Antibiotics

Ampicillin is a beta-lactam antibiotic that inhibits bacterial cell wall synthesis. Bacteria need some time to replicate and divide, and ampicillin interferes with this process. It may take some time for ampicillin to affect bacterial growth and demonstrate antibiotic resistance.

Kanamycin is an aminoglycoside antibiotic that disrupts bacterial protein synthesis. It acts relatively quickly by binding to the bacterial ribosomes. The incubation time required to observe antibiotic resistance with kanamycin may be shorter compared to ampicillin.

One possible reason why your cells are not forming colonies could be the loss of selective pressure. In a selective medium containing antibiotics or other inhibitory substances, bacterial colonies will only form from cells that have acquired resistance to the selective agent. If the selective pressure is reduced or eliminated, non-resistant cells can also grow on the plate but won't form distinct colonies.

To help address this challenge, we have developed a rigorous performance growth testing process for our agar plates, handpicking 27 bacterial and 8 fungal strains to establish proprietary predictive growth patterns that include a variety of antibiotic combinations. We perform tests daily, screening the first and last plates in every lot for sterility and performance to help ensure batch-to-batch consistency so you get reproducible results. We also maintain retention samples for the lifespan of the product, so we can continually test its integrity.

Antibiotic susceptibility testing is a crucial process in determining the effectiveness of antibiotics against specific bacterial strains. There are several methods used to test antibiotic susceptibility, and your choice of method will depend on the type of bacteria being tested, and the timing of the test. Mueller-Hinton media is typically used for these tests due to its standardized composition and pH.

Here are some common methods for testing susceptibility:

Source: Patel, 2021

Many antibiotics are sensitive to heat and could potentially degrade if the media is too hot. These antibiotics are typically not recommended to be added until the media has cooled to an appropriate temperature (usually around 45-50°C or 113-122°F).

The degradation rates obtained for the model within a liquid matrix (water) at 100°C observed among different antibiotic classes and can be summarized as follows:

β-lactams = tetracyclines (most heat-labile) > lincomycin > amphenicols > sulfonamides > oxfendazole > levamisole (most heat-stable)

To help address this challenge we ofter a wide variety of pre-mixed solutions that already contain the anitbiotics you need. With over 27 years of experience making complex formulations, we have a well-established process in place to safely manufacture agar plates that include heat sensitive antibiotics.

Source: Tian L., Khalil S., Bayen S. Effect of Thermal Treatments on the Degradation of Antibiotic Residues in Food. Crit. Rev. Food Sci. Nutr. 2017;57:3760–3770

It has been demonstrated that no significant degradation occurs from UVA irradiation alone, however pH does have a substantial impact on antibiotic degradation. To address this and ensure consistent and accurate pH in our products, we follow a rigorous quality testing procedure for every lot to ensure variability is less than 0.04 pH from batch to batch. You can learn more in our guide for optimal pH.

Source: Elmolla, E. S., & Chaudhuri, M. (2010). Photocatalytic degradation of amoxicillin, ampicillin and cloxacillin antibiotics in aqueous solution using UV/TiO2 and UV/H2O2/TiO2 photocatalysis. Desalination, 252(1-3), 46-52.

Antibiotics used for selection in mammalian cells are toxic to mammalian cells lacking a particular resistance gene or marker. This selectivity ensures that only mammalian cells with the desired genetic modification survive and proliferate.

Antibiotics commonly used for mammalian cell selection include:

●      Zeocin (Blasticidin S): Mammalian cells expressing the zeocin resistance gene can survive and proliferate in the presence of Zeocin.

●      Puromycin: Puromycin is a protein synthesis inhibitor that is toxic to mammalian cells but has a limited effect on bacterial cells.

●      Hygromycin B: Hygromycin B is effective against a broad range of bacteria and fungi but can be safely used with mammalian cells expressing the hygromycin resistance gene. It interferes with protein synthesis in bacterial cells while allowing resistant mammalian cells to grow.

●      G418 (Geneticin): G418 is an antibiotic that selectively inhibits the growth of mammalian cells. Cells expressing the G418 resistance gene can survive and proliferate in the presence of G418.

●      Bleomycin: While Bleomycin can affect some bacterial strains, it is used less frequently for bacterial selection. It causes DNA damage in mammalian cells but can be tolerated by those expressing the bleomycin resistance gene.

Source: Curr.Protoc.Mol.Biol. 86:9.5.1-9.5.13. (2009)

The antibiotic kill curve is a dose-finding experiment in which mammalian cells are exposed to increasing concentrations of an antibiotic to determine the lowest and the most effective antibiotic concentration that kills the untransfected cells. To make a kill curve, typically, untransfected cells are plated in a 96-well plate at a low density so that on the day of antibiotic treatment, they reach ~50% confluency. Following the recommended concentration range for the antibiotic, the cells are treated at increasing concentrations in triplicates. The cells are then regularly observed under a light microscope over a 7- to 10-day period, replacing the medium every 2 to 3 days. Viable cells in each well are quantified directly (e.g., trypan blue) or indirectly (e.g., MTT, CellTiter Glo) and plotted against antibiotic concentrations to determine the lowest concentration effective at killing all the cells.

Sample dose-response plot with a non-linear regression fit, showcasing the cell viability of DU1245 and PC3 cells

Source: adapted from Oseni, 2021

Yes, you can absolutely have more than one antibiotic selection marker. However, you should be aware that some pairs of antibiotics can exhibit cross-reactivity due to shared resistance mechanisms or overlapping target sites in bacterial cells.

Some common examples of antibiotics with cross-reacting pairs include:

●      Ampicillin, Amoxicillin and Carbenicillin: both are β-lactam antibiotics that target bacterial cell wall synthesis and share similar resistance mechanisms. Bacterial strains resistant to one of these antibiotics may exhibit partial resistance to the other.

●      Kanamycin, Streptomycin, Gentamicin and Neomycin: all belong to the aminoglycoside class of antibiotics and can exhibit cross-resistance. They target bacterial protein synthesis.

●     Tetracycline and Doxycycline and Minocycline: cross-reactivity between these in bacterial culture is related to their shared mechanism of action, it does not imply a uniform response among all bacterial strains.

To ensure you get the right combinations of antibiotics, explore our wide variety of ready-made pre-poured plates that include anywhere from 1-5 antibiotics.

Both antibiotics share the same mechanism of action as they belong to the beta-lactam group. Ampicillin, a commonly used antibiotic in molecular biology, offers stability and cost-effectiveness but may lead to the formation of satellite colonies. On the other hand, Carbenicillin exhibits greater stability due to its enhanced tolerance for heat and acidity, making it a preferred choice when maintaining selective pressure. Furthermore, Carbenicillin is associated with a lower occurrence of satellite colonies, owing to its heightened stability and reduced susceptibility to inactivation by beta-lactamase enzymes. It's worth noting that Carbenicillin has a narrower antibacterial spectrum and is typically more expensive than Ampicillin.

The choice between Gentamicin and Streptomycin depends on the specific needs of your experiment or application. Gentamicin is preferred for its stability at low pH and effectiveness in controlling bacterial growth in tissue culture, especially when acidic conditions are involved, but it has a broader spectrum of activity. Streptomycin is a cost-effective option, suitable when increased stability is not a priority.