Effective laboratory sterilization methods are essential for working with isolated cell lines. For obvious reasons, you don’t want bugs from the environment growing in your nice culture medium, and, equally, cultures must be sterilized before disposal. Unless you are using microbiology microscopes to view your cultures, you might not be aware of any unwanted microbe guests. Time to buff up on the various laboratory sterilization methods, and how they work to keep your cells healthy, happy, and bug-free.
6 Common Laboratory Sterilization Methods
1. Wet Heat (Autoclaving)
The laboratory sterilization method of choice in most labs is autoclaving: using pressurized steam to heat the material to be sterilized. This is a very effective method that kills all microbes, spores, and viruses, although, for some specific bugs, especially high temperatures or incubation times are required.
Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is efficiently achieved by intense heat in the presence of water.
The intense heat comes from the steam. Pressurized steam has a high latent heat; at 100oC it holds 7 times more heat than water at the same temperature. This heat is liberated on contact with the cooler surface of the material to be sterilized, allowing rapid delivery of heat and good penetration of dense materials.
At these temperatures, water does a great job of hydrolyzing proteins… so those bugs don’t stand a chance.
2. Dry Heat (Flaming, Baking)
Dry heating has one crucial difference from autoclaving. You’ve guessed it – there’s no water, so protein hydrolysis can’t take place.
Instead, dry heat tends to kill microbes by oxidation of cellular components. This requires more energy than protein hydrolysis so higher temperatures are required for efficient sterilization by dry heat.
For example, sterilization can normally be achieved in 15 minutes by autoclaving at 121oC, whereas dry heating would generally need a temperature of 160oC to sterilize in a similar amount of time.
Filtration is a great way to quickly sterilize solutions without heating. Filters, of course, work by passing the solution through a filter with a pore diameter that is too small for microbes to pass through.
Filters can be sintered glass funnels made from heat-fused glass particles or, more commonly these days, membrane filters made from cellulose esters. For the removal of bacteria, filters with an average pore diameter of 0.2um are normally used.
But remember, viruses and phages can pass through these filters so filtration isn’t a good laboratory sterilization method if these are a concern.
Ethanol is commonly used as a disinfectant, but isopropanol is a better solvent for fat and is probably a better option.
Both solvents work by denaturing proteins through a process that requires water, so they must be diluted to 60–90% in water to be effective.
Again, it’s important to remember that although ethanol and IPA are good at killing microbial cells, they have no effect on spores.
UV, x-rays, and gamma rays are all types of electromagnetic radiation that have profoundly damaging effects on DNA, so make excellent tools for sterilization.
The main difference between them, in terms of their effectiveness, is their penetration.
UV has limited penetration in air so sterilization occurs in only a fairly small area around the lamp. However, it is relatively safe and is quite useful for sterilizing small areas, like laminar flow hoods. (It’s really important to remember to sterilize your equipment too.)
X-rays and gamma rays are far more penetrating, which makes them more dangerous but very effective for large-scale cold sterilization of plastic items (e.g. syringes) during manufacturing.
6. Gas Sterilization
Ethylene oxide can be used to sterilize equipment that is sensitive to heat or moisture and is often used to sterilize medical equipment such as catheters and stents. Ethylene oxide essentially prevents cell metabolism and replication by alkylation. Because ethylene oxide is easily absorbed, equipment must be aerated after sterilization to remove any residue. Ethylene oxide is also highly toxic and can present a number of health risks. As it’s generally used for healthcare products, you’re not very likely to be using it in the lab.
So, those are some of the main laboratory sterilization methods. If we’ve missed any, please feel free to let us know in the comments section.
Originally published March 28, 2012. Reviewed and updated February 2021.