An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment

Begin by seeding a bacterial culture into an assay plate. Then, incubate the plate.

The bacterial cells have surface adhesins and pili that enable them to adhere to each other, forming small aggregates.

Add varying antibiotic concentrations to the wells, leaving one untreated for a control.

The antibiotic molecules interact with sensitive bacteria, affecting their ability to grow and reproduce, leading to their death.

The antibiotic-resistant bacteria remain metabolically active, producing ATP molecules.

Use a sonicator to disrupt the bacterial cells, releasing their intracellular contents, including ATP.

Add ATP-utilization glow reagent containing luciferin, luciferase enzyme, and other co-factors.

Luciferase utilizes ATP and oxygen to convert luciferin to an excited oxyluciferin.

The oxyluciferin subsequently transitions to its ground state, emitting luminescent light.

Measure the absorbance using a plate reader.

The decrease in absorbance at higher antibiotic concentrations suggests lower ATP production, indicating a reduced bacterial survival rate.