Basic Microbiology Techniques - Sterilization

Basic Microbiology Techniques - Sterilization

• Sterilization is a method of microbial control that aims to eliminate all forms of microbial life, including vegetative cells, endospores, and viruses.
• Sterilization is not the same as disinfection or anti-microbial or microbiostats or microbicidal agents,
• The four main sterilization types focus on heat, radiation, chemical and sterile/membrane filtration.
• Heat sterilization is the most effective and widely used method. It works through the destruction of enzymes and other essential cell constituents, operating more efficiently in a fully hydrated state.

Modes of Microbial Control

• Microbicidal: Kills microbes and/or reduces the cell number.
• Microbiostatic: Stops or inhibits microbial growth, maintaining a constant viable cell count.

Classification of Sterilization Methods

• Physical: Heat and Radiation
• Chemical: Liquid and Gaseous Sterilants
• Mechanical: Filtration

Heat Sterilization - Physical Method

• Most effective and widely used method.
• Heat sterilisation is microbicidal, destroying enzymes and other cellular components.
• Efficiency is enhanced in a fully-hydrated state, needing lower temperatures and shorter durations.

Different Modes of Heat Sterilization

• Moist Heat: Steam under pressure.
• Dry Heat: Red Heat, hot air (oven), ethanol flaming.

Moist Heat Control

• Boiling: Not sterilization, kills vegetative cells of pathogens at 100°C for at least 20 min, but does not destroy all endospores.
• Pasteurization: Not sterilization, heats food to kill vegetative cells (135°C for 15 seconds or 63°C for 30 minutes).
• Steam Under Pressure (Autoclaving): Sterilisation, kills all viable microorganisms (including endospores) at 121°C under 15 psi for 15 min.

Steam Under Pressure/Steam Sterilization/Autoclaving

• Water boils at 100°C under normal atmospheric pressure (760 mm Hg).
• Boiling point increases with increased pressure.
• Autoclave: pressure at 15 PSI (775 mm Hg) for water to boil at 121°C; higher penetrating power kills all microbes including endospores and some viruses.
• Condensed steam ensures moist killing.

Dry Heat Sterilization

• Destroys microorganisms by denaturing and lysing proteins.
• Appropriate for moisture-sensitive substances (and materials practically impossible to sterilize with moist-heat).
• Examples include powders, oily materials, and laboratory equipment like Petri dishes and pipettes.

Hot Air Oven - Dry Heat Sterilization

• Temperature and duration requirements vary (e.g., 170°C for 30 min, 160°C for 60 min, 150°C for 150 min).
• Suitable for sterilizing glassware, Petri dishes, and powder samples.

Red Heat/Ethanol Flaming - Dry Heat Sterilization

• Methods covered in streak and spread plate topics

Comparison of Moist vs Dry Heat

• Moist Heat: More effective, uses lower temperatures and shorter durations; can corrode some materials like metal. Suitable for materials like rubber, plastic, textiles, etc and materials through which steam penetrates easily.
• Dry Heat: Requires higher temperatures and longer durations; doesn't corrode metals; may cause thermal shock to glassware; good for powders, oils, etc; not suitable for heat-sensitive materials.

Radiation - Physical Method

• Non-ionizing: Infrared, Ultraviolet
• Ionizing: X-rays, gamma rays, high-speed electrons, cosmos rays.
• UV light (around 260 nm) leads to abnormal DNA bond formation, used for biological safety cabinets.
• Ionizing radiation effective against vegetative cells and endospores.
• Radiation is used for food, medicine, and heat-sensitive products (plastics), but does not always affect viruses. It damages DNA, causes chemical changes in cell organelles, and produces toxic substances.

Infrared (IR) Radiation

• Absorbed and converted to heat energy.
• Used in a tunnel that is used to sterilize instruments and glassware, which are placed in a tray on a conveyor belt moved through the tunnel.

Chemical Sterilization: Gaseous or Liquid

• Gaseous Sterilization: Ethylene oxide, formaldehyde, nitrogen dioxide (NO2), ozone.
• Liquid Sterilization: Hydrogen peroxide, glutaraldehyde, hypochlorite.
• Used for heat-sensitive materials like plastics, and/or biological specimens.

Hypochlorite

• Sodium hypochlorite (bleach) is a disinfecting agent.
• Submerging in hypochlorite for 20-24 hours is needed to reach sterilization level.
• Acts by oxidizing organic compounds; corrosive to metal.
• Can be used at appropriate concentrations to disinfect surfaces and clean blood spills.

Sterile/Membrane Filtration - Mechanical Method

• Removes rather than destroys microorganisms.
• Used for heat-sensitive culture media in a vacuum filter tower.
• Filters have tiny pores to allow liquid to pass but prevent larger particles, thus is good for clearing fluids and sometimes gases.

Membrane Filtration - Sterilization of Liquids

• Assembled pressure-operated filter holders are used for syringe mounting and in-line use.
• Vacuum filtration tower devices are used for filtration.
• Filtration under pressure is suitable.
• Filtrate becomes sterile after filtration.

Summary of Physical Antimicrobial Methods

• Moist Heat: Boiling, Pasteurization, Steam under pressure
• Dry Heat: Red heat, Ovens, Ethanol Flaming
• Radiation: Ionising, Non-ionising