- Antibiotics are frequently used in cell culture to prevent or control microbial contamination. They can be helpful during the early establishment of cultures or when working with primary tissues that carry a higher risk of infection. However, their routine use is controversial and should be approached with caution, as overreliance on antibiotics can mask underlying aseptic technique issues and contribute to antibiotic resistance or cellular changes.
- The most commonly used antibiotics in cell culture include penicillin, streptomycin, gentamicin, amphotericin B, and kanamycin, often combined in formulations known as antibiotic-antimycotic solutions. These are typically added to the culture medium at standard concentrations and provide broad-spectrum coverage against bacteria and fungi.
- Penicillin is a β-lactam antibiotic that inhibits bacterial cell wall synthesis, mainly targeting Gram-positive bacteria. Streptomycin is an aminoglycoside that interferes with protein synthesis by binding to the 30S ribosomal subunit of bacteria, primarily affecting Gram-negative organisms. The combination of penicillin and streptomycin is one of the most commonly used antibiotic mixes in mammalian cell culture.
- Gentamicin is another aminoglycoside with broad-spectrum activity against both Gram-negative and Gram-positive bacteria. It is often preferred over penicillin-streptomycin in some cultures due to its broader range and reduced allergenic potential. Kanamycin is similarly used for its broad-spectrum antibacterial activity but is less common due to its potential toxicity to eukaryotic cells at higher concentrations.
- To prevent fungal contamination, amphotericin B or nystatin is often added to culture media. Amphotericin B binds to ergosterol in fungal cell membranes, increasing permeability and leading to cell death. However, it can also affect mammalian cells at high concentrations and should be used carefully.
- A particularly persistent and challenging contaminant in cell cultures is mycoplasma, which lacks a cell wall and is resistant to many standard antibiotics. Specialized anti-mycoplasma agents such as Plasmocin, BM-Cyclin, or MycoZap are used to detect and eliminate these organisms. These treatments are often reserved for known infections, as they can be expensive and may stress or alter the cultured cells.
- Despite their usefulness, the routine use of antibiotics in cell culture is discouraged by many experienced researchers. Continuous antibiotic use can:
- Mask poor aseptic technique.
- Allow low-level contamination to persist undetected.
- Promote the emergence of resistant strains.
- Potentially alter cell behavior, metabolism, or gene expression.
- It is generally recommended to culture cells without antibiotics once good aseptic technique is established, and to use antibiotics selectively—for example, during the initial culture of primary cells or tissues from potentially contaminated sources. Periodic testing for contamination, particularly for mycoplasma, remains critical even in antibiotic-supplemented cultures.
- In genetic selection protocols, antibiotics can also serve as selection agents, such as G418 (geneticin), puromycin, hygromycin B, and blasticidin, which are used to maintain or select for cells carrying specific resistance genes. These antibiotics function differently from antimicrobial agents and should not be confused with general-purpose antimicrobials.
