Overview
Disinfection and sterilisation are essential to ensure that medical and surgical instruments do not transmit infectious pathogens to patients. Because sterilising every patient-care item is unnecessary and impractical, health-care policies must decide — primarily based on intended use — whether cleaning, disinfection, or sterilisation is required.
This lesson covers Earle H. Spaulding's classification of patient-care items, the physical, chemical and gaseous methods of sterilisation, quality monitoring of sterilisation cycles, the major classes of chemical disinfectants, and standard methods for testing disinfectant efficacy.
Learning Objectives
After this lesson you will be able to…- Define terms related to sterilisation and disinfection
- Classify items as critical, semi-critical or non-critical for sterilisation/disinfection purposes
- Discuss the different physical, chemical and gaseous methods of sterilisation
- Describe evaluation and in-process monitoring of sterilisation procedures
- Discuss the major classes of chemical disinfectants and their mechanisms of action
- Describe standard methods for testing disinfectant efficacy
Clinical Story
Why This MattersA patient is scheduled for minor day-care surgery. The surgical instruments must enter sterile tissue, making them "critical items" under Spaulding's classification — any microbial contamination could cause a life-threatening infection. The central sterile supply department autoclaves the instrument tray at 121°C, and a biological indicator strip with Geobacillus stearothermophilus spores is included in the load. Only when that spore strip fails to show growth after incubation can the department release the tray — a single missed step in this chain could turn a routine procedure into a surgical site infection.
Core Concepts
Sterilisation destroys or eliminates all forms of microbial life. Disinfection eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects. Cleaning removes visible soil. Decontamination removes pathogenic microorganisms so objects are safe to handle.
Earle H. Spaulding classified patient-care items into three categories: Critical items (enter sterile tissue or the vascular system — must be sterile, e.g. surgical instruments, catheters, implants); Semi-critical items (contact mucous membranes or non-intact skin — require high-level disinfection, e.g. endoscopes, laryngoscope blades); Non-critical items (contact only intact skin — e.g. bedpans, blood pressure cuffs).
Dry heat sterilisation (incineration, red heat, flaming, hot air oven) uses 160–180°C for up to 2 hours; it is good for glassware and metal instruments and destroys bacterial endotoxins/pyrogens.
Moist heat sterilisation uses steam at 121–134°C, most commonly via the autoclave, which uses pressurised steam and is the most dependable system for decontaminating laboratory waste and sterilising glassware, media and reagents. Porous loads are typically autoclaved at a minimum of 134°C for one hour; bottled fluids at 121°C. Autoclaves are not suitable for rubber, plastics or heat-sensitive equipment.
Radiation sterilisation uses gamma rays (from a cobalt-60 source) or accelerated electrons, targeting microbial DNA to cause ionisation and free radical production, and is useful for heat-sensitive products such as sutures and plastic syringes. UV light is used for air and surface sterilisation but has poor penetrability.
Filtration removes rather than destroys microorganisms, using depth filters (diatomaceous earth, sintered glass) or membrane filters (0.22 µm pore size for liquids). HEPA filters remove up to 99.97% of particles >0.3 µm and are used for air supplied to aseptic areas.
Disinfectants can be classified by consistency (liquid/gaseous), spectrum of activity (high/intermediate/low level), or mechanism of action. Major classes include alcohols (dehydrate cells, denature protein — 70% ethyl/isopropyl alcohol for skin/surfaces), aldehydes (alkylate proteins/nucleic acids — formaldehyde, glutaraldehyde, kill spores), phenols (disrupt membranes — chlorhexidine, chloroxylenol/Dettol), halogens (oxidise sulfhydryl groups — iodine, hypochlorite), heavy metals, surface active agents (quaternary ammonium compounds), dyes, hydrogen peroxide, and beta-propiolactone.
No single ideal disinfectant exists — an ideal agent would have a wide spectrum, act quickly, remain active in organic matter, be stable, non-toxic, and inexpensive.
Disinfectants lose activity on standing and in the presence of organic matter, so periodic testing is essential. Methods include Koch's method (spores dried on silk thread), the Rideal-Walker method (phenol coefficient in water), the Chick-Martin test (phenol coefficient in the presence of organic matter), the capacity use dilution (Kelsey-Sykes) test, and the routine in-use test — if more than five of ten drops of diluted disinfectant show growth, the disinfectant is considered a failure.
Laboratory Principle
Autoclaving works because saturated steam under pressure carries far more thermal energy than dry air at the same temperature, and delivers it efficiently to microbial cell surfaces, causing rapid denaturation of proteins and enzymes. This is why moist heat sterilises at a much lower temperature (121–134°C) and shorter time than dry heat (160–180°C) — hydrolysis and denaturation occur far more readily in the presence of water. Efficient air removal from the autoclave chamber is essential, because trapped air layers prevent steam from reaching all surfaces and can leave pockets of the load unsterilised.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Ethylene oxide gas | 800–1200 mg/L, 45–63°C | Gaseous sterilisation of heat-sensitive devices | Pressurised cylinder, ventilated area |
| Formaldehyde (formalin) | 40% / 15–100 mg/L vapour, 70–75°C | Fumigation, surface disinfection | Cool, ventilated, away from ammonia |
| Glutaraldehyde | 2% alkaline | Sterilises thermometers, endoscopes, bronchoscopes | Sealed container, alkaline activator added before use |
| Peracetic acid | Low concentration, sporicidal | Low-temperature liquid sterilant for immersible devices | Single-use sealed cup, no pre-mixing |
| Hydrogen peroxide | 3–6% aqueous / vapour plasma | Skin disinfection (3%); instrument sterilisation (6%, plasma) | Dark bottle, away from light |
| Sodium hypochlorite | 0.5% / 1:10 dilution | Serology/virology disinfection, spillage decontamination | Fresh dilution daily, dark container |
| Bacillus stearothermophilus spore strips | Standardised biological indicator | Verifies autoclave (moist heat) efficacy | Per manufacturer instructions |
Step-by-Step Procedure — Autoclaving
Clean and wrap items appropriately; arrange the load in the chamber to allow free steam circulation and avoid overpacking.
Inspect and clean the drain screen at the base of the chamber; ensure sufficient water is present to generate adequate steam.
Allow steam to flush air out of the chamber completely — trapped air prevents efficient heat transfer to the load.
Bring the chamber to the required temperature and pressure (121°C for bottled fluids, 134°C for porous loads/dressings for one hour), hold for the exposure time, then allow controlled cooling.
Confirm chemical indicator colour change and check the biological indicator (e.g. G. stearothermophilus spores) after incubation before releasing the load for use.
Flow Diagram — Autoclave Cycle
Quality Control
Every sterilisation cycle should be monitored with physical (temperature/pressure recording charts), chemical (colour or melting-point indicators such as Browne's tube), and periodic biological indicators (standardised spore preparations — Clostridium species for dry heat, Geobacillus stearothermophilus for moist heat, B. subtilis var. niger for gas sterilisation). A load should not be released until these confirm satisfactory conditions were achieved.
Central sterile supply and infection control departments should participate in accreditation programmes and external validation of sterilisers, including periodic third-party testing of biological indicators and calibration of temperature/pressure gauges.
Reference Values
Standard Sterilisation Parameters⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Biological indicator shows no spore growth after incubation | Sterilisation cycle achieved adequate conditions | Load may be released for use |
| Biological indicator shows spore growth after incubation | Sterilisation cycle failed — load is not sterile | Do not release load; investigate cause (air trapping, incorrect load, equipment fault) and reprocess |
| In-use disinfectant test — growth in more than 5 of 10 drops | Disinfectant has lost potency or is contaminated | Discard and replace disinfectant; review dilution and storage practice |
Common Errors & How to Avoid Them
Cause: Overloading prevents steam from reaching all surfaces of the load, leaving pockets unsterilised.
Prevention: Load items loosely, following manufacturer guidance on maximum chamber capacity and arrangement.
Cause: Debris blocking the drain screen allows a layer of air to form at the bottom of the chamber, preventing efficient steam penetration.
Prevention: Inspect and clean the drain screen before every autoclave cycle.
Cause: Disinfection does not eliminate bacterial spores; using it on critical items (entering sterile tissue) risks transmitting infection.
Prevention: Apply Spaulding's classification correctly — critical items must always be sterilised, never simply disinfected.
Cause: Disinfectants rapidly lose potency once diluted and standing, especially in the presence of organic matter.
Prevention: Prepare fresh working dilutions as needed and perform routine in-use testing.
Laboratory Tips from the Bench
Never rely on temperature/pressure gauges alone — always include a biological indicator in every load to directly confirm microbial kill, especially for implant loads.
Chlorinated plastic bags should never be incinerated — burning them releases toxic dioxins and violates biomedical waste incineration norms.
Remember Spaulding's classification with "Critical Cuts, Semi touches Skin-mucosa, Non touches skiN only" — Critical = sterile tissue, Semi-critical = mucous membrane, Non-critical = intact skin.
Important Notes
Both gases are alkylating agents that are potentially mutagenic and carcinogenic, and cause acute toxicity including skin, conjunctival and nasal mucosal irritation. Adequate aeration time and exhaust systems are mandatory after use.
Disinfectants are applied to inanimate surfaces; antiseptics can be safely applied to skin and mucous membranes; some chemicals used at the correct concentration and duration act as sterilant liquids (e.g. peracetic acid, 2% glutaraldehyde). The same chemical class may fall into more than one category depending on concentration.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgeDuring a routine audit, three surgical trays from the same autoclave batch are found to have a slightly cloudy appearance on the chemical indicator tape, though the temperature chart recorded the correct 134°C cycle.
Despite the correct temperature reading, a blocked drain screen allowed a layer of trapped air to remain at the bottom of the chamber, preventing steam from reaching all instrument surfaces — resulting in an incomplete chemical indicator change and biological indicator failure.
- →Physical (temperature/pressure) monitoring alone is not sufficient — chemical and biological indicators must always be checked together.
- →The drain screen must be inspected and cleaned before every autoclave cycle to prevent air entrapment.
- →A failed biological indicator always requires withholding the load and reprocessing, regardless of other monitoring results.
Frequently Asked Questions
In the presence of water, microbial proteins undergo hydrolysis and denaturation much more readily than under dry conditions, where oxidative changes require higher heat input. This is why autoclaving (121–134°C) achieves the same sterilising effect as dry heat at 160–180°C.
No. Disinfection does not reliably eliminate bacterial spores, so it can never substitute for sterilisation on critical items that enter sterile tissue or the vascular system. Disinfection is appropriate only for semi-critical and non-critical items per Spaulding's classification.
Disinfectants at working concentration rapidly lose potency on standing and are further inactivated by organic matter such as blood, pus and soil. Periodic testing (e.g. the in-use test) verifies that the disinfectant in actual clinical use is still effective.
Quick Revision
10-Minute ReviewKey Takeaways
- Sterilisation destroys all forms of microbial life; disinfection eliminates most pathogens except spores.
- Spaulding's classification (critical, semi-critical, non-critical) guides the appropriate level of reprocessing.
- Physical sterilisation methods include heat (dry/moist), radiation and filtration.
- Chemical and gaseous methods include alcohols, aldehydes, phenols, halogens, ethylene oxide and formaldehyde.
- Sterilisation cycles must be monitored with physical, chemical and biological indicators together.
- Disinfectants require periodic efficacy testing because they lose potency on standing and with organic matter.
Competency Checklist
Track Your MasteryReferences
- NIOS Microbiology Module — Lesson 4: Sterilisation and Disinfection.
- Spaulding EH. Chemical disinfection of medical and surgical materials.
- Collee JG, Fraser AG, Marmion BP, Simmons A. Mackie & McCartney Practical Medical Microbiology. 14th ed.