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 antibiotic serially diluted with 100% deuterium containing MHB medium to the 300 microliters of antibiotic pre-treated bacteria in the same antibiotic concentration. Homogenize the mixture by pipetting up and down several times. Add 700 microliters of antibiotic-free 100% deuterium containing MHB medium to 300 microliters of antibiotic-free bacteria as a positive control.

Add 700 microliters of antibiotic-free 0% deuterium containing MHB medium to 300 microliters of antibiotic-free bacteria as a negative control. Incubate all the microtubes at 37 degrees Celsius and 200 rotations per minute for 30 minutes. After incubation, centrifuge the 1 milliliter of antibiotic and deuterium-treated bacterial sample at 6,200 times g for 5 minutes at 4 degrees Celsius. Then, wash the pellet twice with purified water. Fix the samples in 10% volume by volume formalin solution, and store them at 4 degrees Celsius.

For the sample preparation, wash 1 milliliter of fixed bacteria solution with purified water, and centrifuge the washed bacteria solution at 6,200 times g for 5 minutes at 4 degrees Celsius.

Remove the supernatant and enrich the bacterial solution to about 20 microliters with sterilized water. Deposit the bacterial solution on a poly-L-lysine coated cover glass, sandwich with another cover glass, and seal the sample. Put the bacterial sample on the sample stage under the 60x water immersion objective. Start the SRS imaging of bacterial samples. Image at least three fields of view for each sample.