is more effective than absolute (100%) alcohol because water is needed for protein denaturation.

Phenol coefficient (Rideal–Walker test)

A perennial favourite.

High-yield: Phenol coefficient = ratio of the dilution of the test disinfectant to the dilution of phenol that kills the test organism (Salmonella Typhi, Staphylococcus aureus) under standard conditions in the same time. Phenol is the reference standard with a coefficient of 1. A coefficient >1 means the agent is more potent than phenol; <1 means less potent. The Chick–Martin test modifies this by adding organic matter (yeast/faeces) to mimic real soiled conditions.

Levels of disinfection (classification by activity):

• High-level: glutaraldehyde, peracetic acid, hydrogen peroxide, chlorine dioxide — kill all microbes except large numbers of spores.
• Intermediate-level: alcohols, iodophors, phenolics — kill Mycobacterium, most viruses/fungi, vegetative bacteria; not spores.
• Low-level: QACs — kill most vegetative bacteria, some fungi/viruses; not TB or spores.

Sterilisation of special / resistant agents

Prions are the most resistant infectious agents — standard autoclaving and routine disinfectants fail.

High-yield: Prion decontamination → autoclave at 134°C for 18 minutes (or 121°C for ≥1 h) PLUS 1 N NaOH or sodium hypochlorite (≥2% available chlorine). Glutaraldehyde, alcohol, formalin and standard autoclaving are ineffective against prions and may even fix infectivity.

Quality control & validation — summary table

Process	Standard condition	Biological indicator
Autoclave	121°C, 15 psi, 15 min	Geobacillus stearothermophilus
Hot air oven	160°C, 60 min	Bacillus atrophaeus / Cl. tetani
Ethylene oxide	55–60°C, several hours	Bacillus atrophaeus
Ionising radiation	25 kGy (2.5 Mrad)	Bacillus pumilus
LTSF / formaldehyde	73°C	Geobacillus stearothermophilus

Key differentials & traps

• Sterilisation vs disinfection vs antisepsis — only sterilisation guarantees spore kill; antiseptic = living tissue.
• Dry vs moist heat — moist heat (autoclave) kills by coagulation; dry heat (oven) by oxidation. Moist is faster/lower temperature.
• Boiling vs autoclaving — boiling (100°C) is disinfection, not sterilisation (spores survive). Autoclaving sterilises.
• Tyndallisation vs inspissation — both are intermittent over 3 days, but Tyndallisation uses free steam at 100°C (for sugar media); inspissation uses 80–85°C (for serum/egg media like LJ).
• 0.22 µm vs 0.45 µm membrane — 0.22 µm is the sterilising grade.
• Pasteurisation methods — Holder 63°C/30 min vs Flash 72°C/15 s.

Recently asked / exam angle

• Temperature–time pairings are the bread and butter: autoclave 121°C/15 psi/15 min, hot air oven 160°C/1 h, pasteurisation 63°C/30 min or 72°C/15 s, inspissation 80–85°C × 3 days.
• "Which method kills spores?" → autoclave, hot air oven, EO, glutaraldehyde, ionising radiation, formaldehyde. Boiling, alcohols, QACs and pasteurisation do not.
• Biological indicator matching: autoclave → Geobacillus stearothermophilus; hot air oven → Bacillus atrophaeus. This single-best-answer pairing recurs.
• Mechanism MCQs: EO/glutaraldehyde/formaldehyde = alkylation; halogens & H₂O₂ = oxidation; alcohol = protein denaturation; QAC = cationic surfactant/membrane disruption; moist heat = coagulation; UV = thymine dimers.
• Bowie–Dick test checks air removal/steam penetration in pre-vacuum autoclaves (not the sterilising temperature itself).
• Method of choice for heat-labile fluids (serum, antibiotics, vaccines) → filtration.
• Method of choice for plastic disposables (syringes, gloves) at industrial scale → gamma/ionising radiation.
• Endoscope sterilisation → glutaraldehyde or low-temperature H₂O₂ plasma/EO.
• Coxiella burnetii is the index organism for pasteurisation standards (most heat-resistant non-sporing organism).
• Phenol coefficient definition and the Chick–Martin modification (organic load).

Rapid revision

1. Autoclave = 121°C, 15 psi, 15 min; kills by protein coagulation; control organism Geobacillus stearothermophilus.
2. Hot air oven = 160°C for 1 hour; kills by oxidation; control Bacillus atrophaeus; for glassware, powders, oils.
3. Pasteurisation: Holder 63°C/30 min, Flash 72°C/15 s — kills Coxiella, Brucella, TB; spores survive (it is disinfection).
4. Tyndallisation = free steam 100°C, 20 min × 3 days; inspissation = 80–85°C × 3 days for LJ/Loeffler media.
5. Boiling (100°C) is disinfection, not sterilisation — spores survive.
6. Ethylene oxide = alkylating, sporicidal, penetrating, for heat-sensitive plastics; explosive & carcinogenic; needs aeration.
7. Glutaraldehyde 2% (Cidex) = sporicidal liquid for endoscopes; 10 h to sterilise.
8. 70% alcohol > absolute alcohol; alcohols and QACs are not sporicidal.
9. Membrane filter 0.22 µm sterilises heat-labile fluids but does not stop viruses/mycoplasma; HEPA for theatre air.
10. UV = non-ionising, thymine dimers, surface only; gamma (Co-60) = ionising "cold" sterilisation of disposables.
11. Phenol coefficient: ratio vs phenol (standard = 1); Chick–Martin adds organic matter.
12. Prions = most resistant → 134°C/18 min autoclave plus 1 N NaOH or hypochlorite; resistance order: Prions > Spores > Mycobacteria > Naked viruses > Fungi > Vegetative bacteria > Enveloped viruses.