Sterilization and Disinfection

In order to work safely in a microbiology laboratory and to maintain aseptic conditions to avoid contamination of cultures, it is essential that you have an understanding of how materials can be sterilized or disinfected. These processes are defined as:

Disinfection: application of a chemical, usually to a surface such as a bench or glassware, to reduce the numbers of micro-organisms. This process does not necessarily kill ALL organisms on the surface, but must be able to kill possible pathogens. Common disinfectants can include 70% ethanol, bleach (chlorine and chlorine compounds), quaternary ammonium salts, phenolics etc. They are not suitable for use on living tissues. The chemical should be applied to the surface and left for several minutes to enable it to work. Times vary depending on chemical.

Sterilization: killing of all micro-organisms on or in a material. Can be achieved via several methods of heat sterilization or by filtration for fluids. Materials must be heated to above 100^oC to ensure killing of all organisms including heat resistant spores. This level of heating can be achieved by application of moist heat under pressure (autoclaving) or by dry heat in an oven. Media/reagents and plastics are commonly sterilized by autoclaving whereas glassware and other equipment can be sterilized by dry heat in an oven.

Sterilization by heat

For dry heat sterilization : seal or wrap materials in brown paper or aluminium foil and heat in a hot air oven at 160^oC for 1 hour.

Autoclave: times and temperatures vary depending on materials. Most applications utilize 121^oC for 15 minutes, but lower temperatures may be required for certain media. Containers of media should not be filled completely and all closures should be loosened in the autoclave. Pressure sensitive tape is used to monitor whether the required pressure (and hence temperature) has been achieved. Non-liquids should be in containers that allow the entry of steam, otherwise sterilization will not be achieved.

NOTE: autoclaves and hot air ovens should only be operated by trained technical staff.

Sterilization by filtration for small volumes

For small volumes, a syringe and syringe filter can be used, but for larger volumes, a filtration apparatus is required.

Syringe filter method

Note: cultures of bacteria may need to be centrifuged at 5000 rpm for 10 minutes to remove the majority of organisms prior to filtration. Uncentrifuged cultures will quickly block the filter, making the process very difficult and increasing the risk of breaking the filter. Only the supernatant is filtered. 

• Prepare a clean, sterile container for the filtrate.
• Take a syringe of an appropriate volume (10-20ml) and remove the plunger.
• Fit a sealed, sterile syringe filter* to the bottom of the syringe barrel and place over the fresh sterile container.
• Pour the liquid to be filtered into the barrel of the syringe.
• Replace the plunger and gently depress to expel the liquid through the filter. Be careful not to force the liquid as the filter may break.
• When completed, replace the closure on the container and dispose of the filter appropriately, considering that it may be contaminated.

*Filters can be of several pore sizes. Select a pore size appropriate to the application. The most commonly used pore size is 0.22µm which should exclude most bacteria and hence should be used for general sterilization where the nature of the organisms present is not known. Also available is 0.45µm which may be suitable for some organisms and hence can be used for specific applications where the organism present is known to be larger than the pore size.