The Sterile Workspace
AuthorAlexander KellySpray your hands with 70% ethanol or 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.
Note: If you're using a portable Bunsen, the final 15 minutes of flame as the canister runs out are quite weak. Consider emptying the canister fully and then replacing it before continuing work - or just be mindful that your cone of protection has shrunk to a much smaller area. Do not try to unscrew the canister before letting all the gas burn - one small spark and you'll be missing a hand.
Fill bottles with the liquid (water, media, biological waste, etc) you wish to sterilise. Try use large bottles and only fill to a maximum of 40% of total volume for the best outcomes.
Tighten the lids of all bottles, then loosen the lid by half a turn. Failure to leave space for air to travel and the 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.
Meow will grace us with his protocol soon (tm).
I'm going to do some experiments with this and get back to you - Alex
Ingredients
Directions
Spray your hands with 70% ethanol or 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.
Note: If you're using a portable Bunsen, the final 15 minutes of flame as the canister runs out are quite weak. Consider emptying the canister fully and then replacing it before continuing work - or just be mindful that your cone of protection has shrunk to a much smaller area. Do not try to unscrew the canister before letting all the gas burn - one small spark and you'll be missing a hand.
Fill bottles with the liquid (water, media, biological waste, etc) you wish to sterilise. Try use large bottles and only fill to a maximum of 40% of total volume for the best outcomes.
Tighten the lids of all bottles, then loosen the lid by half a turn. Failure to leave space for air to travel and the 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.
Meow will grace us with his protocol soon (tm).
I'm going to do some experiments with this and get back to you - Alex
