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Autoclave Procedure: Four Rules for No-Mess Liquid Sterilization

photo of a researcher at work

As we’ve discussed before, the best autoclave procedure for running liquid loads is trickier than just loading up the autoclave and choosing the “liquid cycle.” Follow these four rules and you’ll largely avoid trashing your autoclave chamber, plumbing, and drain while losing your liquids to evaporation, boil over, or burst bottles:

1. Never fill the bottles more than half way

With containers bigger than 1L, you can consider going over 50%, but never do so for LB agar, and think twice before reducing the headspace when processing other agars, broths, or growth media.  Never, ever, ever fill any container more than 75%; always assume you’ll get at least ~20% boil up.

2. Always use a tray!

 Some days you will mess up, or the barometric pressure will be especially high, or the autoclave vent will stick, or there will be a power surge, or … whatever. It’s very easy to dump out and rinse a tray, and very, very hard to clean an autoclave’s internal plumbing.

3. Always put at least .25″ of water in that tray

Water absorbs and releases heat more slowly than air or steam; having your bottles rest in a water bath acts as a buffer and smooths out your temperature and pressure ramps.  Added bonus: If a flask does overflow or burst, media diluted in a water bath cleans up very easily.

4. Best autoclave procedure: Assume the worst will happen

Run every load with the assumption that every bottle will, somehow, both boil over and burst:  Make sure your tray is deep enough to accommodate the water bath, all of the liquid from all of the flasks, and all of the broken glass without overflowing. For more tips