Benzylpenicillin Assay 2

Reagents and Materials

• 96-well plate
• Bromothymol Blue pH Indicator
• 60 mg/mL Benzylpenicillin
• Purified β-lactamase 1µg/µL
• Commercial β-lactamase 1µg/µL
• pH 8 Water

Protocol

1. Dilute β-lactamase to the appropriate concentrations in 100 µL of pH 8 water. Add this to the wells.
2. Add an additional 50 µL of pH 8 water to the wells.
3. Add 10 µL of bromothymol blue pH indicator to these wells.
4. Add 100 µL of 60 mg/mL benzylpenicillin to the wells.
5. Measure the change from blue to yellow using a spectrophotometer at an absorbance wavelength of 616 nm.