Autoclaving (How to Sterilize)

Introduction

Autoclaving is a means of heat-sterilizing media and equipment without combustion or oxidation (if you had to sterilize your growth media by burning it, the media wouldn't do you much good). You might ask, "Why not simply sterilize by boiling?" A lot of the time that might work. However, there are several species of bacteria form spores in response to stress, and those spores can often survive temperatures above 100^oC.¹ To ensure errant spores don’t germinate in your media, you need to sterilize at even higher temperatures (around 121 ^oC).

How is this accomplished? Contrary to popular belief, you cannot simply crank up the heat on your stove and boil the water hotter. Adding more heat to a liquid will make it boil off faster, but it will not change the boiling point of a liquid. A phase transition (solid to liquid, liquid to gas, solid to gas, etc.) happens at exactly one temperature, and that depends on the substance and the surrounding pressure.

Thus, we can increase the temperature at which water boils by increasing the pressure (Figure 1). A good way to do that is to start by boiling water. When the water goes from a liquid to a gas phase, it expands. If the steam is trapped, it puts pressure on the rest of the container, including the water that hasn’t yet boiled off. Over time the enclosed system increased in temperature and pressure.

Figure 1: Phase diagram of water

(image adapted from http://www.earth.northwestern.edu/people/seth/202/new_2004/H2Ophase.html)

This is the idea behind a pressure cooker, and why for generations people have used canning as a very successful method of preserving food. And while you can purchase an autoclave for several hundred to several thousand dollars, a decent substitute can be procured for around $20.

Procedure

1. Put your media/instruments in an autoclave-safe container. To autoclave media, mix up the reagents in a jar and cap it loosely so the jar doesn't over-pressurize (if your media has agar - if you're pouring plates, for example - the agar won't completely dissolve until autoclaving anyway). To autoclave instruments, you can either put them in a dry jar and cap loosely, or put them in disposable autoclave bags. Just make sure to put the sealed autoclave bags on top of something dry (like an uncapped glass jar) to keep it out of the water.

2. Put water in the pressure cooker. You'll need the water level to be high enough so that the water doesn't boil off before you've been sterilizing for at least 15 minutes. You also don't want the level of the water to go above the pressure cooker's limit (there will be a line inside the vessel). It may take some experimentation to hone in on the ideal water volume; I fill the pressure cooker about 5 centimeters from the bottom.

3. Seal the pressure cooker and heat it on the stove top. As pure your pressure cooker's instructions, the water will need a few minutes of boiling before there's enough steam to build up pressure. When the pressure is high enough to be "autoclaving," the top piece will bob up and down to off-gas excess steam (it usually takes 10 minutes before my pressure cooker enters "sterilization phase"). Once it does, let the pressure cooker continue to cook for at least another 15 minutes (the minimum sterilization time I've seen for any autoclave cycle). 

4. Cool. Once you're sterilization phase is complete, simply turn off the stove's heat and allow the whole pressure cooker to cool. If you're sterilizing media with agar (e. g., for plates), you'll want to crack the pressure cooker lid (be careful not to get a faceful of steam!). Media with agar needs some tending; with oven mitts, gently swirl the jar so the agar remains well mixed. This will also help you gauge when the jar of agar is cool enough to handle without an oven mitt. For more, see the section on pouring plates.

References

1. Abraham, G.; Debray, E.; Candau, Y.; Piar, G. 1990. Mathematical model of thermal destruction of Bacillus stearothermophilus spores. Appl Environ Microbiol. 56(10):3073-80