Ampicillin

Mode of Action

Inhibits the formation of cross-links in the peptidoglycan layer (which provides rigidity to the cell wall). Most effective against cells in log phase growth (since this is when new cross-links are being formed), and has little effect on cells in stationary phase.

Mechanism of Resistance

Expression of β-lactamase neutralizes ampicillin. The resistance gene is named bla or amp^R. When this enzyme is expressed on a high-copy number plasmid there is significant diffusion into the extracellular medium. As a result non-resistant satellite colonies may form around larger resistant colonies.

Satellite colonies on an Ampicillin plate

Stock Solution

Typical concentrations of ampicillin are 50 ug/ml for low copy plasmids and 100 ug/ml for high copy plasmids. Stock solutions are typically at 100 mg/ml, so that 1 ml of antibiotic can be added to 1 liter of broth or agar. Stock solutions made in 50% alcohol remain liquid at -20 C and are easy to pipet. Cool agar to 55C or below prior to adding antibiotic.

Ampicillin available from Sigma A-9518 (Ampicillin sodium salt), FW 371.39. To make 100ml of 100 mg/ml stock solution, dissolve 10 g of ampicillin in 50 ml of water and 50 ml of 100% ethanol.

Stability

Culture plates with ampicillin can be stored at 2-8 °C for up to two weeks. Stock solutions may be stored at 2-8 °C for up to 3 weeks. For long term storage (4-6 months), stock solutions should be stored at -20 °C. At 37 ° C in culture, ampicillin is stable up to 3 days. Sigma reference

Usage Notes

A 1990 paper by Bill Studier discusses how the secreted β-lactamase can quickly consume all the ampicillin in a culture (even at 20μg/ml Amp). A stationary culture of ampicillin resistant cells can have such a concentration of β-lactamase that even a 1/200 to 1/1000 dilution will still contain enough β-lactamase to consume all the fresh ampicillin before all the non-resistant cells from the stationary phase culture have been killed. The authors recommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid.

• Barry Canton & Matt Gethers have seen this occur when using the BioBrick plasmid, pSB1A3 expressing a high level of mCherry.

Carbenicillin is much more resistant and would be preferred except for cost. Mixtures of ampicillin and carbenicillin are often used.

References

Molecular Cloning: A Laboratory Manual, Vol 1.