Source: Ketelboeter, L. M. et al., Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress. J. Vis. Exp. (2015)
This video demonstrates a spot plate assay tailored to assess the oxidative stress responses of bacterial strains. Some strains produce pyomelanin, an antioxidant pigment. Conversely, strains treated with a test agent, leading to reduced pyomelanin production, exhibit increased susceptibility to oxidative stress, resulting in decreased cell viability compared to pyomelanin-producing strains.
1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)
2. Spot Plate Assay for Oxidative Stress Response
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.
Final Concentration of H2O2 (mM) | Amount of 9.79 M H2O2 (30% wt) to add (µl) |
0 | 0 |
0.2 | 2.04 |
0.4 | 4.09 |
0.6 | 6.13 |
0.8 | 8.17 |
1 | 10.21 |
The authors have nothing to disclose.
2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC) | Sigma-Aldrich | SML0269-50mg | Also called nitisinone. Soluble in DMSO. |
H2O2 | Sigma-Aldrich | 216763-100ML | 30 wt.% in H2O. Stabilized. |