- Penicillin is one of the most commonly used antibiotics in cell culture, primarily employed to prevent bacterial contamination during the maintenance and propagation of animal cells. It was among the first antibiotics discovered and remains widely used due to its effectiveness against Gram-positive bacteria, ease of use, and low cytotoxicity to eukaryotic cells at standard concentrations.
- The mechanism of action of penicillin involves the inhibition of bacterial cell wall synthesis. It binds to penicillin-binding proteins (PBPs), which are essential enzymes for the cross-linking of peptidoglycan, a major component of the bacterial cell wall. This interference compromises the structural integrity of the bacterial wall, ultimately causing osmotic lysis and cell death. Since Gram-positive bacteria possess a thick peptidoglycan layer and lack an outer membrane, they are particularly susceptible to penicillin. Gram-negative bacteria, by contrast, are less affected due to their outer lipid membrane, which limits penicillin access to PBPs.
- In cell culture, penicillin is typically used at a concentration of 50–100 units/mL and is most often combined with streptomycin, an aminoglycoside antibiotic that targets bacterial protein synthesis in Gram-negative organisms. This combination, known as Pen-Strep, provides broad-spectrum coverage and is widely included in commercial culture media. It is especially useful during the initial establishment of primary cultures from tissue explants, where microbial contamination risk is elevated.
- Despite its widespread use, there are limitations to penicillin. It is ineffective against mycoplasma—bacteria that lack a cell wall and are a frequent, hard-to-detect contaminant in cell cultures. Additionally, overuse of penicillin can mask poor aseptic practices and lead to the emergence of antibiotic-resistant bacterial strains. For this reason, many experienced researchers recommend limiting antibiotic use to high-risk situations and avoiding their inclusion in routine long-term culture unless necessary.
- Penicillin is relatively stable in aqueous solutions at 4°C for up to a week, but for longer-term storage, concentrated stocks should be kept at –20°C. Care should be taken to avoid repeated freeze-thaw cycles, which can reduce antibiotic potency. While penicillin helps maintain culture sterility, its presence should not replace strict aseptic technique and regular screening for contamination, especially for organisms like mycoplasma that evade routine antibiotic coverage.
