A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)

1. Make Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, 0.25% sodium chloride, NaCl in H₂O) and aliquot into appropriate volumes. Sterilize by autoclave. Store at room temperature.
2. Make 100 ml LB agar (1% tryptone, 0.5% yeast extract, 0.25% NaCl, 1.5% agar in H₂O) in 250 ml flasks. Sterilize by autoclave and store at room temperature. Ensure that the agar is melted before pouring into plates.
   NOTE: Flasks containing 100 ml of LB agar will yield 4 plates. The amount of LB agar can be altered to correspond to the number of plates needed for the assay.
3. Make phosphate-buffered saline, PBS (137 mM NaCl, 2.7 mM potassium chloride, KCl, 10 mM Disodium hydrogen phosphate, Na₂HPO₄, 2 mM potassium dihydrogen phosphate, KH₂PO₄). Sterilize by autoclave or filtration and store at room temperature.
4. Prepare the antibiotic stock solutions for gentamicin, kanamycin, and tobramycin.
   1. Prepare appropriate antibiotic stock concentrations for Pseudomonas aeruginosa strains containing 100 mg/ml gentamicin, 30 mg/ml kanamycin, and 10 mg/ml tobramycin. Dissolve the antibiotics in water, filter sterilize (0.2 µm), and store at 4 °C. Alter the antibiotics and concentrations depending on the bacterium studied.

2. Spot Plate Assay for Oxidative Stress Response

1. Set up overnight cultures of the strains to be tested. Add 2 ml LB broth to 16 x 150 mm test tubes (one per strain) and inoculate with 1 isolated colony from each strain. Incubate overnight at 37 °C with aeration on a tissue culture rotator in an air incubator.
2. Measure the OD₆₀₀ of the overnight cultures. Wash cultures before taking OD₆₀₀ readings to eliminate pyomelanin present in the media.
3. Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant and any loosely pelleted cells with a micro pipettor, and resuspend the solid cell pellet in 1 ml LB.
4. The next day, prepare LB agar plates containing H₂O₂ as an oxidative stressor. A range of H₂O₂ concentrations from 0 to 1 mM is a good starting point.
   1. Melt the LB agar flasks. Cool media to approximately 50 °C at room temperature.
   2. Add H₂O₂ directly to the cooled media at the desired concentrations. Swirl flasks to mix. See Table 1 for concentrations of H₂O₂ and volumes of concentrated H₂O₂ to add. These values are based on 100 ml of LB agar.
   3. Pour plates immediately after adding H₂O₂ and flame the surface to remove bubbles. The yield is 4 plates per 100 ml of LB agar. Mark the plates with the H₂O₂ concentration.
   4. Place the plates uncovered in a biological flow hood with the fan running for 30 min to remove excess moisture from the plates.      
      NOTE: Use the plates the same day they are prepared. Failure to do so may result in inconsistent data.
      NOTE: Oxidative stressors such as paraquat can be substituted for H₂O₂ in this assay. The concentrations used for other oxidative stressors may be different than those used for H₂O₂.
5. Wash and measure the OD₆₀₀ of the overnight cultures.
6. Normalize the OD₆₀₀ of all the overnight cultures to the lowest value for the set of strains being tested. P. aeruginosa generally has an OD₆₀₀ of approximately 2.5 when grown overnight in LB.
   1. Determine the volume of culture needed to dilute the culture to the lowest OD₆₀₀ in a total volume of 1 ml. For example, if a culture has an OD₆₀₀ of 3 and the lowest OD₆₀₀ for the set of strains is 2.5, perform the following calculation: (2.5)(1 ml) = (3)(x). x = 0.833 ml. 0.833 ml of culture will be placed in a microfuge tube.
   2. Calculate the amount of LB + DMSO needed to bring the culture volume to 1 ml. For the example in step 4.4.1, the amount of LB + DMSO added to the culture would be 0.167 ml (1 ml total volume – 0.833 ml culture). Make stock solutions of LB + DMSO to use for these dilutions based on the volume needed for diluting all strains.
   3. Mix the culture and LB + DMSO by vortexing.
7. To maintain cultures in a constant concentration of DMSO, perform tenfold serial dilutions of the normalized overnight cultures in PBS + DMSO.
   1. Make a stock solution of PBS + DMSO. For one set of dilutions for one strain, mix 300 µM volume of DMSO with PBS to yield a total volume of 720 µl. Scale these stocks up or down depending on how many strains are tested.
   2. Label microfuge tubes for 10^-1 through 10^-7 serial dilutions. Add 90 µl of PBS + DMSO to the appropriate tubes. Use PBS + DMSO for strains grown in LB + DMSO.
   3. Add 10 µl of culture to the appropriate 10^-1 dilution tube. Mix by vortexing and transfer 10 µl of the 10^-1 dilution to the 10^-2 dilution tube. Repeat until all dilutions have been performed. Change pipet tips between dilutions.
8. Spot 5 µl of the 10^-3 through 10^-7 dilutions on LB + H₂O₂ plates in duplicate for each strain. Use one pipet tip if spots are plated from most dilute to least dilute (10^-7 to 10^-3). Do not tip or tilt the plate until the liquid has dried into the plate.
9. Incubate the plates for 24-48 hr at 37 °C (air incubator), depending on the strain.
   NOTE: P. aeruginosa PAO1 will have good-sized colonies on LB after 24 hr of incubation. Incubate strains until they have colonies approximately the same size as PAO1.
10. Photograph the plates using a CCD camera above a transluminator. Optionally, edit the photos for contrast and crop them to the same size. Count the number of colonies in each spot to determine changes in sensitivity to oxidative stress.

Table 1: Concentrations of H₂O₂ to add to LB agar for the oxidative stress spot plate assay. This table gives various H₂O₂ concentrations and the corresponding amount of concentrated H₂O₂ stock to add to 100 ml LB agar.