Absolutely correct John..
Basic steps of sterilisaton
1) Start with a sterile environment - whether you're using a glove box, fume hood, class II biocabinet the principle is the same. Use Isopropyl Alcohol! mix up a 70/30 blend: 70% Isopropyl Alcohol & 30%
distilled water (referred to as IPA from here on in). Make sure you use gloved hands - never try and do this without gloves (it may work but it's poor aseptic technique and will increase your contamination risk - dead skin cells which, no matter how good your aseptic technique is, contain millions of bacteria and spores. Always use good quality
sterile gloves (they come individually packed and are usually gamma irradiated. After having put the gloves on coat your hands liberally in the 70/30 IPA and allow to air dry (your body heat will evaporate the IPA off pretty quickly) Have a look at this tutorial for good aseptic gloving technique:
. Liberally (with a fine mist) coat all of the interior surfaces of your 'cabinet'.
2) Spray the bench near your cabinet with the IPA and use a sterile wipe
to wipe it down
3) Place all of the equipment you need to use on that sterile bench ensure that a bunsen burner is operating near the cabinet and have a container of alcohol nearby (use common sense - alcohol is flammable). Give all of the equipment a good spray with the IPA, turn it over after having coated one side, allow it to air dry. Give your hands a liberal spray of IPA (
allow to dry fully. Ensure you have a resting block that you can put in the cabinet - prior to flaming anything pick up that block with a sterile wipe (think of the wipe as a barrier between contamination and sterility - your hands are contamination - the block is sterility) and give the block a good wipe with the sterile wipe as you place it in to the cabinet. Then pick up the metal equipment that can be dipped in alcohol and flamed. Dip the surfaces you desire to be sterilised i.e. with a scalpel it would be the blade. Get it glowing white hot and then place it on the resting block so that the sterile surface is not contacting the walls. Do that will all of the equipment that you can flame sterilise
4) Then you have the items you cant flame sterilise - you need to wipe these as you place them in to the cabinet. Use the barrier technique utilising a sterile wipe to keep you from the sterile equipment and give them a good wipe as you place them in to the cabinet
5) Last but not least sterilise in the media - some may laugh - but you definitely need to take care of the outer surfaces of the media containers as you place them in to the cabinet. These should have been sitting on your bench with your other equipment when you gave it a good liberal spray with IPA. A technique that i found worked for me is to double wrap things. When you make the media and pour it in to your containers make sure you leave the lid a little loose. Wrap in
two layers of aluminium foil. With a freshly IPA glove take the container and, as your placing it in the cabinet, peel off the outer layer of aluminium foil. This should leave your media jar with one layer of foil on it. Prior to your using the container you peel that final layer of foil off. After everything is sterilised in take your sterilised seed pod and again wipe it in to the cabinet with a sterile wipe
6) the very last step, after having taken 6 hours to get setup, is to take off your gloves, put a fresh pair on and let the fun begin...remember to IPA them well before you put your hands in to the cabinet. Once they are well wetted put your hands in the cabinet and let them dry in there as this is now a 'sterile' area
Oh, also, 70/30 isopropyl alcohol is not highly flammable as such - you could spray it in to a bunsen burner flame and you will get a flare up but not an explosion - just use common sense
Other equipment you will need:
Quote:
Originally Posted by John
Ok I am a little confused. I don't know if you are talking about a glove box or flow hood. If you are using a flow hood, then spray the inside with alcohol and run it for 15 to 30 minutes and let it dry before lighting the bunsen burner, if you are not careful, you may get a flash from the alcohol. If you are using a glove box, then you probably can't use a burner and everything will need to soak in alcohol or bleach solution. I was also told not to use gloves that are powdered. The powder will fall off and contaminate the culture. So either wash well with the alcohol or don't use them at all and use good proceedure to avoid contamination.
John
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