In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

1. Preparation of growth medium, ofloxacin solution and E. coli cell stocks

1. Regular Luria-Bertani (LB) medium: Add 10 g/L of tryptone, 10 g/L of sodium chloride (NaCl), and 5 g/L of yeast extract in deionized (DI) water. Sterilize the medium by autoclaving.
2. LB agar plates: Add 10 g/L of tryptone, 10 g/L of NaCl, 5 g/L of yeast extract, and 15 g/L agar in DI water and sterilize the medium by autoclaving. At the desired temperature (~55 °C), pour ~30 mL of agar medium into square plates (10 x 10 cm). Dry the plates and store them at 4 °C.
3. Modified LB medium: Add 10 g/L of tryptone and 5 g/L of yeast extract in DI water. Sterilize the medium by autoclaving.
4. Modified LB medium including an osmolyte: Mix 2x modified LB medium with a 2x osmolyte solution in equal volumes. To prepare 2x modified LB medium, add 20 g/L of tryptone and 10 g/L of yeast extract in DI water and sterilize by autoclaving. To prepare the 2x osmolyte solution, dissolve the osmolyte of interest (2x amount) in DI water, and then filter sterilize the solution. 
   NOTE: The osmolyte of interest and its concentrations can be determined from the Phenotype Microarray screening assays (see Protocol 4). However, the tested osmolyte and the associated 2x concentration can be adjusted depending on the nature of the research. For example, to study the impact of 1 mM sodium chloride, a 2x osmolyte solution would be a 2 mM sodium chloride solution.
5. Ofloxacin stock solution (5 mg/mL): Add 5 mg of ofloxacin (OFX) salt in 1 mL of DI water. Add 10 µL of 10 M sodium hydroxide to increase the solubility of OFX in water, and then filter sterilize the solution. Prepare aliquots and store at -20 °C.
   NOTE: Ofloxacin is a quinolone antibiotic that has been widely used for both growing and non-growing bacterial cells14,21. The minimum inhibitory concentration (MIC) of OFX for E. coli MG1655 cells is within the range of 0.039–0.078 μg/mL. Also, note that other antibiotics such as ampicillin and kanamycin are commonly used in persister research. The choice of antibiotics is dependent on the nature of the study.
6. E. coli MG1655 cell stocks:  Inoculate 2 mL of regular LB medium with a single colony in a 14 mL test tube (snap-capped) and culture the cells in an orbital shaker at 250 rpm and 37 °C. When the cells reach the stationary phase, mix 500 μL of the cell culture with 500 μL of 50% glycerol (sterile) in a cryogenic vial and store at -80 °C.

2. Propagation of cells to eliminate pre-existing persisters

1. To prepare an overnight culture, scrape a small amount of cells from a frozen cell stock with a sterile pipette tip (do not thaw the glycerol cell stock), and inoculate the cells in 2 mL of modified LB medium in a 14 mL snap-capped test tube. Culture the cells in an orbital shaker at 250 rpm and 37 °C for 12 h.
2. First propagation
   1. After 12 h, transfer 250 µL of overnight culture to 25 mL of fresh modified LB medium in a 250 mL baffled flask covered with sterile aluminum foil.
   2. Grow the cell culture in an orbital shaker at 250 rpm and 37 °C until cells reach the mid-exponential phase (OD₆₀₀ = 0.5).
   3. Measure the optical density at 600 nm (OD₆₀₀) using a microplate reader every 30 min.
3. Second propagation
   1. At OD₆₀₀ = 0.5, dilute 250 µL of cell culture from the first flask into 25 mL of fresh modified LB medium in a 250 mL baffled flask.
   2. Grow the second cell culture in an orbital shaker at 250 rpm and 37 °C until OD₆₀₀=0.5.
   3. Measure the optical density every 30 min.
      NOTE: An overnight culture may have a significant amount of persister cells. The dilution/growth cycle method described above can be used to eliminate these pre-existing persisters before transferring the cells to microarrays. Their elimination can be validated by quantifying persister levels of overnight cultures with the assay described in Protocol 3.

3. Validating the elimination of pre-existing persister cells

1. After the second propagation (see step 2.3), dilute 250 µL of the cell culture (OD₆₀₀ = 0.5) in 25 mL of fresh modified LB medium in a 250 mL baffled flask.
   NOTE: For controls, dilute 250 µL of overnight culture (see step 2.1) in 25 mL of fresh modified LB medium in a 250 mL baffled flask. Before transferring the cells to the flask, the cell density of the overnight culture should be adjusted in a fresh modified LB medium to obtain OD₆₀₀ = 0.5.
2. Add 25 µL of OFX stock solution (5 mg/mL) into the cell suspension and shake the flask gently to make the assay culture homogeneous. The final concentration of OFX is 5 µg/mL in the assay culture.
3. Incubate the assay culture in an orbital shaker at 250 rpm and 37 °C.
4. At every hour during the treatment (including 0 h, the time point before adding OFX into the assay culture), transfer 1 mL of the assay culture from the flask to a 1.5 mL microcentrifuge tube.
5. Centrifuge the assay culture in the microcentrifuge tube at 17,000 x g for 3 min.
6. Carefully remove 950 µL of the supernatant without disturbing the cell pellet.
7. Add 950 µL of phosphate-buffered saline (PBS) solution into the microcentrifuge tube.
8. Repeat the washing steps 3.5-3.7 for 3x in total until the antibiotic concentration is below the minimum inhibitory concentration (MIC).
9. After the final wash, resuspend the cell pellet in 100 µL of PBS solution, resulting in a 10x concentrated sample.
10. Take 10 µL of the cell suspension and serially dilute it six times in 90 µL of PBS solution using a 96-well round bottom plate.
11. Spot 10 µL of diluted cell suspensions on antibiotic-free fresh agar plates. To increase the limit of detection, plate the remaining 90 µL of cell suspension on a fresh agar plate.
12. Incubate the agar plates at 37 °C for 16 h, and then count the colony-forming units (CFUs). Account for the dilution rates while calculating the total number of CFUs in 1 mL of assay culture. Kill curves are generated by plotting the logarithmic CFU values with respect to the duration of antibiotic treatment.
    NOTE: A 6 h OFX treatment is sufficient to obtain a biphasic kill curve for E. coli cells. The persister level of propagated cells (as measured by CFU counts at 6 h) should be significantly less than that of the overnight culture. The procedures described in Protocol 2 and 3 can be performed with regular LB depending on the research design. 

4. Microarray plate screenings

1. Preparing microarray-cell cultures
   1. Transfer 250 µL of exponential-phase cells to 25 mL of fresh modified LB medium in a 50 mL centrifuge tube. Gently mix the cell suspension to make it homogeneous.
      NOTE: The exponential-phase cells (OD₆₀₀ = 0.5) are obtained from the second propagation step (see step 2.3).
   2. Transfer the diluted cell suspension into a sterile 50 mL reservoir.
   3. Using a multichannel pipette, transfer 150 µL of the cell suspension to each well of a microarray, i.e., a 96-well plate containing various osmolytes. 
      NOTE: Wells that do not have osmolytes serve as controls.
   4. Cover the microarray with a gas-permeable sealing membrane.
   5. Incubate the plate in an orbital shaker at 37 °C and 250 rpm for 24 h.
      NOTE: In these experiments, commercially available plates were used, such as Phenotype Microarrays (PM-9 and PM-10) that include a wide range of osmolytes, pH buffers, and other chemicals in a dried state at various concentrations. These microarrays are in half-area 96 well plate formats. Culture volumes should be adjusted depending on the type of plates being used. Microarrays can also be generated manually.
2. Manual preparation of microarray plates
   1. Transfer 75 μL of 2x osmolyte solutions into the wells of a half-area 96 well plate.
   2. Transfer 500 µL of exponential-phase cells to 25 mL of 2x modified LB medium in a 50 mL centrifuge tube. Gently mix the cell suspension to make it homogeneous.
      NOTE: The inoculation rate was adjusted to be consistent with the microarray screening protocol described in 4.1. 
   3. Add 75 μL of the cell suspension into each well of the half-area 96-well plate containing 2x osmolyte solutions.
   4. Incubate the plate in an orbital shaker at 37 °C and 250 rpm for 24 h.
3. Preparing persister assay plates
   1. Prepare 25 mL of modified LB medium containing 5 µg/mL of OFX in a 50 mL centrifuge tube and transfer this medium to a sterile reservoir.
   2. Transfer 190 µL of modified LB medium with OFX from the reservoir into each well of a generic flat-bottom 96 well plate (persister-assay plate) using a multichannel pipette.
   3. Remove the microarray from the shaker (after culturing for 24 h) and transfer 10 µL of cell cultures from the microarray to the wells of the persister-assay plate, containing modified LB medium with OFX.
   4. Take 10 µL of cell suspensions from the persister-assay plate and serially dilute three times in 290 µL of PBS solution using a round-bottom 96 well plate and a multichannel pipette.
   5. Following the serial dilution, spot 10 µL of all serially diluted cell suspensions on antibiotic-free fresh agar plates using a multichannel pipette. 
   6. Incubate the persister-assay plate (prepared in step 4.3.3) in an orbital shaker at 37 °C and 250 rpm for 6 h after covering the plate with a gas-permeable sealing membrane.
   7. After 6 h incubation in a shaker, take the persister-assay plate out and repeat the steps 4.3.4-4.3.5.
   8. Incubate the agar plates for 16 h at 37 °C, and then count CFUs. The CFU levels before and 6 h after the antibiotic treatment enable to calculate the persister fraction in each well. The CFU counts before the OFX treatment also help assess the effects of osmolytes and on E. coli viability.