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1. D2O incorporation treatment in the presence of antibiotics (Figure 1a)
- Check the bacterial concentration by measuring the optical density (OD) with a photometer at a wavelength of 600 nm.
- Dilute the bacterial solution using the normal Mueller-Hinton broth (MHB) medium, which does not contain deuterium, to reach a final cell concentration of 8 x 105 CFU/mL. Vortex gently to mix the bacterial cells.
- Prepare 300 µL aliquots of the bacterial solution in seven 1.5 mL micro tubes, and 600 µL aliquots of the bacterial solution in one 1.5 mL micro tube.
- Add 4.8 µL of antibiotic (gentamicin or amoxicillin) stock solution (1 mg/mL) into the micro tube containing 600 µL of the bacterial solution, to make the final antibiotic concentration to 8 µg/mL.
- Take 300 µL of solution out of the 8 µg/mL of antibiotic-containing bacteria solution, and add to another 300 µL of bacterial solution, to make two-fold diluted antibiotic- (4 µg/mL) containing bacteria solution.
- Repeat the two-fold serial dilution of the test antibiotics, gentamicin, or amoxicillin, until the micro tube with the lowest concentration (0.25 µg/mL) is reached, and discard 300 µL from the tube. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 µg/mL - 8 µg/mL.
- Leave one tube with no antibiotics for blank control. This will be the positive control to inspect the bacterial metabolic activity without antibiotics treatment but with D2O treatment.
- Leave one tube with no antibiotics and no D2O for the negative control.
- Incubate the bacterial aliquot with a certain antibiotic (gentamicin or amoxicillin) containing MHB medium for 1 h.
- During incubation, prepare a serial dilution of antibiotics with 100% D2O containing medium with the same concentration gradient of antibiotics prepared in step 1.6. For both gentamicin and amoxicillin, the serial concentrations range from 0.25 µg/mL - 8 µg/mL.
- After 1 h of antibiotic treatment, add 700 µL of serially diluted antibiotic and 100% D2O-containing MHB medium to the 300 µL of antibiotic-pretreated bacteria in the same antibiotic concentration (prepared in step 1.6), respectively.
- For example, add 700 µL of 100% D2O-containing MHB medium (containing 8 µg/mL of antibiotic) to the 300 µL of 8 µg/mL antibiotic-pretreated bacteria. In the same manner, transfer to the corresponding tubes of the next concentration, and homogenize by pipetting up and down several times.
- Add 700 µL of antibiotic-free 100% D2O-containing MHB medium to 300 µL of antibiotic-free bacteria (prepared in step 1.6.1) as a blank control.
- Incubate at 37 °C in an incubation shaker at 200 rpm for an additional 30 min.
NOTE: In this step, the final concentration of D2O in the medium for the test is 70%.
- First centrifuge the 1 mL of antibiotic and D2O-treated bacterial sample at 6200 x g for 5 min at 4 °C, and then wash twice with purified water. Finally, fix samples in 10% formalin solution and store them at 4 °C.
2. stimulated Raman scattering (SRS) imaging of D2O metabolic incorporation in a single bacterium
- Wash 1 mL of fixed bacteria solution with purified water and then centrifuge at 6200 x g for 5 min at 4 °C. Remove the supernatant. Enrich the bacterial solution to about 20 µL.
- Deposit the bacterial solution on a poly-L-lysine coated coverglass. Sandwich and seal the sample for SRS imaging.
- Image bacteria at the C-D vibrational frequency at 2168 cm-1 using an SRS microscope.
- Input and tune the pump wavelength to 852 nm using the control software on a computer.
- Measure the laser power using a power meter. Set the power of pump laser at the sample to ~8 mW and the power of Stokes laser at the sample to ~40 mW by adjusting the half-wave plate in front of the laser output.
NOTE: In the SRS microscope, a tunable femtosecond laser with an 80-MHz repetition rate provides the pump (680 to 1300 nm) and Stokes (1045 nm) excitation lasers.
- By adjusting the screws of the reflection mirrors, spatially align the pump and Stokes beams and direct the two beams into an upright microscope equipped with a 2D galvo mirror system for laser scanning.
- Use a 60x water immersion objective to focus the pump and Stokes lasers on the sample.
- Use an oil condenser to collect the signals from the sample in the forward direction.
- Use a bandpass filter to filter out the Stokes laser before directing it into a photodiode.
- Extract the stimulated Raman signal by a lock-in amplifier and detect the signals by a photodiode.
- Set each SRS image to contain 200 x 200 pixels and the pixel dwell time for 30 µs in the software's control panel. The total acquisition time for one image is ~1.2 s. Set the Step size to 150 nm, so the image size is about 30 x 30 µm2. Image at least three fields of view for each sample.