Lab Sterilization, Sanitation, and Decontamination

No matter how hard-pressed you are in the lab, ensuring that your cleanliness and sterilization procedures are up to scratch is essential. Cloudy media, ruined samples and strange results caused by contaminants are just some of the problems that can be caused by failure to keep your workspace, equipment and samples sanitized. And each time it happens it costs you valuable time and resources. Here are some of the most common ways to keep contamination under control, and save you a lot of grief down the road:

General Lab Bench Sterilization

For quick sterilization of surfaces and equipment, cleaning with a solvent such as ethanol or isopropanol is a convenient option. These solvents work by rupturing the cells and denaturing the cellular proteins. The rupturing is caused by osmosis and requires water, which is why diluted rather than neat solvent is used. Solvents are good for decontaminating microbes, and viruses, but are not effective against spores or tough enzymes like RNases. Autoclaving is a good option for sterilizing solutions and heat-resistant equipment. Autoclaves kill microbes by hydrolysis and coagulation of cellular proteins, using intense heat from pressurized steam. They do an excellent job of killing microbes, spores and viruses but are only partially effective against RNases and can cause problems with certain solutions containing sugars, phosphates, metals and amino acids.

Autoclaving Alternatives

The latest option for sterilizing small pieces of equipment is the CoolClave™ Benchtop Sterilizer. It uses ozone gas to eliminate 98% of pathogenic organisms from everything from plasticware, pipettes to safety glasses, and sensitive electronics. The sterilization cycle takes just 8 minutes and since it does not use any corrosive solvents or damaging heat, it is very gentle on the items being sterilized. RNases are ubiquitous, tough enzymes that can cause endless problems for researchers working with RNA, so ensuring that work-area, equipment and solutions used for RNA work are RNase-free is essential. The first step is to set aside a dedicated RNase-free work area and regularly decontaminate it. This can be done by washing with 0.5% SDS, then RNase-free water and then ethanol and allowing it to dry. RNases are very thermostable so autoclaving is not sufficient to completely eliminate them. Instead, plasticware should be cleaned with 0.1 M NaOH, 1 mM EDTA and rinsed with RNase-free water. Heat-resistant equipment, like glassware, should be baked at 240oC for 4 hours. Certified nuclease-free tubes, tips, gloves and other consumables are available and are a worthwhile investment.

Autoclaving

RNase-free solutions can be made by adding a 0.1M dilution of DEPC prior to autoclaving. The DEPC will covalently modify and strongly (but not completely) inhibit RNases in the solution. But no matter how thorough you are with all of the above preparations, it is almost impossible to completely eliminate RNases. So a good catch-all is to use an RNase inhibitor in your solutions, and there are many of these on the market.

For siRNA transfection, most RNase inhibitors are unsuitable since they can interfere with the transfection so siGuard™, an RNase inhibitor that is specifically designed for siRNA transfection, is a must-have.

Mycoplasma Decontamination

Mycoplasma infections can cause huge problems in eukaryotic cell cultures. The threat of mycoplasma contamination can be minimized by practicing good sterile technique, quickly identifying and decontaminating infected cultures. The most prudent way to limit the possibility of mycoplasma contamination is to use gloves and lab coats and do all work in a laminar flow hood. A regular cleaning schedule is essential; the hood and incubators should be thoroughly disinfected with 70% ethanol or bleach once per week. The hood should also be disinfected between each use.

Mycoplasma Detection

Early detection of any mycoplasma-infected cultures is essential to prevent it from spreading to other cultures and worsening the problem, and this can be done using mycoplasma detection kits like the MycoScope PCR Detection Kit, which can quickly detect even trace amounts of mycoplasma in your cell culture quickly and easily.

Mycoplasma Treatment

Once detected, mycoplasma-infected cells should be treated immediately. Traditionally, this has involved discarding the culture after disinfecting with Virkon®, glutaraldehyde or similar. However antibiotic cocktails such as MycoGONE Mycoplasma Antibiotics Cocktail are now available, which can completely eliminate mycobacterial infection even in heavily contaminated cultures, without having any effect on the cultured cell line.