Q2 LONG ANSWER 10M ★ High-yield

Define sterilisation and disinfection. Describe the physical and chemical methods with their control.

Microbiology · 2nd Professional — Para-Clinical

Definitions

Term	Definition
Sterilisation	Complete destruction or removal of all forms of microbial life, including spores. An absolute term — an object is sterile or it is not
Disinfection	Destruction of most pathogenic organisms on inanimate objects; spores usually survive
Antisepsis	Application of a chemical to living tissue to reduce microbial numbers
Sanitisation	Reduction of microbial numbers to a level judged safe by public health standards
Asepsis	The technique of preventing organisms from reaching a site at all
Bactericidal / bacteriostatic	Kills organisms / inhibits their multiplication

Physical Methods — Heat

Method	Conditions	Uses and comments
Red heat	Held in a flame until red	Inoculating loops, wires, forceps tips
Flaming	Passed through a flame	Mouths of tubes, scalpels, slides
Incineration	Complete burning	Soiled dressings, animal carcasses, pathological and sharps waste
Hot air oven (dry heat)	160°C for 2 hours	Glassware, metal instruments, oils, greases, powders, glass syringes — things that steam cannot penetrate or would damage. Slower and needs a higher temperature than moist heat
Pasteurisation	Holder 63°C for 30 min; flash 72°C for 15–20 sec	Milk — kills Mycobacterium bovis, Brucella, Salmonella and Coxiella without sterilising; the phosphatase test confirms adequacy
Boiling	100°C for 10–20 min	Disinfection only — spores survive
Autoclave (moist heat under pressure)	121°C at 15 lb/in² for 15 minutes (or 134°C at 30 lb for 3 min)	The standard hospital method — surgical instruments, dressings, linen, culture media, rubber goods

CLINICAL PEARL

Moist heat kills at a much lower temperature than dry heat, and the reason is the mechanism. Moist heat coagulates and denatures protein, and water conducts heat efficiently while steam releases a large amount of latent heat as it condenses on the load. Dry heat must work by oxidation, which is far slower. That is why 121°C for 15 minutes in an autoclave achieves what needs 160°C for 2 hours in an oven.

The Autoclave in Detail

Principle — water boils at 100°C at atmospheric pressure; raising the pressure raises the boiling point, so saturated steam at 15 lb/in² reaches 121°C
All air must BE removed — this is the single most important requirement. Air is a poor conductor, and a pocket of trapped air is cooler than the surrounding steam, so items within it are not sterilised. Modern autoclaves use a pre-vacuum to remove it
Steam must be saturated and must condense on the load, releasing latent heat and wetting the surface; superheated or wet steam both perform poorly
Loading matters — packs must not be overpacked and must allow steam to penetrate; the holding time begins only when the whole load has reached temperature
Not suitable for oils, powders, greases (steam cannot penetrate), or heat-labile plastics and endoscopes

Sterilisation Control

Type of indicator	Example	What it tells you
Physical	Thermometers, pressure gauges, time and temperature charts	That the machine reached the stated conditions — but not whether the inside of the load did
Chemical	Autoclave (Bowie-Dick) tape, BROWNE tubes which change from red to green, Class 6 emulating indicators	That the pack was exposed to the process; the BOWIE-DICK test specifically checks air removal and steam penetration and is done daily
Biological — the definitive test	Spore strips: autoclave — Geobacillus stearothermophilus; hot air oven — Bacillus subtilis subsp. niger; ethylene oxide — Bacillus atrophaeus; ionising radiation — Bacillus pumilus	That organisms were actually killed — the only indicator that tests the outcome rather than the conditions

Bacterial spores are used because they are the most resistant form of life to the process being tested; if the spores are dead, everything else is

Other Physical Methods and Chemical Agents

Radiation — non-ionising (UV, 254 nm) causes thymine dimers but has very poor penetration, so it disinfects surfaces and air (operating theatres, safety cabinets) and cannot sterilise a wrapped object; it also damages the eyes and skin. ionising (gamma rays, electron beam) penetrates deeply and is "cold sterilisation" used industrially for disposable syringes, gloves, catheters, sutures and plastics
Filtration — membrane filters of 0.22 micrometre pore size for heat-labile fluids (sera, antibiotic solutions, vaccines); viruses and mycoplasma pass through. HEPA filters remove particles above 0.3 micrometre from air in operating theatres, laminar flow cabinets and isolation rooms
Gaseous sterilisation — ethylene oxide for heat-sensitive items (endoscopes, prosthetic heart valves, plastics); it is an alkylating agent, effective but highly toxic, mutagenic, carcinogenic and explosive, and requires prolonged aeration afterwards. Alternatives are hydrogen peroxide plasma and formaldehyde vapour
Chemical disinfectants — glutaraldehyde (2%) for endoscopes, being sporicidal with prolonged contact; hypochlorite for surfaces and spills, and sporicidal, which is why it is used against Clostridioides difficile; 70% alcohol, chlorhexidine, povidone-iodine for skin
Spaulding classification — critical items entering sterile tissue or the vascular system require sterilisation; semi-critical items contacting mucous membranes (endoscopes) require high-level disinfection; non-critical items touching intact skin require low-level disinfection

Choosing a Method

Item	Appropriate method	Reason
Surgical instruments, dressings, linen	Autoclave	Steam penetrates and is reliable
Glassware, metal, oils, powders	Hot air oven	Steam cannot penetrate oils and powders
Heat-labile fluids — sera, antibiotic solutions, vaccines	Membrane filtration (0.22 micrometre)	Heat would destroy the product; note that viruses pass through
Endoscopes and heat-sensitive instruments	Glutaraldehyde (high-level disinfection) or hydrogen peroxide plasma	Heat would damage optics and plastics
Prosthetic valves, catheters, plastics (industrial)	Ethylene oxide or gamma irradiation	Effective at ambient temperature; gamma rays penetrate sealed packs
Operating theatre and safety cabinet air	UV light and HEPA filtration	Surface and air disinfection only — UV has poor penetration
Milk	Pasteurisation	Kills pathogens while preserving nutritional and organoleptic quality
Infectious laboratory waste	Autoclaving followed by incineration	Must be decontaminated before it leaves the laboratory

Applied Aspects

Use a biological indicator regularly, not merely tape; autoclave tape shows only that the pack met steam, not that sterilisation was achieved
Perform the Bowie-Dick test daily on a vacuum autoclave; failure of air removal is the commonest cause of sterilisation failure
Clean before you disinfect — organic matter (blood, pus, faeces) inactivates hypochlorite and quaternary ammonium compounds and shields organisms; this is the most frequently neglected step
Endoscopes are a recognised vehicle of transmission, and outbreaks have been traced to inadequate cleaning of channels; the process is manual, tedious and easily short-cut
Prions resist all routine methods — standard autoclaving does not destroy them, and extended cycles (134°C for 18 minutes), sodium hydroxide, or disposal by incineration are required
Reuse of single-use devices is common in India for reasons of cost, and is a real infection risk; where it is unavoidable, validated reprocessing is essential rather than informal cleaning
Boiling is disinfection, not sterilisation, and the distinction is frequently misunderstood in smaller facilities where boiled instruments are treated as sterile
Check the expiry and integrity of sterile packs; a pack that is damp, torn or has been stored badly is no longer sterile whatever the indicator shows
Disinfectant solutions can themselves become contaminated — outbreaks of Pseudomonas and Burkholderia have been traced to diluted or topped-up containers, which is a real hazard where cost drives dilution

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