This study investigates the effects of varying antibiotic concentrations on bacterial survival rates using luminescent ATP detection. The methodology involves seeding bacterial cultures, incubating them, and measuring ATP production to assess the impact of antibiotics.
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.
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.
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