Basic Microbiology Techniques - Sterilisation

Basic Microbiology Techniques - Sterilisation

• Sterilisation is a method of microbial control, but not all microbial control is sterilisation
• Four main types of sterilisation are covered in this lecture: heat, radiation, chemical and sterile / membrane filtration
• Sterilisation is the complete removal of all living microorganisms, including spores.

Objectives

• Understand common terms used in microbial growth control
• Identify various sterilisation methods (heat, chemical, radiation, filtration)
• Describe procedures for different sterilisation methods
• Outline the principles of different sterilisation methods

Some Common Terms

• Sterilisation: complete removal of all living organisms
• Disinfection: removal of pathogenic organisms
• Antimicrobial agents: agents that kill microorganisms
• Microbiostatic agents: agents that prevent microbial growth
• Microbicidal agents: agents that kill microorganisms
• Physical, chemical and mechanical methods: different approaches to sterilisation

Modes of Microbial Control

• Microbicidal: kills microbes, reduces cell number
• Microbiostatic: stops or inhibits growth, viable cell number remains constant

Classification of Sterilisation Methods

• Physical: heat, radiation
• Chemical: liquid sterilants, gaseous sterilants
• Mechanical: filtration

Heat Sterilisation - Physical Method

• Most effective and widely used method
• Microbicidal through enzyme and essential cell constituent destruction.
• Effectivity depends on hydration level (lower temperature required with higher hydration)

Different Modes of Heat Sterilisation

• Moist heat: steam under pressure
• Dry heat: red heat, hot air (oven), ethanol flaming

Moist Heat Control

• Boiling: not sterilisation, 100°C for at least 20 minutes, kills vegetative cells of pathogens, not endospores
• Pasteurization: not sterilisation, heat treatment for food (e.g., 135°C for 15 seconds or 63°C for 30 minutes), kills vegetative cells of most pathogens
• Steam under pressure (autoclaving): sterilisation, 121°C at 15 psi for 15 minutes, kills all viable microorganisms, including endospores

Steam Under Pressure / Steam Sterilisation / Autoclaving – Moist Heat Sterilisation (above 100°C)

• Water boils at 100°C under normal atmospheric pressure.
• Boiling point increases with increased pressure
• Autoclave: pressure at 15 PSI (775 mm Hg), water boils at 121°C
• Steam under pressure has higher penetrating power, kills all viable microbes, including endospores and viruses
• Condensed liquid ensures moist killing of microbes
• Used for sterilisation of culture media, glassware etc
• Decontamination of instruments and media contaminated by microorganisms

Dry Heat Sterilisation

• Process of destroying microorganisms by applying moisture-free heat
• Based on the principle of conduction
• Destroys microorganisms by denaturing and lysing proteins
• Suitable for moisture-sensitive substances and materials that cannot be sterilised by moist heat
• Examples: powders, oily materials, petri dishes, pipettes

Hot Air Oven – Dry Heat Sterilisation

• Examples of temperatures and duration needed: 170°C for 30 min, 160°C for 60 min, 150°C for 150 min (lower temperature = longer duration)
• Can be used for sterilisation of glassware, petri dishes, and powder samples

Red Heat / Ethanol Flaming – Dry Heat Sterilisation

• Sterilisation by red heat and ethanol flaming are covered under other topics (streak plate and spread plate)

Comparison of Moist vs Dry Heat

• Moist heat: more effective, requires lower temperatures and shorter durations, may corrode some materials (like metal)
• Dry heat: requires higher temperatures and longer durations, does not corrode metals, may cause thermal shock to glassware, suitable for powders and oils, not suitable for heat-sensitive materials

Radiation - Physical Method

• Includes non-ionising and ionising radiation
• Non-ionising: infrared, ultraviolet -UV Light (around λ = 260nm, most effective): leads to abnormal bond formation in DNA which may lead to mutation. Used for surfaces of biological safety cabinets (BSC), air and water. Blocked by glass, water and dirt.
• Ionising: X-rays, gamma rays, high-speed electrons: effective against vegetative cells and endospores, but not always against viruses. Used on food, medicine and heat sensitive products (plastics), causes damage and mutation to DNA, chemical changes in cell organelles and production of toxic substances.

Infrared (IR) Radiation

• Radiation is absorbed and converted into heat energy
• A tunnel containing an IR source sterilises instruments and glassware kept in a tray, passed through the tunnel on a conveyor belt.
• During this movement, the instruments reach 180°C for about 8 minutes
• Applicable for mass sterilisation of packaged items like syringes and catheters

Example of an Ionising Radiation Facility

• Shows a schematic of a facility for ionising radiation
• Includes parts like irradiation rooms, storage pools, conveyor systems, radiation shield, loading/unloading areas and control console.

Chemical Sterilisation: Gaseous or Liquid

• Gaseous: Ethylene oxide, formaldehyde, nitrogen dioxide (NO2), ozone
• Liquid: hydrogen peroxide, glutaraldehyde, hypochlorite

Chemical Sterilization

• The process removes microorganisms using chemical microbicidal agents.
• Used for heat-sensitive materials like plastics, fibre optics, and biological specimens
• Chemicals can be in liquid or gaseous states; commonly categorised as liquid or gaseous sterilisation

Gaseous Sterilization

• Exposes equipment/devices to gases (ethylene oxide, formaldehyde, nitrogen dioxide (NO2), ozone) in a sealed, heated/pressurised chamber.
• More effective than liquid sterilization, as gases penetrate small spaces.
• Often used along with heat treatment.

Liquid Sterilization

• Submerges equipment in liquid sterilants (hydrogen peroxide, glutaraldehyde, hypochlorite) to kill viable microorganisms and spores.
• Not as effective as gaseous sterilisation, but appropriate where contamination level is low.

Hypochlorite

• Sodium hypochlorite (bleach): frequently used disinfecting agent.
• Submersion for 20-24 hours is required to reach sterilisation.
• Oxidising agent, which oxidizes organic compounds.
• Corrosive to metallic equipment.

Sterile/Membrane Filtration – Mechanical Method

• Mechanical method of physically removing microorganisms, not destroying them.
• Used for heat-sensitive culture media.
• Membrane filter with small pores (microfiltration) prevents larger particles (microorganisms) from passing through. Filters liquid and gases.,
• Used in filtration of liquids like injections and ophthalmic solutions, as well as air.

Membrane Filtration - Sterilization of Liquids

• Assembled pressure-operated filter holders, syringe mounting, and in-line use are methods that aid in sterilisation.
• Vacuum filtration tower devices for liquid filtration.
• Filtration under pressure is suitable.
• The filtrate becomes sterile after filtration.

4 Main Types of Sterilization

• Heat - Physical
• Radiation - Physical
• Chemical - Physical
• Sterile/Membrane Filtration - Mechanical