Microbiology chapters
Sterilisation and disinfection
The difference between sterilisation, disinfection and antisepsis, the physical methods (autoclave, hot air oven, incineration, filtration, radiation, pasteurisation) with their settings and uses, the chemical disinfectants and what they kill, sterilisation controls, and how prions and spores are handled, with D-value and dilution worked out by hand.
- Sterilisation kills all microbes including spores; disinfection kills most pathogens on objects but not necessarily spores; antisepsis is disinfection of living tissue; resistance runs from prions (most resistant) > bacterial spores > mycobacteria > non-enveloped viruses > fungi > vegetative bacteria > enveloped viruses (least resistant).
- The autoclave (moist heat under pressure: 121 °C for 15 minutes at 15 psi, or 134 °C for 3 minutes) is the most reliable method and kills spores (checked with Geobacillus stearothermophilus spores); the hot air oven (dry heat 160 °C for 2 hours) is for glassware, powders and oils; heat-sensitive items use ethylene oxide, gamma radiation (disposable plastics), filtration (sera, antibiotic solutions) or glutaraldehyde (2%: endoscopes).
- Disinfectants: 70% alcohol (skin, hands; not sporicidal), chlorine/hypochlorite (blood spills, water; inactivated by organic matter), povidone-iodine and chlorhexidine (skin antisepsis), hydrogen peroxide, phenolics; prions need extended autoclaving (134 °C for 18 minutes) plus sodium hydroxide or hypochlorite, and instruments from high-risk tissue are often destroyed.
On this page
Definitions
| Term | Meaning |
|---|---|
| Sterilisation | Killing or removing all forms of microbial life, including spores |
| Disinfection | Killing or removing most pathogens on non-living objects; may not kill spores |
| Antisepsis | Disinfection of living tissue (skin, mucosa) without harming it |
| Asepsis | Practices that prevent contamination (aseptic technique) |
| Sanitisation / decontamination | Lowering microbial numbers to safe levels (food utensils; making items safe to handle) |
| -cidal vs -static | Kills vs stops growth |
Order of resistance (most to least)
- Prions
- Bacterial spores (Bacillus, Clostridium)
- Mycobacteria (waxy wall)
- Non-enveloped (small) viruses (polio, hepatitis A, norovirus)
- Fungi
- Vegetative bacteria
- Enveloped viruses (HIV, hepatitis B, influenza, herpes): least resistant (lipid envelope destroyed by alcohol and detergents)
Prions > spores > mycobacteria > non-enveloped viruses > fungi > vegetative bacteria > enveloped viruses.
Spaulding classification (what level a medical device needs)
| Category | Contact | Requirement | Examples |
|---|---|---|---|
| Critical | Enters sterile tissue or blood | Sterilisation | Surgical instruments, implants, needles, urinary catheters |
| Semi-critical | Touches mucous membranes or broken skin | High-level disinfection (at least) | Endoscopes, laryngoscope blades, vaginal probes |
| Non-critical | Touches intact skin | Low-level disinfection or cleaning | BP cuffs, stethoscopes, bed rails |
Cleaning (removing organic matter) must always come first; organic material shields microbes and inactivates many disinfectants.
Physical methods
Heat
Moist heat kills by denaturing and coagulating proteins; it penetrates better and works at lower temperatures than dry heat. Dry heat kills by oxidation.
| Method | Settings | Uses and notes |
|---|---|---|
| Autoclave (steam under pressure) | 121 °C, 15 psi (about 103 kPa above atmospheric), 15 minutes; or 134 °C, about 30 psi, 3 minutes | Most reliable; kills spores; surgical instruments, linen, dressings, culture media, lab waste. Not for heat-sensitive plastics, oils and powders (steam cannot penetrate) |
| Flash autoclave | 134 °C for 3 minutes (unwrapped) | Urgent instruments in theatre |
| Boiling | 100 °C for 10–30 minutes | Not sterilisation: spores and some viruses survive |
| Pasteurisation | Holder: 63 °C for 30 minutes; flash (HTST): 72 °C for 15–20 seconds | Milk; kills vegetative pathogens (M. bovis, Brucella, Coxiella, Salmonella, Listeria); not spores. Phosphatase test checks adequacy |
| Tyndallisation (intermittent steaming) | 100 °C for 20 minutes on 3 successive days | Media with sugars or serum (spores germinate between cycles) |
| Inspissation | 80–85 °C on 3 days | Egg and serum media (LJ, Loeffler) |
| Hot air oven (dry heat) | 160 °C for 2 hours (or 170 °C for 1 h, 180 °C for 30 min) | Glassware, syringes (glass), sharp instruments (no rusting or blunting), powders (talc), oils, grease, petroleum jelly. Not for plastics, rubber or fabrics |
| Incineration | High temperature burning | Hospital waste, soiled dressings, pathological waste, carcasses |
| Red heat / flaming | Bunsen flame | Inoculating loops, needles; flaming of glass mouths |
Filtration
- Membrane filters (0.22 µm pore size) remove bacteria from heat-sensitive liquids: sera, antibiotic and drug solutions, vaccines, sugar solutions. Viruses and mycoplasma may pass through.
- HEPA filters clean air in operating theatres, laminar flow cabinets and isolation rooms.
Radiation
| Type | Action | Uses |
|---|---|---|
| Ionising (gamma rays from cobalt-60, electron beams) | Free radicals, DNA breaks; penetrates deeply; "cold sterilisation" | Disposable plastics (syringes, catheters, gloves), sutures, some foods and drugs |
| Ultraviolet (non-ionising, about 260 nm) | Thymine dimers; poor penetration | Surfaces, air in operating rooms and labs, biosafety cabinets, water |
Chemical agents
| Agent | Spectrum and level | Uses | Notes |
|---|---|---|---|
| Glutaraldehyde 2% | High-level; sporicidal with long contact (several hours: chemical sterilant) | Endoscopes, heat-sensitive instruments | Irritant; alternatives: ortho-phthalaldehyde, peracetic acid |
| Formaldehyde (formalin 37–40%) | High-level; sporicidal | Tissue preservation, fumigation of rooms; inactivating toxins (toxoids) and vaccines | Irritant, carcinogen |
| Ethylene oxide (gas) | Sterilant (alkylating); sporicidal | Heat-sensitive equipment: plastics, heart-lung machines, catheters, sutures | Toxic, explosive, carcinogenic; long aeration time |
| Alcohols (70% ethanol or isopropyl) | Intermediate; kill bacteria, mycobacteria, fungi, enveloped viruses; not spores | Hand rubs, skin before injection, thermometers | 70% works better than 100% (water needed for protein denaturation); evaporates fast |
| Chlorine compounds (sodium hypochlorite, bleach) | Broad; sporicidal at high concentration | Blood and body fluid spills (1% available chlorine: 10,000 ppm), water treatment, surfaces, hepatitis B and HIV contamination | Inactivated by organic matter, corrosive |
| Iodine (tincture, povidone-iodine) | Broad; some sporicidal action | Skin antisepsis (pre-operative), wounds | Povidone-iodine is an iodophor (slow release, less irritant) |
| Chlorhexidine | Bacteria (Gram-positive best), enveloped viruses; not spores, poor against mycobacteria | Surgical hand scrub, skin prep (with alcohol), mouthwash, catheter care | Persistent action on skin; inactivated by soap |
| Hydrogen peroxide | Broad; sporicidal at high concentration | Wound cleaning (3%), contact lenses, vapour for rooms and devices | Catalase-positive organisms resist low concentrations |
| Phenolics (phenol, cresols, Lysol) | Bacteria, mycobacteria (some), fungi | Floors, surfaces, excreta | Toxic to skin; phenol is the reference for the phenol coefficient |
| Quaternary ammonium compounds (cetrimide, benzalkonium) | Low-level; Gram-positives, enveloped viruses | Surfaces, skin (with chlorhexidine as Savlon) | Inactivated by soap and organic matter; Pseudomonas can grow in them |
| Peracetic acid | High-level; sporicidal | Endoscope reprocessing, dialysers | (none commonly tested) |
| Heavy metals (silver nitrate, mercury compounds) | Bacteriostatic | Silver in burn dressings; old eye prophylaxis in newborns | (none commonly tested) |
Testing sterilisation
| Method | Indicator |
|---|---|
| Autoclave | Biological: spores of Geobacillus stearothermophilus (heat-resistant); chemical: autoclave tape, Browne's tubes; Bowie-Dick test (air removal in pre-vacuum autoclaves) |
| Hot air oven | Spores of Bacillus atrophaeus (formerly B. subtilis var. niger); Browne's tubes |
| Ethylene oxide | Bacillus atrophaeus spores |
| Ionising radiation | Bacillus pumilus spores |
| Filtration | Brevundimonas diminuta (Serratia marcescens formerly) |
Kinetics: the D value
The decimal reduction time (D value) is the time at a given temperature needed to kill 90% of organisms (a 1-log reduction).
Survivors after time t: N = N₀ × 10^(−t/D)
Spore load 10⁶, D₁₂₁ = 1.5 minutes
After 15 minutes: number of log reductions = 15 ÷ 1.5 = 10
N = 10⁶ × 10⁻¹⁰ = 10⁻⁴
→ chance of one surviving spore = 1 in 10,000 items
Sterility assurance level 10⁻⁶ needs 6 + 6 = 12 log reductions:
time = 12 × 1.5 = 18 minutes
Preparing hypochlorite
C₁V₁ = C₂V₂
Bleach is about 5% available chlorine; you need 1 litre of 1% (for blood spills)
5% × V₁ = 1% × 1000 mL → V₁ = 1000 ÷ 5 = 200 mL bleach
Add water to 1000 mL (200 mL bleach + 800 mL water)
0.1% (for general surfaces): 1000 ÷ 50 = 20 mL bleach + 980 mL water
Make fresh daily; hypochlorite loses strength over time.
Prions
Prions (Creutzfeldt-Jakob disease) resist normal autoclaving, formaldehyde, alcohol, radiation and UV. Recommended: disposable instruments for high-risk tissue (brain, spinal cord, posterior eye), or extended autoclaving (134 °C for 18 minutes) combined with sodium hydroxide or concentrated hypochlorite.
Expect: definitions (sterilisation vs disinfection vs antisepsis), resistance order, Spaulding categories, autoclave settings and indicator organism, hot air oven settings, uses and indicator, pasteurisation temperatures and organisms killed, tyndallisation and inspissation, filtration for sera, gamma rays for disposables, UV, glutaraldehyde for endoscopes, ethylene oxide, 70% alcohol, hypochlorite for spills, chlorhexidine and povidone-iodine, and prion decontamination.
Quick revision
- Sterilisation kills spores; disinfection may not; antisepsis = living tissue.
- Prions > spores > mycobacteria > non-enveloped viruses > fungi > bacteria > enveloped viruses.
- Autoclave 121 °C, 15 psi, 15 min (or 134 °C, 3 min); G. stearothermophilus.
- Hot air oven 160 °C, 2 h; glassware, powders, oils; B. atrophaeus.
- Pasteurisation 63 °C/30 min or 72 °C/15–20 s.
- Filtration: sera, drugs. Gamma rays: disposable plastics. UV: surfaces, air.
- Glutaraldehyde: endoscopes. Ethylene oxide: heat-sensitive devices. 70% alcohol: hands. Hypochlorite: spills.
- Prions: 134 °C for 18 min + NaOH or destroy instruments.
Written by The House Job from standard microbiology references (Levinson's Review of Medical Microbiology and Immunology).
FAQ
What is the difference between sterilisation and disinfection?
Sterilisation destroys all microorganisms including spores; disinfection kills most disease-causing organisms on objects but may leave spores alive.
What are the standard autoclave settings?
121 °C at 15 psi for 15 minutes, or 134 °C for about 3 minutes.
How are glassware and powders sterilised?
In a hot air oven, usually at 160 °C for 2 hours.
Which disinfectant is used for endoscopes?
A high-level disinfectant such as 2% glutaraldehyde (or ortho-phthalaldehyde or peracetic acid).