Preparation of stock solution of ampicillin


  • Ampicillin belongs to a group of beta-lactam antibiotics.
  • Ampicillin is used to treat many different types of infections caused by bacteria, e.g., bladder infections, ear infections, gonorrhea, pneumonia and E. coli or salmonella infection.
  • Ampicillin is one of the synthetic derivatives of penicillin. It was generated by adding an amino group side chain on the penicillin.
  • In contrast to penicillin which is active only against gram-positive bacteria, ampicillin is active against both gram-positive and gram negative bacteria.
  • Ampicillin inhibits the synthesis of cell wall of bacteria by stopping the peptidoglycan cross-linking.
  • Ampicillin functions as a competitive inhibitor of the enzyme transpeptidase which is required for the synthesis of bacterial cell wall.
  • Due to inhibition of cell wall synthesis, bacteria do not grow and have risk to die due to lysis in the presence of ampicillin. However, as the ampicillin concentration decreases below critical level in the surroundings, live bacteria resume the cell wall synthesis and growth.
  • Ampicillin is one of the most extensively used as selection marker in molecular biology and bacteriology. Many commonly used vectors have ampicillin resistance gene.
  • 50 – 100 mg/ml stock solution of ampicillin is generally prepared in water. Depending on the need, stock solution is diluted to final concentration.


  • Reagents
    • Ampicillin Sodium salt
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • 15 ml screw-capped tube / Conical flask / Beaker / 15 ml screw-capped tube


  • 50 mg/ml ampicillin


  • Preparation of 10 ml of 50 mg/ml ampicillin stock solution in water


Step 1: To prepare 10 ml of 50 mg/ml stock solution of ampicillin, weigh out 500 mg of ampicillin. Transfer it to 15 ml screw-capped tube / Conical flask / Beaker. Add 9 ml of deionized / Milli-Q water. Mix until all ampicillin is completely dissolved.


  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml deionized / Milli-Q water and mix the content by inverting the tube number of times or using rotator. Once the content is mixed, make up the volume 10 ml. Since the tube is marked, you don’t need to transfer the content to measuring cylinder.
  • Use autoclaved sterile deionized / Milli-Q water.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

Step 3: Filter sterilize by passing the solution through 0.2 micron filter paper under aseptic condition.


  • Store the solution at -20°C for long time.


  • It is recommended to store the ampicillin stock solution as small aliquots (1 ml). Repeated freeze-thaw cycle can cause ampicillin degradation.
  • Aliquoting must be done under sterile condition.


  • A selection marker for bacterial culture during gene cloning experiments.
Follow the table to prepare ampicillin solution of different concentration and volume.
Conc. / Volume 5 ml 10 ml 25 ml 100 ml
50 mg / ml 0.25 g 0.5 g 2.5 g 5 g
100 mg / ml 0.5 g 1 g 5 g

10 g