An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment

Measure the optical density at 650 nanometers or OD₆₅₀ to determine the concentration of the suspended bacteria, and adjust the GC concentration to about 1 x 10⁸ colony-forming units per milliliter. Next, add 99 microliters of the GC suspension into individual wells of a 96-well plate, and allow the bacteria to aggregate at 37 degrees Celsius and 5% carbon dioxide for 6 hours.

At the end of the incubation, add 1 microliter of serially diluted ceftriaxone to each well, leaving some wells untreated to serve as controls, and return the plate to the incubator for another 24 hours. The next day, sonicate the suspension in each well three times at 144 watts and 20 kilohertz for 5 seconds per sonication, and add 100 microliters of commercially available ATP utilization glow reagent to each well.

Carefully transfer 150 microliters of mixture from each well into individual wells of a new 96-well black microplate and measure the absorbance of each well at 560 nanometers on a plate reader. Then, calculate the survival rate by the ratio of the reading obtained after serial antibiotic treatment to the reading from the untreated wells.