Mammalian cell cultures can become contaminated with microbial contaminants (e.g., fungi, bacteria, and yeast) as well as viruses. An estimated 25% of all cell cultures are contaminated with mycoplasma bacterium alone. Contamination can affect cell viability, inhibit normal cellular processes, and lead to inconsistent results and experimental failures. 

Sources of contamination include working surfaces, reagents, personnel, and instrumentation. Water is excellent at conducting heat and equally great at becoming contaminated. Thus, water baths and CO₂ incubators are notorious for fostering the growth of bacteria, yeast, algae, and fungi, which can result in contaminated cultures. 

Water Baths 

Water baths are used to warm mammalian cell culture reagents before use, but they are also often the source of cell culture contamination. Below we’ve outlined best practices to ensure your water bath does not ruin your next mammalian cell or tissue culture experiment. 

Best practices to prevent water bath contamination

1. Replace the water frequently with distilled water to avoid contamination in your water bath. Tap water may contain contaminants. 
2. Consider the use of a biocide additive in your water bath to prevent the growth of microbes. In most cases, these prevent contamination for weeks or months in between water changes. 
3. Clean the water bath regularly with soap and water or a mild laboratory detergent. For more on how to keep a water bath clean, see our previous post. 
4. In between cleanings, the bath can be disinfected by heating it for 30 minutes to the highest temperature possible in your water bath. Alternatively, a chemical disinfectant can be added to the water within the bath. There are several products designed specifically for this purpose.  Do not use bleach; make sure the disinfectant is suitable for use with stainless steel and follow the instructions indicated on the product for the amount to add. 
5. Wipe down and sanitize containers (and the work surfaces they rest on) that will come in contact with the water. A spray bottle filled with 70% ethanol is a great choice. Note: label tubes or bottles with water and ethanol-resistant markers!
6. Just as you do when working with cell cultures, keep your gloves clean and change frequently or as needed. 
7. Keep your samples and reagents from being submerged, both to avoid their contamination and to avoid contaminating the water. Float the sample using floating foam tube racks so the lid stays dry. Use a sample rack to prevent the samples from tipping and contaminating the water.

Alternatives to Water Baths

Many scientists opt for bead baths filled with metal LabArmor beads for cell culture use. There are several pros and cons to using beads compared to water baths. Metal beads are resistant to contamination and thus useful for mammalian cell culture purposes to help prevent contamination. 

Note: A water bath and a bead bath are not interchangeable. For more on that, see our previous blog post. 

CO₂ incubators

CO₂ incubators used for mammalian cell culture maintain a growth environment suitable for cell growth, with tightly regulated temperature, humidity, and CO₂  levels. The conditions that are conducive to mammalian cell growth are also optimal for the growth of many contaminants, like bacteria, mold, yeast, and fungi. Today, CO₂ incubators come equipped with a variety of features that aid in the prevention of contamination. Below we’ve outlined best practices to help avoid incubator contamination and highlighted some features to look for when selecting an incubator.  

Best practices to prevent incubator contamination

1. Always use filtered, distilled water in the incubator reservoir. There are also additives that you can add to the water to prevent contamination, including copper sulfate or biocide solutions such as Aquaguard-1.
2. Minimize door opening time to avoid aerosolized contaminants from entering. 
3. Always use clean gloves when opening the incubator and disinfect any culture vessels or equipment that will enter the incubator with 70% ethanol. 
4. Thoroughly clean any spills immediately.
5. If the incubator has a HEPA filter, make sure to replace it regularly according to the manufacturer's protocol. 
6. Remove and clean racks regularly. If possible, autoclave incubator racks and the water reservoir. Schedule periodic cleaning and disinfecting of the entire incubator.

Features to look for in CO₂ incubators

• Many incubators come with decontamination programs that perform high-heat decontamination of the entire incubator with a pre-programmed protocol. 
• Some CO₂ incubators include HEPA filters. HEPA filters are great for larger microorganisms but do not protect against mycoplasma contamination as mycoplasma are too small to be filtered out (0.1-0.3 µm). However, the trade-off is that HEPA filtration is fan-assisted. Incubators with fan-assisted circulation have higher rates of contamination and incubator fans can spread contaminants to other cultures within the incubator. 
• Bacteria-resistant copper surfaces and water trays help prevent the growth of microbes.   
• If you will need to open the incubator frequently, consider a model with split inner doors to reduce exposure to all cultures. 

Other resources:

AddGene Water Bath Protocol

Keeping a Clean Water Bath

Cell Culture Contamination Troubleshooting from Millipore Sigma

Effective Contamination Control with CO₂ Incubators from Eppendorf