Preventing Contamination in Mammalian Cell Culture

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.

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.

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.

Best practices to prevent incubator contamination

• Always use filtered, distilled water in the incubator reservoir.
• Consider adding a water-tray antimicrobial to suppress microbial growth between water changes (e.g., copper sulfate or a biocide solution).
• Recommended option: Biofargo MycGuard-1 Solution (100×) — a non-toxic, non-volatile, non-corrosive antimicrobial/fungicidal agent designed to disinfect CO₂ incubator water trays. Use at 1:100 and treat/replace every 7–10 days.
• Minimize door opening time to reduce entry of aerosolized contaminants.
• Always use clean gloves when opening the incubator and disinfect any culture vessels or equipment with 70% ethanol.
• Thoroughly clean spills immediately.
• If the incubator has a HEPA filter, replace it regularly.
• 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

• ✔ High-heat decontamination programs
• ✔ Anti-microbial copper surfaces
• ✔ Split inner doors
• ✔ Optional HEPA filtration

Water Baths

Water baths are often the source of cell culture contamination. Below we’ve outlined best practices to ensure your water bath does not ruin your next experiment.

Best practices to prevent water bath contamination

• Replace the water frequently with distilled water.
• Consider adding a biocide additive to prevent the growth of microbes.
• Clean the water bath regularly with soap or mild laboratory detergent.
• In between cleanings, heat the bath for 30 minutes at max temp or use a stainless steel–compatible disinfectant. Do not use bleach.
• Wipe down and sanitize containers with 70% ethanol.
• Keep your samples from being submerged: use floating foam tube racks.

Alternatives to Water Baths

Many scientists opt for bead baths filled with metal thermal beads (e.g., Aluminum Beads). Metal beads are more resistant to contamination and are ideal for workflows where water baths are repeatedly implicated as a contamination source.

A bead bath can accommodate metal thermal beads such as Aluminum Beads.

Quick Action Checklist

• ✅ Use distilled/filtered water in incubator trays and baths.
• ✅ Limit incubator door openings; disinfect items before entry.
• ✅ Adopt a routine schedule for cleaning racks, trays, and chambers.
• ✅ Control bioburden with MycGuard-1 Solution.
• ✅ Consider bead baths for warming steps.