---+ Autoclave Sterilization ---++ Overview Autoclaves heat their contents to 121°C, which is 21°C over the boiling point of water. To prevent the solutions from boiling over/vaporizing, the autoclave chamber is pressurized during this process, most commonly to 15 psi. <br> Most buffers and other solutions used in any lab are sterilized before use to prevent bacterial and fungal growth during storage. There are two basic techniques for sterilizing solutions: autoclaving and sterile filtration. Many buffers and other salt solutions are autoclaved because filtration of large volumes is time-consuming and expensive. Solid containers and biowaste are autoclaved because they cannot be filtered. Before autoclaving any solution you should always check whether it contains any heat-labile ingredients ([[ProtocolsMediaRecipes][Media Recipes]]). If it does, the heat-labile substance will usually have to be prepared separately, filter-sterilized, and added to the remainder of the solution after autoclaving. Also, certain combinations of compounds that are stable when autoclaved on their own can react at the high temperatures in the autoclave to produce compounds that inhibit microbial growth (e.g. Mg and glucose) <br> | *Autoclave* | Most buffers and salt solutions<br>Rich bacterial and yeast media broth | | *Do Not Autoclave* | Buffers with detergents (SDS) – they will boil over<br>Organic solvents that are volatile and/or flammable (ethanol, acetone, phenol, chloroform, DMF, etc.)<br>Heat labile ingredients (vitamins, hormones, antibiotics, proteins), HEPES solutions<br>DTT (dithiothreitol) or BME (beta-mercaptoethanol)<br> *Among many others...* | Be sure the containers you are autoclaving things in are also safe: bottles should be made from borosilicate glass or autoclavable plastic – be sure that any plastic items you put in the autoclave are really autoclavable (polypropylene is OK, polystyrene is NOT). Pipette tips, microcentrifuge tubes, and their storage containers are generally autoclavable. If unsure, please ask about other plastics before you experiment and make a mess.<br> ---++ General autoclave use: 1 Leave at least a quarter of the container volume as free space (otherwise your solutions will boil over). 1 Place containers in autoclavable pans to catch liquids from any breaking containers. 1 Make sure all caps are loose. 1 Use autoclave tape to tape on caps (including tinfoil caps), and put a small bit of autoclave tape on any other items you autoclave. The tape will change color during autoclaving, signaling to any future user that the item has been sterilized. <br> * Note: Autoclave tape only shows that the tape has been exposed to heat (80°C). No information on time, steam penetration or temperature/pressure can be inferred. 1 Use a strip of lab tape (with a tape tab!) to carefully label your item(s) with date, initials, and contents. This can instead be done after autoclaving if you make sure you know which solution is yours throughout the autoclaving process. 1 Tighten autoclave doors thoroughly but not so much that you can’t get it back open; note that this only really applies to older autoclaves, most newer autoclaves will seal themselves. 1 Select the appropriate program: * minimum sterilization time should be 20 min * use longer times for larger volumes * use the “liquid cycle” for any load containing liquids * use the “gravity cycle” only if there are no liquids in the load (autoclave will exhaust faster with this program, causing liquids to boil over) 1 Rooms with autoclaves often have binders allowing you to log in each use of the autoclave. Write in the date, contents and weight of your item(s), pressure and time used, principal investigator, and initials. 1 Make sure the pressure is down completely (check gauge) before opening. 1 Open the door VERY SLOWLY and STAND BACK unless you want your face scalded 1 Wear heat-resistant gloves to unload the autoclave 1 CLEAN UP ANY SPILLS, empty any liquids from autoclave pans down the drain, rinse well <br> ---++ Other considerations: _Secondary Containers:_ <br> Only use polypropylene plastic plans or stainless steel containers as secondary containers. *Do not use polyethylene or polystyrene as these plastics will melt when autoclaved.* Note that plastic containers increase the time needed for perfect sterilization as plastic is a good insulator. As a rule, add 5 min to run when using a plastic container. _Volume:_ <br> An important consideration for liquid media is the higher the volume, the longer the sterilization time. Generally, the volume of liquid per container is more important than the total volume per load. For example, a 2 liter flask containing 1 liter of liquid takes longer to sterilize than four 500ml flask each containing a volume of 250ml of liquid. _Packing the autoclave:_ <br> Avoid overloading the chamber with material; space must be available for the heat/steam to penetrate everything. Room should be left between flasks, bottles, and other containers to allow proper steam circulation. _General time guidelines:_ <br> The following are the recommended sterilization times for autoclaving liquids (volume is per container used): | 75-200ml | 20 minutes | | 200-500ml |25 minutes | | 500–1000ml | 30 minutes | | 1000-1500ml | 35 minutes | | 1500-2000ml | 40 minutes | Remember to modify these times as needed. When in doubt, autoclave for 1 hour. <br> The amount of real time it takes for the autoclave to finish sterilizing items will be longer than the selected sterilization time due to the time it takes for pressure to build and release before and after sterilization. If you want to set a timer for your autoclaved materials, add at least 30 minutes to the sterilization time to account for this. ---++ Autoclave locations: <br> Our lab is authorized to use the autoclaves in MBB and NMS, though we generally stick to the ones in MBB. If there are any problems with these autoclaves please notify senior lab personal and/or the building manager.
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Topic revision: r9 - 2025-06-13 - AlexaMorton