Detection of Bacterial Antibiotic Susceptibility Using a Stimulated Raman Scattering Microscope

Begin with bacterial samples pretreated with increasing concentrations of an antibiotic that eliminates susceptible bacteria.

Add a growth medium containing deuterium oxide and the same antibiotic concentration to the corresponding tubes.

Incubate the tubes under shaking conditions.

Deuterium oxide enters bacterial cells, where deuterium is incorporated into newly synthesized biomolecules, forming carbon–deuterium bonds.

Spin down the bacteria, discard the supernatant, and wash.

Add a fixative to preserve the bacterial structure.

Add water, centrifuge, and remove the supernatant.

Resuspend the bacteria and transfer the suspension onto a poly-L-lysine-coated coverglass, then seal it.

Place the sample on the stage of a pre-calibrated stimulated Raman scattering or SRS microscope. Add water and lower the objective.

Acquire images of multiple fields to assess the carbon–deuterium vibrational frequency.

At higher antibiotic concentrations, the carbon–deuterium signal decreases, indicating reduced bacterial viability.

This enables rapid assessment of antibiotic susceptibility by correlating the SRS signal intensity with bacterial viability.

After 1 hour of incubation, add 700 microliters of antibiot