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

To begin this procedure, set up overnight cultures of the strains to be tested in LB, with and without NTBC. On the following day, prepare LB agar plates containing hydrogen peroxide as an oxidative stressor. First, melt the LB agar flasks and cool the media to approximately 50 degrees Celsius at room temperature.

Next, add hydrogen peroxide directly to the cooled media at the desired concentrations. Swirl the flask to mix. A range of hydrogen peroxide concentrations from 0 to 1 millimolar is a good starting point. Pour the plates immediately, and flame the surface to remove bubbles. The yield is four plates per 100 millimeters of LB agar. Mark the plates with the hydrogen peroxide concentration.

Place the plates uncovered in a biological flow hood, with a fan running for 30 minutes to remove excess moisture from the plates. The plates should be used on the same day they are prepared. While the plates are drying, wash and measure the OD600 of the overnight cultures.

Normalize the OD600 of all the overnight cultures to the lowest value for the set of strains being tested. To do this, determine the volume of culture needed to dilute the culture to the lowest OD600 in a total volume of 1 milliliter.

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, which will result in 0.833 milliliters as the volume of culture needed. Place 0.833 milliliters of culture in a microfuge tube. Next, calculate the amount of LB plus NTBC or LB plus DMSO needed to bring the culture volume to 1 milliliter. In this example, the amount would be 0.167 milliliters. Mix by vortexing.

To maintain cultures in a constant concentration of NTBC or DMSO, perform 10-fold serial dilutions of the normalized overnight cultures in PBS plus NTBC or PBS plus DMSO. Make stock solutions of PBS plus NTBC and PBS plus DMSO scaling up or down, depending on how many strains are tested.

For one set of dilutions for one strain, mix 300 micromolar of NTBC or an equivalent volume of DMSO with PBS to yield a total volume of 720 microliters. Add 90 microliters of PBS plus NTBC or PBS plus DMSO to the appropriate microfuge tubes labeled for 10 to the minus 1 through 10 to the minus 7 serial dilutions. Add 10 microliters of culture to the appropriate 10 to the minus 1 dilution tube.

Mix by vortexing, and transfer 10 microliters of the 10 to the minus 1 dilution to the 10 to the minus 2 dilution tube. Changing pipette tips between dilutions, repeat until all dilutions have been performed.

Spot 5 microliters of the 10 to the minus 3 through 10 to the minus 7 dilutions on LB plus hydrogen peroxide plates in duplicate for each strain. Use one pipette tip if spots are plated for most dilute to least dilute. Do not tip or tilt the plate, until the liquid has dried into the plate. Incubate the plates at 37 degrees Celsius. For 24 to 48 hours, depending on the strain. Once good-sized colonies have appeared, photograph the plates using a CCD camera above a transilluminator.