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

Abstract

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.

Protocol

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 amo....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.