30L of insect cells hopefully chock full of my protein to harvest meant that this morning I spent a lot a lot of time in the lab “kitchen” washing bottles and flasks. Which meant I spent a lot a lot of time hanging out near the autoclave – a sort of super high heat & pressure oven we use to disinfect labware. Which got me to wonder – how do those things even work? And when I wonder, I research. And, when I do some research on this sort of thing, I like to share what I learn with you all because somewhere out there someone might be wondering too… So, here goes – an ode to the autoclave!
Autoclaves are aka “steam sterilizers” because they use steam (aka water vapor aka the gas form of water) to sterilize things (kill any microbes like bacteria, viruses, fungi, etc. living on/in them). The idea is to get the things so hot that they cause those microbes’ proteins, etc. to degrade. And steam is really good at this because of something called the heat of vaporization.
The only difference between the molecules in ice, liquid water, and steam is how much energy they have. Water in each of these “states of matter” consists of molecules of H₂O – 2 hydrogens “permanently” stuck to an oxygen. In addition to those strong, covalent, bonds connecting the Hs to the O, water molecules can form weak, temporary bonds called hydrogen bonds to other water molecules. This makes water “sticky” and leads to strong intermolecular water networks that give you things like the surface tension that allows insects to walk on water.
That’s convenient for insects, but water molecules, like all molecules, would rather be free to roam and aren’t thrilled by the idea of having to hang out with other water molecules. So, why do it? Molecular peer pressure’s strong! In order to roam freely, a water molecule has to have enough energy to break free from bonds to other water molecules and escape to a less crowded environment where there are fewer water molecules to have to avoid.
In a solid, like ice, the molecules barely have enough energy to wiggle in place; in a liquid, like liquid water, the molecules have enough energy to kinda glide past one another and they can break some linkages, but quickly get latched onto by another molecule when they do so; it’s only in a gas, like steam, where molecules have sufficient energy to break free *and* stay broken free.
So, how do they get that energy? One form of energy is heat. Heat is related to, but distinct from, temperature. Heat is the actual energy that gets transferred, whereas temperature is the average kinetic energy of a group of molecules. Kinetic energy refers to energy of motion. So if you give molecules a bunch of energy as heat, but they use that heat energy to do things other than wiggle, the temperature won’t increase. And this is what happens when you go between states of matter, such as when you go from liquid water to steam. As you add heat to liquid water at the boiling point, the water molecules use that heat to break free from the other water molecules. And, since they’re using up this heat, the temperature doesn’t rise.
Basically, the initial breaking free takes a lot of energy (the energy of vaporization), so the temperature doesn’t change when you go liquid->gas. So the gas has a bunch more energy than the liquid at the same temp. These liquid<-> gas interconversions are reversible, so you can also go from a gas back to a liquid (a process called condensation which dewy leaves can tell you all you about). This condensation often occurs when gas molecules meet a cooler surface – like a leaf or, in my case, an Erlenmeyer flask. And when you go back to a liquid, you release all that energy – which gets transferred to the surface the gas condenses on as heat. And this heat destroys microbes. Tada!
(it also leaves your stuff all “dewy” so we have drying ovens we can stick things in afterwards)
The autoclave (at least ours) looks like a giant oven kinda like the ones they have in the walls of pizza parlors. To generate the needed steam, the oven-y part you stick your “dirty” stuff in (pressure chamber) is surrounded by a steam jacket (water compartment) with some different valves that let new steam and air and old steam out. It’s important to get all the air out in the beginning so that the chamber can get completely filled with steam – we don’t need all of air’s oxygens, nitrogens, etc. hanging out. Air can be removed in a couple of different ways depending on the type of autoclave. Gravity displacement autoclaves basically just pump a bunch of steam in and use it to push the air out. Vacuum displacement autoclaves use vacuum pumps to suck the air out before they start sending in steam.
An electrical heater hooked up to the steam jacket provides the heat energy needed to get the water to boil into steam that enters the chamber. You might think that the water needs to get to 100°C – that’s the boiling point of water, right? But actually, you need to get it to ~121°C (at least once the pressure builds to where you want it)! Before you go WTF? let me explain. 100°C is the boiling point of water at sea level. But pressure in an autoclave is much higher, which makes it much harder for water molecules to break free from one another. They have to wiggle a lot harder in order to overcome the pressure, and the harder they wiggle, the higher the temperature. So autoclaves are able to get really hot without just having all the liquids you’re trying to sterilize evaporate into nothingness. This is a similar concept to how pressure cookers work.
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 recombinant protein expression in insect cells: http://bit.ly/bevsinsect
more on boiling: http://bit.ly/boilingpoints
more on gases: http://bit.ly/idealgaslaws
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0