Ampicillin

Mode of Action

Inhibits the formation of cross-links in the peptidoglycan layer (which prvides rigidity to the cell wall). Most effctive 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. 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.

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 reccommend not allowing cultures to reach stationary phase if you need a high proportion of cells to contain your plasmid.

References

Molecular Cloning: A Laboratory Manual, Vol 1.