Unit 3: Sterilization and disinfection
General Microbiology notes · PTU syllabus (BMLS102-18)
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Unit summary
Sterilisation kills all microbes; disinfection reduces them to safe levels. This unit covers the definition, types and principles of sterilisation — dry and moist heat, autoclaving, radiation and filtration — testing sterilisers, antiseptics and disinfectants, and biomedical waste management.
After this unit you can
- Distinguish sterilisation, disinfection and antisepsis
- Explain heat, radiation and filtration methods
- Test the efficiency of sterilisers
- Use disinfectants and manage biomedical waste
PTU syllabus topics
- Definition
- types and principles of sterilization — heat (dry/moist, autoclave)
- radiation
- filtration
- efficiency testing of sterilizers
- antiseptics and disinfectants — types
- properties
- mode of action
- in-house preparation and efficiency testing
- biomedical waste management in a microbiology lab
Autoclave (moist heat)
121 °C, 15 psi, 15–20 min
Media, gowns, lab waste
Hot air oven (dry heat)
160 °C for 2 hours
Glassware, metal instruments
Filtration
0.22 µm membrane filter
Heat-sensitive liquids, antibiotics
Radiation
Gamma rays or UV
Disposables, surfaces
Topic 1
Definitions
- Sterilisation
- Killing or removing all microorganisms including spores
- Disinfection
- Destroying pathogens (not necessarily spores) on objects
- Antisepsis
- Disinfection of living tissue
- Bactericidal
- Kills bacteria
- Bacteriostatic
- Stops bacterial growth
- Decontamination
- Making items safe to handle
Topic 2
Sterilisation by heat
Mechanism
Oxidation of cell components
Coagulation and denaturation of proteins
Efficiency
Less; needs higher temperature and longer time
More efficient; steam penetrates
Methods
Red heat (loops), flaming, incineration, hot air oven (160 °C, 2 hours)
Boiling, pasteurisation, tyndallisation, autoclave
Used for
Glassware, oils, powders
Media, dressings, discarded cultures
- Pasteurisation
- 63 °C for 30 minutes (holder) or 72 °C for 15 seconds (flash) — milk
- Boiling
- 100 °C; kills vegetative cells, not all spores
- Tyndallisation
- Steaming at 100 °C for 20 minutes on three successive days — sugar and gelatin media
- Inspissation
- 80–85 °C on three days — Löwenstein–Jensen and Loeffler's serum media
- Autoclave
- Steam under pressure, 121 °C at 15 lb/in² for 15 minutes
Topic 3
The autoclave
- 1
Load items loosely; loosen caps
- 2
Add water and close the lid
- 3
Let air escape through the vent until steam flows
- 4
Close the valve; pressure rises to 15 lb/in² (121 °C)
- 5
Hold for 15–20 minutes
- 6
Let pressure fall to zero before opening
Topic 4
Radiation and filtration
- Ionising radiation (gamma rays from cobalt-60): cold sterilisation of syringes, catheters, gloves and sutures. Non-ionising (UV, 260 nm): surface disinfection of rooms and biosafety cabinets — poor penetration.
- Filtration: heat-labile fluids (sera, antibiotics, sugar solutions) passed through membrane filters (0.22 µm), Seitz, sintered glass or candle filters; HEPA filters clean air in cabinets and operating theatres.
Topic 5
Testing the efficiency of sterilisers
Biological indicator
Spores of Geobacillus stearothermophilus — must not grow after the cycle
Autoclave
Biological indicator
Spores of Bacillus atrophaeus (subtilis)
Hot air oven, ethylene oxide
Chemical indicator
Autoclave tape or Browne's tube changes colour
Autoclave, oven
Physical indicator
Thermometers, pressure gauges, recorders
All
- Bowie–Dick test: checks air removal in pre-vacuum autoclaves.
Topic 6
Antiseptics and disinfectants
Alcohols (70% ethanol or isopropanol)
Denature proteins, dissolve lipids
Skin, thermometers; not sporicidal
Aldehydes (2% glutaraldehyde, formaldehyde)
Alkylate proteins and nucleic acids
Endoscopes (cold sterilant), fumigation
Halogens (hypochlorite, iodine, povidone-iodine)
Oxidation
Spills (1% hypochlorite), skin antisepsis
Phenolics (phenol, cresol, Lysol)
Damage membranes
Floors, discarded slides
Quaternary ammonium compounds
Disrupt membranes
Surfaces, instruments
Chlorhexidine
Membrane damage
Hand wash, surgical scrub
- Broad spectrum
- Kills all microbes
- Fast acting
- Short contact time
- Active with organic matter
- Works in blood or pus
- Non-toxic and non-corrosive
- Safe for users and materials
- Stable and economical
- Long shelf life, cheap
- In-house preparation: make working dilutions from stock daily, e.g., 1% sodium hypochlorite from 5% stock: 1 part stock + 4 parts water. Efficiency testing: the in-use test (Kelsey–Sykes) and phenol coefficient (Rideal–Walker).
Topic 7
Biomedical waste management
Yellow
Human and animal anatomical waste, soiled dressings, discarded medicines, microbiology cultures (after pre-treatment)
Incineration or deep burial
Red
Contaminated recyclable plastics — tubing, bottles, gloves, syringes without needles
Autoclave or microwave, shredding, recycling
White (translucent)
Sharps — needles, blades
Autoclave, shredding, encapsulation
Blue
Glassware and metallic implants
Disinfection, recycling
- Microbiology cultures and stocks must be pre-treated (autoclaved) on site before disposal.
Key terms
- Sterilisation
- Removal of all microbial life including spores
- Autoclave
- Pressurised steam steriliser
- Tyndallisation
- Intermittent steaming on three days
- Biological indicator
- Spore preparation used to test sterilisation
- Antiseptic
- Disinfectant safe for living tissue
Quick revision
- Sterilisation, disinfection, antisepsis.
- Dry heat (oven 160 °C, 2 h) vs moist heat (autoclave 121 °C, 15 min); pasteurisation, tyndallisation, inspissation.
- Gamma and UV radiation; membrane filtration 0.22 µm.
- G. stearothermophilus and B. atrophaeus indicators; Bowie–Dick.
- Disinfectant groups; ideal properties; BMW colour codes.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Distinguish sterilisation and disinfection.
- Q2.Give the conditions for hot air oven sterilisation.
- Q3.What is tyndallisation?
- Q4.Which organism is used to test an autoclave?
- Q5.Name two disinfectants and their uses.
- Q6.Which colour bag is used for contaminated plastics?
Long-answer questions
- Q1.Describe methods of sterilisation by heat.
- Q2.Explain the autoclave and its efficiency testing.
- Q3.Describe antiseptics and disinfectants.
- Q4.Explain biomedical waste management in a microbiology laboratory.
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