Autoclave Training & FAQ

Recommended sterilization times by volume: (from AC manual)

Sterilization time depends on following key variables:

1. Volume - The greater the volume of liquid, the longer it takes for the product to reach temperature. 10x 200ml heats faster than 1x 2L.
2. Viscosity - Thicker, more viscous solutions absorb heat more slowly than products such as water.
3. Material of the container - Different containers with the same volume of liquid will reach temperature at different rates. Metal containers conduct heat more rapidly than glass or PP.
4. Load volume - The greater the physical size of the load, the longer it will take to reach exposure temperature. 1x 1L heats faster than 1x 10L.
5. Load density - If the bottles are jammed together, it essentially becomes one large mass. So 10x 200ml, if packed tightly together, will become 1x 2L and you will need to select a 55min liquid cycle. If the bottles are separated enough to allow steam to envelope each bottle in the load, each 200ml bottle will come up at approximately the same time as long as the variables mentioned in 1-3 are consistent and a 30min liquid cycle will suffice.
6. Location in the autoclave - Bottles that are positioned nearest the heated jacket of the autoclave chamber will tend to reach temperature faster than those in the center of the load.

Since all of the variables mentioned above can affect the come-up time of the product, when processing mixed liquid loads, the total exposure time selected for the load should be based on the largest volume of liquid in a bottle, placed in the most-difficult-to-sterilize location in the chamber.

Most Divisional autoclaves have three standard sterilization cycles:

Autoclave liquids in a liquid cycle only. Glassware can be added to a liquid cycle but your bottle of media cannot be added to your gravity or pre-vac cycle. It will boil over and you will lose media.

Before assuming the autoclave is not doing its job, go down this checklist

1. Make sure your glassware truly clean before you put it in the autoclave
   Is your glassware washing procedure leaving your glassware free of all chemical residue? Autoclaves sterilize but do not clean. Review your manual and/or automated washing protocols (detergent, water temp, number of pre-rinse, wash & rinse cycles, duration of each, etc.). Ensure your glassware is properly cleaned before sterilizing in the autoclave.
2. Did you receive official autoclave training by the Safety Office?
   Instruction is provided by the School's Safety Office. Please send a request to autoclave@biology.ucsd.edu for your 30min Autoclave & Biohazardous Waste Management training before you start using the autoclaves.
3. Make sure you the sterilization times you are selecting are sufficient for the load volume
   See above for “How long should I sterilize my LIQUIDS?” and “What AUTOCLAVE CYCLE should I select?”
4. Check your loading technique
   These loading techniques will aid the sterilization process:
   • Do not overfill autoclave chamber. For non‐liquid items, only surfaces that are contacted by steam will be sterilized. Arrange/package items such that steam can penetrate and reach all surfaces. For liquid items, load bottles/flasks as spread out as possible, place bottles/flasks with larger volumes towards the chamber walls, containers with smaller volumes towards the middle. Items along the chamber walls heat faster.
   • Arrange items such that dense dry air will not be trapped. Autoclave glassware in a tipped position or fill your secondary container with 1-2” of water to displace trapped air between glassware.
   • Do not overfill bags, containers or liquids in vessels. Never fill a vessel to greater than 2/3 of its capacity. Liquid bubbles/boils during sterilization. Steam needs to enter bags.
5. Check the autoclave
   Did your cycle run its full course? Did it abort prematurely due to steam issues or mechanical failure? Did your cycle get aborted manually by someone? (This happens on occasion).
6. Contact the Safety Office for assistance with further trouble shooting
   Please send a message to the Safety Office, autoclave@biology.ucsd.edu with a description of what problems you are having.