What are Satellite Colonies?

Troubleshooting Satellite Colonies article download

During antibiotic selection, you may notice a few small colonies growing around a large colony on the plate.These small colonies are satellite

colonies and the large colony is the colony of interest, containing both the antibiotic resistance gene (selectable marker) and gene insert.

The presence of satellite colonies on the plate sometimes becomes a common problem during the antibiotic selection. These satellite colonies didn’t take up the plasmid vector with the resistance gene and DNA insert. So, you don’t want to pick these small colonies.



Escherichia coli plate containing ampicillin. The large colony secretes β-lactamase enzyme to degrade ampicillin. The degradation of ampicillin around the large colony causes the reduction of ampicillin level in that area. Satellite colonies begin to appear and grow around this area. The presence of satellites colonies on the plate sometimes becomes a common problem during the antibiotic selection.


Troubleshooting Guide for Common Antibiotic Selection Problems

1. Satellite Colonies are Present

  • Check if the antibiotic stock is too old.
  • Check if the antibiotic concentration is too low.
  • Check if the temperature of the plate is not too hot when adding the antibiotic.
  • Use a stirrer to mix the antibiotic evenly in the growth medium.
  • Don’t grow your transformation plate more than 16 hours.

2. No Colonies Grow on the Plate

  • Check if the competent cells is still viable.
  • Check if the correct antibiotic is used for selection.

3. Too Many Small Colonies Grow on the Plate

  • Check if the antibiotic stock is too old.
  • Check if the antibiotic concentration is too low.
  • Use a stirrer to mix the antibiotic evenly in the growth medium.


Tips to Avoid Satellite Colonies

  • Use fresh and sterilized growth medium without antibiotics. You can add antibiotics onto your plate before you grow your colonies.
  • Use the antibiotic concentration recommended in your protocol to prevent unwanted colonies. A higher concentration of ampicillin than recommended may also reduce the presence of satellite colonies.
  • Check if the temperature of the growth medium is not too hot when adding the antibiotic.
  • Use carbenicillin (another b-lactam antibiotic) as an alternative to ampicillin. Carbenicillin is more stable and less susceptible to inactivation than ampicillin in growth media, reducing the formation of satellite colonies.
  • Use only new stock of antibiotics to make sure your antibiotics are still effective. For tips on how to check that, watch our Kirby Bauer Assay video below:


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References

Demain, A. L., & Elander, R. P. (1999). The β-lactam antibiotics: past, present, and future. Antonie van Leeuwenhoek, 75(1), 5-19. doi:10.1023/A:1001738823146

Knudsen, E. T., Rolinson, G. N., & Sutherland, R. (1967). Carbenicillin: a new semisynthetic penicillin active against Pseudomonas pyocyanea. British medical journal, 3(5557), 75-78. doi:10.1136/bmj.3.5557.75

Medaney, F., Dimitriu, T., Ellis, R. J., & Raymond, B. (2016). Live to cheat another day: bacterial dormancy facilitates the social exploitation of β-lactamases. The ISME journal, 10(3), 778.

Rolinson, G., Macdonald, A., & Wilson, D. (1977). Bactericidal action of β-lactam antibiotics on Escherichia coli with particular reference to ampicillin and amoxycillin. Journal of Antimicrobial Chemotherapy, 3(6), 541-553.

Yurtsev, E. A., Chao, H. X., Datta, M. S., Artemova, T., & Gore, J. (2013). Bacterial cheating drives the population dynamics of cooperative antibiotic resistance plasmids. Molecular Systems Biology, 9(1), 683. doi:10.1038/msb.2013.39