If you work in a life science lab, chances are you’ve used an autoclave. Autoclaves are pressure chambers that are used for the sterilization of waste, glassware, instruments, media, etc., so a basic understanding of how they work and how to use them is imperative to both avoid contamination in your experiments and to ensure safety of lab members and the community.
Biohazard waste
Firstly, let’s talk about biohazard waste—the kind that goes in the red trash bags. Biohazard waste includes any items that could be infectious, such as pipettes, petri dishes, gloves, or other materials that may have been contaminated by body fluids or bacteria. As biohazard waste eventually ends up in a landfill outside the lab, it needs to be sterilized to prevent outbreaks of infectious bacteria into the community. You should always be aware of your institution’s policy concerning red bag waste. Sometimes they’ll require you to autoclave it yourself, and sometimes they’ll have it taken by waste management personnel who manage the sterilization themselves. If you autoclave the waste, be sure to look up your state and institution regulations regarding the temperature, pressure, and length of the sterilization cycle for your load.
How do autoclaves work?
The first thing the autoclave does in a cycle is remove all trapped air from the chamber, as steam is a far more effective medium to achieve sterility than air. There are different types of autoclaves, some that use gravity to remove trapped air and others that use vacuum pumps. The vacuum pump method is more effective, especially if the material to be sterilized is porous with plenty of spaces where air can get trapped. Once the air is gone, the autoclave fills with steam until it reaches the target temperature and pressure. It will then automatically carry out the designated cycle. When the cool-down phase begins, the autoclave will maintain the pressure until it reaches a safe temperature so that liquids will not boil over inside the autoclave.
Which autoclave is right for you?
Autoclaves come in many sizes, from small bench-top models to larger machines that take up significant floor space. Additionally, the autoclave may be purchased for single-lab use or for multiple labs to share.
“Finding the appropriate autoclave starts with the size and quantity of one's load. From there, end users must determine the available space in their lab along with available utilities,” says Dennis Lee, director of marketing at Amerex. “At Amerex, we specialize in top-loading autoclaves that occupy minimal floor space and do not require an outside steam generator or plumbing, thereby eliminating costly setup charges. Only water and an electrical outlet are needed to begin operation. We offer 14 self-contained models ranging from 25 to 110 liters, including two models with drying.”
According to Brian Petuch, a corporate biosafety professional from Merck, other things to keep in mind are the organisms that are used and the manipulations that are done in the area.
Quality assurance
In order to be confident that the autoclave is doing its job, you must use indicators that prove either that sterilization has occurred or that the proper parameters to achieve sterilization have been met. “We recommend or require (as per permit) a biological indicator be used for cycle development,” says Petuch. “After that, a Class 5 integrator can be used for each run.”
Biological indicators are used to prove sterility. If the cycle was successful, the spores of a resistant bacterium in the indicator will be prevented from growing out. Class 5 integrators are chemical integrators that will change color when all three conditions required for sterilization—temperature, pressure, and time—have been met. Common autoclave tape is a marker that the autoclave has reached the proper temperature and pressure, but it does not account for time.
In order to keep the autoclave running smoothly, immediately clean any spills in the chamber with appropriate chamber-cleaning solutions, ensure that the drain strainer is free of debris, and drain the generator at the end of each day if your autoclave does not do so automatically. You should also inspect your autoclave on a regular basis to ensure that there are no leaks and that the materials are not warping. Schedule professional inspections at least once a year or more, depending on use. Given the importance of preventing contamination from ruining your work or infecting the public, it is vital to stay on top of autoclave maintenance.
Something else to keep in mind is that your water source should be as pure as possible to prevent the buildup of minerals inside the autoclave pipes and chambers. Also, if your autoclave is made of stainless steel, there are certain materials that you should not sterilize in your autoclave, as they can contribute to the corrosion of the chamber. As a rule of thumb, avoid sterilizing anything with high levels of chlorine or chlorides.
How to choose a cycle
It is important that you consult your state and institution regulations and the manufacturer protocol to ensure that you are using an appropriate cycle for your load. Different materials, packaging, and end goals may require different conditions to be appropriately sterilized.
According to Lee, “Our autoclaves incorporate agar, liquid, solid, warming, drying, and pre-vacuum cycles that are ideal for many uses, including the sterilization of media, waste, glassware, fermenters, bioreactors, biohazard bags, test tubes, pipette tips, and wrapped goods.”
Safety
Autoclaves are designed to promote safety of lab members and the public, but autoclave users need to follow the safety protocol in order to avoid burns or other problems.
“Training programs are important, followed by an instructor-led demo,” says Petuch, “and users should always use thermal-protective PPE.”
When autoclaving liquids, leave the lid loose or cover with foil to prevent explosions. Never autoclave cracked glass or radioactive, corrosive, or flammable materials. At the end of a cycle, verify that the temperature and pressure have dropped all the way and the slowly open the door to let steam escape gradually. Let items stand for at least 10 minutes.
Final notes
Autoclaves are essential tools in research to prevent contamination, but they can only be effective if end users use them appropriately. Make sure you understand which of your materials need to be autoclaved, which cycles to use, and how to operate the machine safely. It may also be worth investing in a training program or contacting technical support for more assistance.