In cell culture experiments, an unexpected microbial contamination may cause weeks or even months of research efforts to be wiped out. However, the scientific and correct use of antibiotics such as penicillin/streptomycin solution is the most basic and critical line of defense in the laboratory.
The core function of double antibody solution
The combination of penicillin and streptomycin, generally called "double antibody", is a routine choice to prevent bacterial contamination during cell culture. The mechanisms of action of these two antibiotics complement each other. Penicillin mainly interferes with the synthesis of bacterial cell walls and is particularly effective against Gram-positive bacteria. Streptomycin relies on inhibiting bacterial protein synthesis and is more effective against Gram-negative bacteria.
Their combined use can broaden the antibacterial spectrum and prevent common contamination more comprehensively. During routine cell passaging or culture operations, even if they are carried out in a clean workbench, microorganisms in the environment may be accidentally introduced. Adding an appropriate concentration of dual antibodies can provide a relatively "clean" growth environment for cells, especially in the early stages of culture or during frequent operations.
Design logic of 100X concentrate
Most of the common dual-antibody solutions in the market are 100-fold concentrated solutions. This design brings great convenience to the daily use of experimenters. The so-called 100X means that 1 ml of concentrated solution can be added to 99 ml of cell culture medium, and after mixing, a final concentration working solution suitable for cell growth can be obtained.
Such a commercial solution that has been mixed and sterilized in advance saves researchers from the complicated steps of weighing, dissolving and filtering two antibiotics separately. It not only ensures precise and accurate concentration, but also avoids possible errors and the risk of cross-contamination during weighing, significantly improving the standardization level and repeatability of the experiment.
Strictly regulated usage concentration
When using double antibodies, it is necessary to strictly adhere to the recommended working concentrations. For this product, the final concentration of penicillin is generally 100 units per milliliter and streptomycin is 0.1 mg per milliliter. If the concentration is too low, it may not be able to effectively inhibit bacterial growth, thus causing "hidden pollution"; if the concentration is too high, it may be toxic to the cells themselves, thus affecting their normal growth and metabolism.
For example, this concentration is safe and effective when cultivating common cell lines such as 293T and HeLa. However, for some primary cells or particularly sensitive cell types, it may be necessary to lower the concentration or not use antibiotics at all. This should be judged according to the specific cell culture manual or literature.
Proper storage and transport conditions
The activity of biological reagents comes from the proper and correct storage method. Penicillin and streptomycin solutions are very sensitive to temperature and must be placed in an environment of minus 20 degrees Celsius and frozen. Its validity period is generally 12 months from the date of production. During the transportation process, suppliers will use wet ice packaging, with the ultimate goal of ensuring that the entire process is kept at low temperature.
When the laboratory receives the product, it should immediately be placed in a refrigerator at a temperature of minus 20 degrees Celsius. In order to reduce the loss of potency caused by repeated freezing and thawing, it is recommended to carry out dispensing operations for large packages of solutions. For example, a 50 ml package can be divided into five smaller portions of 10 ml, and only one part is taken at a time, while the rest remains frozen.
Key precautions during operation
Even if there are no bacteria in the double-antibody solution itself, the sterile operation method must still be adhered to when adding it to the culture medium. All steps should be completed near an alcohol lamp or inside a clean workbench. Before opening the bottle cap, 75% alcohol needs to be used to wipe it. The operator must wear a clean lab coat and disposable sterile gloves.
It should be noted that antibiotics cannot replace rigorous aseptic techniques; they are only a means of remediation and prevention and cannot kill all types of microorganisms, especially mycoplasma, fungi and viruses. Over-reliance on antibiotics may cover up operational irregularities, ultimately leading to more serious contamination.
A reasonable view of the limitations of dual resistance
It is crucial to recognize that dual antibodies have limitations. Long-term use of antibiotics may lead to the emergence of drug-resistant strains, which may change the gene expression profile of cells, thereby affecting the accuracy of experimental results. Therefore, in many rigorous experiments, such as preparing cells for transcriptome sequencing or preparing cells for drug sensitivity testing, researchers often tend to use antibiotic-free culture media.
Developing good aseptic operating habits and regularly cleaning the water bath and carbon dioxide incubator are the keys to preventing contamination. The double-antibody solution is like a "guarantee". It gives extra buffer space to cell culture. However, the prerequisite for its core value to be realized is that researchers follow regulations and adhere to rigorous experimental operations.
Do you routinely use dual antibodies in cell culture to prevent contamination, or do you only use them under specific circumstances? Welcome to share your experience and opinions in the comment area. If you find this article helpful, please like it to support it.
