H₂O₂ Kill Assays of Planktonic Stationary Phase Bacteria

Materials and Reagents

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

Equipment

1. 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 Laboratories, model: 680 )
   
5. Cuvettes for OD₆₀₀ reading
   
6. Shaker-incubator at 37 °C, 250 rpm
   
7. Static incubator at 37 °C
   
8. Sterile glassware: 150 ml Erlenmeyer flask, capped or foiled
   
9. Sterile 15 mm glass test tubes and plastic caps
   
10. Sterile wire-loops (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. Inoculate 15 ml liquid LB medium in a 150 ml flask with 1:100 of overnight bacterial culture. Grow cells to for 16-18 hours at 37 °C, with shaking at 250 rpm.
   
4. Day 3. Determine the OD₆₀₀ of the culture and dilute the bacterial suspension to a starting concentration of ~2.5 x 10⁶ cells/ml (in total volume 1 ml LB). 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. To confirm the correct starting bacterial density (at ~2.5 x 10⁶ cells/ml), aliquot 100 μl of the above bacterial suspension into 96-well plate. Serially dilute cells 1:10 in PBS to approximately ~2.5 x 10² cells/ml, then plate 100 μl on LB agar plates for CFU count. This will also be the CFU count for time zero measurement.
   
6. Set up ~2.5 x 10⁶ cells/ml x 1 ml per sample in sterile glass tubes, with at least 3 replicates per strain per condition. For H₂O₂ treated samples, add H₂O₂ (1 mM (2 μl) to 5 mM (10 μl) or other desired final concentration) to each samples in test tubes. Include untreated controls that are challenged with PBS. Each condition should be done at least in triplicates. Incubate cells for 2 hours with shaking at 250 rpm at 37 °C.
   
7. 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.
   
8. To determine the viable cell count in H₂O₂ or PBS treated samples, aliquot 100 μl of bacterial samples 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.
   
9. Day 4. Count CFU on LB agar plates and calculate the viable CFU per biofilm based on the dilution factors applied.
   
10. 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. LB agar plates
   LB medium with 1.5% agar
   Dissolved in ddH₂O and autoclaved
   

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.