How to Sanitize and Sterilize in an Autoclave
"Clean" represents an inadequate standard for surgical tools and laboratory materials; they must be entirely free of any microbial contamination. An autoclave sterilizes materials by raising the pressure inside the autoclave chamber to 15 pounds per square inch and heating to 121 degrees C, which is the boiling point of water at this pressure. Exposing materials to these conditions for at least 15 minutes destroys bacteria and viruses. Proper use of the autoclave and preparation of the materials ensures that the materials are sterile and intact when the autoclave cycle completes.
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Things You'll Need
- Drain pan
- Deionized water
- Autoclave tape
- Autoclave trays
- Aluminum foil
- Heat-resistant gloves
Instructions
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Position the drain pan under the drain hose and open the drain valve. Allow any waste water from previous use to drain out, then close the drain valve and discard the water.
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Add deionized water to the autoclave chamber until the water level reaches just under the tray in the bottom of the autoclave.
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Prepare the materials to be autoclaved. For glass containers, unscrew the lids slightly or place the caps on loosely so pressure does not build up inside them. Cover the tops of bottles, flasks or test tubes without caps with aluminum foil. Fill containers with liquids no more than 3/4 full.
Wrap lab apparatus such as scoops and scalpels in aluminum foil, ensuring that the foil is tightly sealed. You can wrap multiple tools in the same piece of foil.
Place biohazardous waste into an autoclavable biohazard bag and twist tie the bag shut.
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Attach caps to test tubes and other containers using autoclave tape. Attach a small piece of autoclave tape to the other materials being autoclaved as well. Autoclave tape changes color or reveals the word "autoclaved" when the materials have been heated to the correct temperature, so everyone who uses the equipment can easily see it has been autoclaved.
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Place an autoclave tray inside the autoclave and load the materials onto the tray. For top loading autoclaves that can accommodate more than one tray, place larger objects on the bottom tray.
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Shut the autoclave lid and press down firmly to lock it. Your autoclave may have a noise or light to indicate when the door is properly latched.
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Select the appropriate settings for your autoclave cycle. For a digital autoclave, select the default sterilization cycle for most loads; larger loads may require a longer cycle. Check the autoclave manual for the recommended cycles.
If the autoclave has manual settings, set the exhaust selector switch to slow if the load contains liquids and fast if it does not. Turn the timer knob to 20 minutes for most loads; 30 minutes for containers with more than a liter of liquid and 60 minutes for more than a gallon. Some machines automatically add the time it takes the autoclave to heat up, but if yours does not, estimate an additional 20 minutes.
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Press start if necessary; some machines automatically begin the cycle once the timer is set.
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Wait for the autoclave to cool and decompress before opening the door. On some autoclaves, the door will remain latched until it is safe to open. For other autoclaves, wait until the pressure gauge has returned to zero and the temperature has dropped below 90 degrees C.
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Open the autoclave door slowly, keeping your face out of the path of any escaping steam. Wear heat-resistant gloves to remove the materials from the autoclave.
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Tips & Warnings
Make sure all your materials are autoclavable. You can safely autoclave borosilicate glass, autoclavable plastics and metal tools, along with many liquids and agars.
If you are autoclaving a mixture that includes a heat-degradable substance, add that ingredient after autoclaving.
Never autoclave hazardous materials, including those that are flammable, corrosive or radioactive.
Follow any autoclaving protocols mandated by your laboratory in addition to the general procedure.
