Why Does Contamination Still Occur After Adding Penicillin-Streptomycin? Expanded Approaches to Contamination Prevention and Control in Animal Experiments

Dual antibiotics are not "all-powerful" antibiotics.

Penicillin-streptomycin is widely used in cell culture, but its antibacterial spectrum is mainly limited to certain bacteria. Penicillin is more effective against Gram-positive bacteria, and streptomycin is effective against some Gram-negative bacteria. For fungi, yeast, mycoplasma, and some drug-resistant bacteria, conventional dual antibiotics are often ineffective.

Contamination in cell culture is not limited to bacteria alone. In many cases, the culture medium does not appear turbid, yet the cells deteriorate and proliferate more slowly, which may indicate mycoplasma contamination. Mycoplasma lacks a cell wall, so penicillin cannot kill it by inhibiting cell wall synthesis; streptomycin's effect on mycoplasma is also unstable. Therefore, even with continuous addition of dual antibiotics, cells may still be chronically infected by mycoplasma.

The stealthiness of mycoplasma contamination

Mycoplasma is small in size and difficult to observe directly under an optical microscope. It can enter the culture system through culture media, serum, operators, shared reagents, and other routes. Mycoplasma contamination does not rapidly make the culture medium turbid, but it consumes nutrients, alters cell metabolism, and ultimately interferes with the reliability of animal experiment data.

Cell proliferation slows down, but the color change of the culture medium is not obvious.

Subtle changes in cell morphology may occur, such as irregular edges and increased granules.

Experimental results such as transfection and drug screening show greater variability.

When mycoplasma contamination is suspected, specialized mycoplasma detection kits can be used for regular screening. For prevention, using filtered serum, aliquoting reagents, and reducing the sharing of culture media are more meaningful than simply increasing the concentration of dual antibiotics.

Identification of fungal contamination

Fungal contamination is usually easy to observe, with flocculent or clumpy floating matter appearing in the culture medium, sometimes white or pale yellow. Fungal spores can spread through air, incubator water pans, and unclean surfaces. Penicillin-streptomycin is basically ineffective against fungi, and antifungal components such as amphotericin B need to be used when necessary. However, antifungal drugs may have certain toxicity to some cells, so concentration and exposure time should be carefully evaluated.

Comprehensive prevention and control approach

In cell culture related to animal experiments, contamination prevention and control is a system, not something that relies on only one type of antibiotic. Generally, the following aspects can be addressed:

Keep the airflow in the biosafety cabinet stable and replace the filter regularly.

Wipe all reagents and consumables entering the workbench with 75% alcohol before use.

Aliquot culture media and serum for use to avoid repeated opening of containers.

Perform regular screening for mycoplasma, bacteria, and fungi.

Isolate and culture newly arrived cell lines first, and only introduce them into the main laboratory after confirming the absence of contamination.

Penicillin-streptomycin can continue to be used as a preventive additive in basic culture, but it should not be regarded as the only line of defense. Only by combining standardized operations and regular testing can deviations caused by microbial contamination in animal experiments be reduced.