Autoclaves and autoclaving advice

[@thebumblingbiochemist]

A great thing about autoclaves is that steam can get places your scrub brush bristles can’t which is great for cleaning awkward-shaped and porous things. BUT the autoclave is kinda like hand sanitizer in that it disinfects but it doesn’t clean - so you’ve gotta do some scrubbing first to get off the grimy gunk. This is especially important when you have tissue culture flasks where you often get a crusty ring of dead cells at the top of where the cells are swirling around. So, I manually scrubbed all 30 of those flasks and then loaded them up in a giant dishwasher. Only after both these washes are they ready to go in the autoclave. Another thing you have to do before you run an autoclave - LOOSEN THE LIDS! You have to keep lids loose for a similar reason to why you don’t want to get hand sanitizer hot - gases take up more space than liquids. When molecules have enough energy to break free from other molecules and become a gas, it has enough energy to run away fast! But if there is no escape, the pressure will build up & potentially burst catastrophically - or at least the lid will get pretty permanently stuck onto your bottle. I found this out the hard way in undergrad when I didn’t loosen a lid quite enough. The lid got stuck on and we ultimately had to smash the bottle open with a hammer…. not good. So then I overcorrected and over-unscrewed the cap - and all the agar (a sort of sugar gel we use for making Petri dish bacterial homes) inside boiled out… not good. So, lids loose but not too loose! Note: for alcohol-based hand sanitizers, the explosion risk is “high” because alcohols are volatile (they have low boiling points so they boil easily). Another another thing to do before running the autoclave is stick on some autoclave tape. This tape has patterns that change color when they get hot. These patterns are usually stripes or tape brand logos, but I’ve always thought they should make autoclave tape with jokes - or at least motivational quotes or something… Kickstarter? Anyways, these color changes typically involve some chemical decomposing in an irreversible reaction. So they tell you something got hot enough. But not for how long it stayed hot enough - typically you need ~15-20 min of time at that heat. So tape’s not a sure-fire thing and there are other indicators you can use if you need to really really make sure - like if you’re sterilizing stuff at a hospital or something. But tape’s cheap, it lets you leave a “note” on each of your things that they’ve been sterilized, and if your autoclave’s pretty reliable you can (hopefully) get away with it - especially when your autoclave digitally monitors what it’s doing. But, kinda like how your microwave heats the outer part of your food better so you might see your hot pocket steaming but then take a bite and the center’s cold, the autoclave heats the outside first because that’s what the steam sees first. So you need to give the heat time to transfer throughout the entire object, and you want to stick the tape close to the center if possible. Unlike your microwave, you *can* stick aluminum foil in an autoclave - and we frequently do because it makes a nice temporary lid for flasks and beakers. But there are some things you should never put in an autoclave - these include waterproof things and easierly meltable things - like things made of polyethylene. Of course, things only remain sterile until they’re exposed to things again. For bottles and flasks, the inside stays clean which is what we care about. But for things like tubing or surgical equipment, you stick them in a sterilization pouch which is just this little disposable paper/plastic pouch that provides a nice little sterile bubble for the things inside when the pouch is exposed to the real world. I’ve been talking so far mainly about cleaning solid things - you can use an autoclave for flasks, beakers, pipet tips, etc.) - but you can also use an autoclave to disinfect liquids - like that agar I was telling you about earlier… or culture media - like the LB & TB we use to grow bacteria in. When you autoclave liquids you have to set it to a different cycle which lets the steam out more slowly at the end so that the pressure doesn’t drop before the temperature gets to the “normal” boiling point. If the liquids are still really hot and the pressure just suddenly drops, all those wiggly molecules will now have an easy escape! (i.e. liquids can boil out) The autoclaves we have here fully automatic which is really awesome. But the one I used in undergrad wasn’t - and it hated me… The exact details vary from autoclave to autoclave, but, here’s the gist - What happens when you stick your stuff in, seal it super tight with one of those submarine-looking doors, and turn on the electrical heater is that the water in the steam jacket starts heating up and it starts boiling into steam, entering the inner chamber, and pushing the air out through a discharge tap. Once all the air is pushed out, the tap is closed and steam keeps getting pumped into the chamber until the pressure reaches where you want it (usually 1.1 kilograms/square centimeter (kg/cm2) [15 pounds/square inch (lb/in2)]). Then a safety valve opens up and, as you keep adding steam, any excess steam is let out. This steady pressure/temperature period is called the holding period (this is the time I was telling you about which you want to be ~15 min). Then the heater shuts off so the chamber can start to cool off and the steam is let out (this is where you have to go slow if you have liquids in there). And that does it for today’s edition of probably way more than you ever wanted to know about a piece of equipment you either know nothing about or are much too familiar with! more on LB agar plates: bit.ly/agarbakery & kzbin.info/www/bejne/a4XPnKOho8x3fNE more on boiling: bit.ly/boilingpoints more on gases: bit.ly/idealgaslaws helpful references: * Steam Sterilization Principles by Marcel Dion & Wayne Parker, Pharmaceutical Engineering, November/December 2013 www.ispe.gr.jp/ISPE/02_katsudou/pdf/201504_en.pdf * Steam Sterilization Principles & Common Mistakes Using Autoclaves by Marcel Dion & Wayne Parker, Pharmaceutical Engineering, November/December 2013 ispe.org/pharmaceutical-engineering/november-december-2013/steam-sterilization-principles-common-mistakes   more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@189643478]

We put the bottles with agar in a hot water bath set at 60°C to cool down before we add things. This is also warm enough so it doesn't solidify.

[@thebumblingbiochemist]

We did that at my old lab too - great suggestion and sorry I forgot to mention!

[@plantsinjars]

This is a great video

[@thebumblingbiochemist]

Thank you!

[@healthbabe2691]

Should have reviewed your video first beforehand. I got my LB broth boiling over in my little autoclave tonight. You pointed out not to "overfill the bottle" and "have the bottle cap too loose". Those are the mistakes I have made. Will try your recommendations for quarter turn of the cap from its tight position and have less then 75% of liquid in the bottle. Need to have thorough cleaning of the autoclave since it still has smell of the LB Broth inside the tank. I guess it is no pain no gain and that's how I learn. Thanks for the quick review.

[@thebumblingbiochemist]

Oh no! Hope you got it cleaned up okay!

[@healthbabe2691]

@@thebumblingbiochemist Thank you. I did clean it up with multiple cycles of dry runs at max temp. The chamber is ok and the water tank still has some smell of LB/Terrific broth. Will need to clean it with manufacture's recommendation during the next maintenance cycle. Hope your students/audiences will read this post and learn from my mistake.

[@PeterHarket]

Is it technically possible to autoclave liquids without a lid or an aluminum foil? In other words, are there any other reasons for autoclaving with a lid besides the fact that you want the content to maintain sterility after it exits the autoclave?

[@thebumblingbiochemist]

I think the lid is just for post-autoclaving sterility