Many labs don’t heat large liquid loads long enough to assure sterilization. The standard autoclave procedure is “hold a load at 121ºC for 20 minutes to assure sterilization.” This is endorsed by everyone, from the United States Pharmacopeial Convention and Food & Drug Administration, to the Department of Health & Human Services and every university on the planet. But that recommendation assumes that steam can fully saturate the load (thus heating the whole load—not just the chamber—to 121ºC). By way of comparison, a dry-heat sterilizer set to 121ºC will take upwards of 12 hours to sterilize the same load. If part of your load is shielded from the steam, then you’re looking at hours to assure sterilization.
While every lab is aware of the hazards of air pockets preventing steam penetration, we’re finding that many autoclave procedures fail think past “get the chamber to 121ºC for 20 minutes” when running liquid and media loads.
Autoclave Procedure: Taking Thermal Inertia into Account
As Philip Berriman, instrumentation technician at California State University (Fullerton), notes, “Most people, as soon as the chamber says 121 degrees, they begin their countdown. But we’ve found that, in some cases, it’s another whole hour to reach sterilization temperatures in the load itself. For example, if you pack a 320 L autoclave with one-liter bottles of media, it adds another whole hour—beyond the initial 20 minutes that it would take to run a load of dry beakers—before spores are killed in a spore test. It takes that long for the fluid to heat up, because you have so much fluid, and water heats really slowly.”
Validate your loads regularly. If running a full load of growth media is a regular task in your lab, then run a validation load quarterly: Load your autoclave up, place a spore test vial near center of mass of the load (or as close as practical), run your load, and make sure your assumptions are valid.