Ampicillin

Ampicillin is one of the most widely used antibiotic in molecular cloning, for the selection of transformants. It belongs to the penicillin group of antibiotics i.e. beta-lactam antibiotics. The only difference between penicillin and ampicillin is the presence of amino group in the latter.

Unlike penicillin which is effective against only gram positive bacteria, ampicillin is also effective against gram negative bacteria, e.g. E. coli. The amino group present in the side chain of the ampicillin renders it to penetrate the outer membrane of gram negative bacteria and hence enter into it. Therefore, it was one of the first broad spectrum penicillins to be used.

Mode of action

Like other beta-lactam antibiotics, ampicillin also inhibits cell wall synthesis in the bacteria. It basically inhibits the formation of glycan moiety of the peptidoglycan layer of the batcerial cell wall. The peptidoglycan is known to provide the rigidity to the bacterial cell wall.

Ampicillin is basically a competitive inhibitor of the peptidoglycan synthesizing enzyme transpeptidase. Inhibition of the enzyme, ultimately results in the lysis of the bacterial cells.

Ampicillin resistance gene

The gene responsible for conferring ampicillin resistance is called bla gene and codes for a TEM1 ?-lactamase enzyme.  The TEM1 ?-lactamase hydrolyzes the ?-lactam ring of the ampicillin. This results in the inactivation of the antibiotic. The enzyme is usually secreted into the periplasmic space where it catalyzes the hydrolysis reaction.

TEM1 ?-lactamase is the major ?-lactamase responsible for ampicillin resistance in various gram negative bacteria including E. coli. As a result, this gene is widely used to confer resistance against ampicillin. There are a variety of vectors using bla gene to confer resistance against ampicillin for the selection of transformed E. coli.

Preparing the ampicillin stock solution

The stock solution of ampicillin is prepared in water. Generally, ampicillin sodium salt is used for its better solubility. The concentration of stock solution is generally in the range of 50-100mg/ml. We usually prepare 100mg/ml stock solutions.

Weigh 100mg of ampicillin sodium salt, dissolve it in 1ml MQ grade water and filter sterilize it by using 0.22µm filter. Store the stock at -20?C.

You should note that pH of the water has great impact on the stability of the antibiotic. It is better to use pH 6.8-7.0, of water for preparing the stock. Moreover the stock can be stored for one month only. After that the antibiotic starts to degrade.

Working concentration of ampicillin in media

For broth or agar plates the concentration of ampicillin is kept between 50-100µg/ml.

Problem of satellite colonies

As the beta lactamase is secreted in the periplasm, it degrades the ampicillin nearby the transformed cells. This results in the growth of small untransformed colonies in close proximity of the transformed one. These colonies are called as satellite colonies.

One way to deal with satellite colonies is to incubate the plates for less than 14 hours. Another way is to increase the concentration of ampicillin in the plates. We generally use 100µg/ml of ampicillin in the plates. But here also it is better to incubate the plates for less than 16 hours.