An ATP Utilization Assay for Determining Bacterial Survival Under Antibiotics Treatment

1. Viability quantification of GC aggregations

1. Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed broth(GCP, Table 1) supplemented with 4.2% NaHCO₃ and 1% Kellogg solutions. Use spectrophotometry at a wavelength of 650 nm (an OD 650 of 1 = ~1 x 10⁹ CFU/mL) to determine the concentration of suspended bacteria.
2. Adjust the concentration of GC to ~1 x 10⁸ CFU/mL.
3. Add 99 µL of adjusted GC suspension into wells of a 96-well plate.
4. Incubate the plate for 6 h at 37 °C with 5% CO₂ to allow the bacteria to aggregate.
5. Add 1 µL of serial diluted ceftriaxone (1000, 100, 50, 25, 12.5, 6.2, 3.1, 1.5, 0.8, 0.4, 0.2 µg/mL) into each well. Leave some wells untreated to serve as controls.
6. Incubate the plate for 24 h at 37 °C with 5% CO₂.
7. Sonicate the suspension 3 times in each well for 5 s at 144 W and 20 kHz.
8. Add 100 µL of commercially available ATP utilization glow reagent into each well, pipette up-and down for 3 times, and incubate for 15 min at 37 °C with 5% CO₂.
9. Carefully transfer 150 µL of mixture from each well into a new well in a 96-well black microplate and avoid introducing bubbles.
10. Measure the absorbance of each well at 560 nm using the plate reader.
11. Calculate the survival rate by the ratio of the reading obtained after serial ceftriaxone treatment to the reading from untreated wells.

2. Fluorescence microscopic analysis of Live/Dead of GC aggregates

1. Collect GC using a sterile applicator. Swab GC from the plate and re-suspend GC in pre-warmed GCP media plus 1% Kellogg supplements.
2. Determine the number of bacteria by spectrophotometry at a wavelength of 650 nm and adjust the concentration of GC to ~1 x 10⁷ CFU/mL.
3. Add 198 µL of GC suspension into in 8-well coverslip-bottom chambers.
4. Incubate the chamber for 6 h at 37 °C with 5% CO₂ to allow aggregation formation.
5. Add 2 µL of ceftriaxone (100 µg/mL or various dilutions) into each well within each aggregation condition. Incubate for the desired time at 37 °C with 5% CO₂.
6. Add 0.6 µL of live/dead staining solution mixture into each well and incubate for 20 min at 37 °C with 5% CO₂.
7. Acquire Z-series images using a confocal microscope (an equivalent microscope can be used).
8. Analyze the images using ImageJ software for measurement of the size of GC aggregates and the fluorescence intensity ratio FIR of live-to-dead staining in each aggregate.

Table 1: Recipe for 1 L of GCP Bacterial Growth Media.