H₂O₂ Kill Assays of Biofilm Bacteria   

Materials and Reagents

1. Phosphate buffered saline (PBS) solution (Sigma-Aldrich, catalog number: P4417-100TAB )
   
2. 30% w/w Hydrogen peroxide solution (undiluted, as sold commercially) (RICCA Chemical,  catalog number: 3821.7-32 )
   
3. Sodium thiosulfate solution (dissolved in ddH₂O) (Sigma-Aldrich,  catalog number: S8503 )
   
4. 0.2 μM Polycarbonate 25 mm membranes (General Electric Company, catalog number: K02BP02500 )
   
5. P.aeruginosa strains in freezer stock
   
6. 25% Lennox broth (LB) medium (Becton Dickinson and Company, Difco^TM, catalog number: 240230 ) (see Recipes)
   
7. 25% LB agar plates (see Recipes)
   

Equipment

1. 6-well and 96-well plates
   
2. Standard petri plates
   
3. Spectrophotometer (cuvette) (Thermo Fisher Scientific, model: GENESYS 10S UV-Vis )
   
4. Spectrophotometer (96-well plate) (Bio-Rad, model: 680 )
   
5. Cuvettes for OD₆₀₀ reading
   
6. Shaking incubator at 37 °C, 250 rpm
   
7. Static incubator at 37 °C
   
8. Sterile glassware: 150 ml Erlenmeyer flasks, capped or foiled
   
9. 1.5 ml and 2 ml microcentrifuge tubes
   
10. Source of UV irradiation
    
11. Sterile wire-loops (sterilized with 70% ethanol and flame)
    
12. Stainless steel forceps (sterilized with 70% ethanol and flame)
    

Procedure

1. Day 0. Streak P.aeruginosa cells from the freezer stock onto a LB agar place and incubate statically overnight at 37 °C.
   
2. Day 1. Pick 4-5 single colonies from the P.aeruginosa agar plate with a sterile wired-loop and inoculate 15 ml liquid LB medium in a 150 ml Erlenmeyer flask. Grow liquid bacterial cultures overnight for 16-18 hours at 37 °C, with shaking at 250 rpm.
   
3. Day 2. Gently place polycarbonate membranes on agar surface of fresh sterile 25% LB agar plates and sterilize the membranes by placing them under UV irradiation for 1 hour. Handle membranes carefully with sterile forceps and use the membranes immediately after sterilization. Use eyes and skin UV protective equipment. Use at least 3 membranes per strain per condition for adequate biological replicates, and up to 6 membranes may be placed on each agar plate.  
   
4. Measure the OD₆₀₀ of the overnight bacterial culture and dilute the bacterial suspension in LB medium to a starting concentration of 10⁸ cells/ml. Depending on the bacterial strain used, the OD₆₀₀ to CFU ratio will differ and needs to be determined for each strain: for example, for the PAO1 wild type strain, 10⁸ cells/ml = ~OD₆₀₀ 0.1.
   
5. Spot 5 μl (5 x 10⁵ cells) onto the sterile membranes and allow the liquid to be absorbed (10-20 minutes).
   
6. Incubate the colony biofilm on agar plates for 24 h at 37 °C.
   
7. Day 3. Using sterile forceps, gently lift the membranes off the agar surface and transfer them (cells side down) into 6-well plates filled with 2 ml of 25% LB liquid medium in each well. Make sure the membranes are spread flat (i.e. not rolled up) and biofilm cells are remain on the membrane.
   
8. For the H₂O₂ treated biofilms, add 30 μl H₂O₂ (150 mM) to each well in pulses every 10 minutes for 30 minutes (for a final concentration of 450 mM H₂O₂ per challenge). The pulsing is done to mimic a continuous exposure of cells to H₂O₂. In between H₂O₂ pulses, incubate cells at 37 °C without shaking. Include untreated controls that are challenged with PBS. Each condition should be done at least in triplicates.  
   
9. After H₂O₂ or PBS challenge, add 0.2% sodium thiosulfate to all samples to neutralize any remaining H₂O₂. Add even when samples are only challenges with PBS as a control.
   
10. To determine the viable cell count in H₂O₂ or PBS treated biofilms, collect biofilm cells by transferring the membranes and 2 ml of liquid from each well into 2 ml microcentrifuge tubes. Membranes are moved by gently lifting and rolling them using sterile forceps, with biofilm cells facing inward. The entire membrane should be submerged in liquid. Ensure to sterilize forceps between membrane transfers. Vortex biofilms in microcentrifuge tubes at maximal speed for at least 1 minute to detach and resuspend cells. Additional pipetting up and down and vortexing may also necessary to make sure there are no cell clumps visible in the bacterial suspension.
    
11. Aliquot 100 μl of bacterial suspension into 96-well plate, serially dilute cells 1:10, then plate 100 μl of each dilution on LB agar plates for CFU count. Incubate CFU count plates at 37 °C overnight.
    
12. Day 4. Count CFU on LB agar plates and calculate the viable CFU per biofilm based on the dilution factors applied.
    
13. Determine hydrogen peroxide killing by comparing the viable CFU count in the PBS treated and the H₂O₂ treated conditions.

Recipes

1. 25% LB medium
   5 g LB powder medium per L
   Dissolved in ddH₂O and autoclaved
   
2. 25% LB agar plates
   25% LB medium with 1.5% agar
   Dissolved in ddH₂O and autoclave

Acknowledgments

We would like to acknowledge CIHR (MOP-102727 to DN) and the Burroughs Wellcome Fund (1006827.01 to DN) for funding. This protocol was adapted from the previously published paper Khakimova et al. (2013).

References

1. Khakimova, M., Ahlgren, H. G., Harrison, J. J., English, A. M. and Nguyen, D. (2013). The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance. J Bacteriol 195(9): 2011-2020.    
   
2. Walters, M. C., 3rd, Roe, F., Bugnicourt, A., Franklin, M. J. and Stewart, P. S. (2003). Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 47(1): 317-323.