The Sterile Workspace
A 70% ethanol spray is all it takes to sterilise solid surfaces, but how can you control the air around you?
Using a Bunsen burner, you will create a ‘Cone of Protection’ to protect your work from any microorganisms that might fall from the sky. The updraft of the flame pushes up and away any bacteria that might fall within your Sterile Workspace.
Your Bunsen burner will double as a source of heat sterilisation when you proceed to Sterile Technique.
If you’re lucky enough to own either a Biosafety Cabinet, or Laminar Flow Hood:
All the same rules apply, but the air flow dynamics differ a little.
It’s possible to build these devices yourself, however I find a portable Bunsen to be good enough for me.
Equipment and Consumables
Or similar device that creates a hot updraft, e.g. blow torch)
Or a microwave
Protocols:
Creating a Sterile Workspace:
Spray your hands (or gloved hands) with 70% ethanol
Don’t forget to apply a moisturising hand sanitiser if you are prone to dry skin.
Note: Gloves are optional until your experiments become sensitive to RNAses. If you are new to working with a Bunsen flame, we recommend you don't use gloves at first. They won't catch fire, but they sure can leave a nasty burn if they melt onto your skin.
Clear your bench of all clutter. Lightly spray the area with 70% ethanol. Wipe it dry with a paper towel.
Check the collar on your Bunsen burner to ensure the air hole is covered (orange safety flame).
Note: The option of a safety flame is one reason why spending the extra money on a Bunsen burner can be worth it. The force of the gas coming out of a blow torch may actually blow out your lighter (Read: Not Safe)
Flick on your lighter or light your match. Once it is lit, turn the gas valve until there is a large orange flame.
Rotate the collar of the Bunsen until you have a solid blue roaring flame.
A typical Bunsen will provide a sterile workspace within a radius of 30-70 cm depending on the strength of the gas feed. The sterile space is created by the updraft of the flame, not the heat. Bacteria are very light and are therefore pushed away from your workspace - but not necessarily killed.
How to Sterilise Equipment & Media:
How to use an Autoclave
Fill bottles with the liquid (water, media, biological waste, etc) you wish to sterilise. Try to use large bottles and only fill to a maximum of 40% of the total volume for the best outcomes.
Tighten the lids of all bottles, then loosen each lid by half a turn. Failure to leave space for air to travel / pressure to equalise is likely to result in shattered glass.
Solid material and equipment that needs sterilising should be placed into an autoclave bag. Many plastics are not autoclave-proof and will melt in all kinds of interesting ways.
Be sure to check your items will survive the autoclave or you'll be spending multiple days scraping melted plastic off the inside.
PCR, Microcentrifuge and centrifuge tubes (aka. PCR, Eppi and Falcon Tubes) can be autoclaved in jars with foil over the top.
Read the product manual of your specific autoclave. Find out which areas require water changes between cycles. Typically this will involve adding water to the central chamber of the centrifuge and removing water from one or more waste containers. DO NOT start an autoclave without balancing the water, else it will burn itself out like an old kettle.
Generally a "Liquid Cycle / 20 minutes" is sufficient for sterilising even the most persistent spores. Don't be fooled by the misnomer, this generally takes 2 hours including heat-up and cool-down. Be sure to plan your day around this lengthy autoclave time and consider sterilising large batches of material in advance.