Autoclaving is one of the most commonly used methods in the laboratory for the purpose of decontamination.
Many scientists, in particular those working with biological samples, need sterilized equipment and media, and autoclaves provide a quick and economical way to decontaminate materials. They use steam at high temperatures and pressure to kill microorganisms. Due to these extreme conditions, though, they must be used carefully.
This video will illustrate how autoclaves work and how to use them correctly.
In an autoclave, materials are heated with steam to temperatures of 121 degrees Celsius at pressures of at least 15 p.s.i. After at least 30 minutes, these conditions will dehydrate and kill cells.
Autoclaves can sterilize both liquid and solid materials, but different operating settings have to be considered. Liquids require longer times than solids to exhaust the steam, in order to prevent flash boiling. Additionally, larger volumes of liquid materials take longer to heat up to the correct temperature.
Not all materials can withstand the temperatures inside an autoclave. Therefore, never autoclave anything that melts, burns, is explosive, or liquids that will evaporate. Also, do not autoclave toxic or radioactive chemicals.
Now, we will show you how to prepare samples for the autoclave and how to operate one.
First, select the materials you will be autoclaving and only use compatible containers and instruments. Pyrex, borosilicate glass, stainless steel, polypropylene, and polycarbonate can be used for autoclaving.
Other common lab materials such as pipette tips and boxes are also compatible. Paper can be autoclaved in autoclave-compatible bags.
Solid waste can also be autoclaved in an appropriate bag. Leave space in this bag to allow steam to circulate and tie up the ends. Lastly, before autoclaving make sure to create a small opening in the bag to allow steam to enter.
For aqueous media and other compatible liquid samples, use an intact container without any cracks. Fill the container about half full and never more than two-thirds full.
To ventilate gasses from a liquid container, loosen the cap. If there is no cap, cover loosely with aluminum foil to prevent contamination of the autoclaved material.
Put all materials in a secondary container made of polypropylene or stainless steel. Separate solid and liquid materials to be autoclaved. Leave space between materials, and don't overfill the container.
Lastly, attach a piece of autoclave tape to all materials. This tape has temperature-sensitive indicators that will turn black if sufficient temperature has been reached.
When using an autoclave wear appropriate personal protective equipment to protect against the high temperature and pressure risks. This includes standard PPE-safety glasses, lab coats, and closed-toe shoes and heat-resistant gloves. Use an apron and face shield when handling liquid samples.
At the autoclave, first open the door and check for any hazards within. Load liquid and dry materials separately, as they require different autoclave cycles.
To prevent melting make sure materials do not touch the walls. Ensure a good flow of heat by keeping sufficient spacing between materials. Lastly, close and seal the door.
Different autoclaves have different operating instructions. Cycles may be set by manually setting the temperature and time, or by selecting from a preset list. In general, use the recommended cycles for the type of material you are decontaminating.
A variety of cycles exist for solids and liquids, as well as for different amounts of material and waste to be autoclaved. When you run a cycle, ensure that the temperature has reached at least 121 degrees Celsius and the pressure at least 15 p.s.i.
After the cycle is over, make sure the pressure has returned to zero and the autoclave has cooled down. Carefully open the door to let out leftover steam. Then, allow the materials to stand for 10 minutes to release any hot gasses. The autoclave tape should now have black stripes or say autoclaved on it, although this is not a guarantee that sterilization has taken place.
Wearing appropriate PPE, remove the materials, making sure to not shake any liquid samples. Note down your usage in the log, including the type of substances and any special circumstances if encountered. Finally, transport the materials to a secure area or a laminar flow hood to cool.
You've just watched JoVE's introduction to autoclaves. You should now understand what materials can be autoclaved, how to prepare them, and how to safely use the instrument. Thanks for watching!