Quantification of Light-Induced Bacterial Inactivation Through Reactive Oxygen Species Generation

Abstract

Source: Cheng, C., et al. Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5′-Phosphate. J. Vis. Exp. (2022).

In this video, a buffered reaction mixture containing L-methionine, nitro blue tetrazolium (NBT), and flavin mononucleotide (FMN) is irradiated with violet light to induce reactive oxygen species (ROS) production. This leads to bacterial inactivation and the formation of spectrometrically detectable formazan.

Protocol

1. Detection of Reactive Oxygen Species (O₂•⁻) in Staphylococcus aureus

1. Prepare the Staphylococcus aureus samples.
2. Dilute the bacterial density of the samples with sterilized water to an optical density of 0.5 at 600 nm (OD (Optical density)₆₀₀, ~6 x 10⁶ CFU (Colony-forming unit) /mL). Transfer 0.5 mL of the culture to a 1.5 mL centrifuge tube. Centrifuge at 14,000 x g for 10 min and decant the supernatant to obtain a cell pellet.
3. Add 0.1093 g of L-methionine, 0.1 g of NBT (Nitro blue tetrazolium chloride), and 25 mL of FMN (Flavin mononucleotide) (400, 240, or 120 µM) to 75 mL of PB (Phosphate buffer). For each 1 mL of the reactant applied, the final concentrations of FMN, methionine, and NBT are 100 (60 or 30) × 10^-6 M, 7.3 × 10^-3 M, and 1.2 × 10^-3 M, respectively.
4. Add 1 mL of each reactant solution (i.e., with varying FMN concentrations) to the cell pellets obtained. Irradiate the solutions by violet light at 10 W/m² for 10 min.
5. Centrifuge the mixture at 14,000 x g for 10 min and decant the supernatant. Resuspend the pellet in 1 mL of dimethyl sulfoxide (DMSO) to extract the reduced NBT. Detect the produced O₂•⁻ species from the absorbance at 560 nm.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dimethyl Sulfoxide	Sigma-Aldrich, St. Louis, MO	190186
Infrared thermometer	Raytek Co. Santa Cruz, CA	MT4
LB broth	Difco Co., NJ
L-Methionine	Sigma-Aldrich, St. Louis, MO	1.05707
NBT (Nitro blue tetrazolium chloride)	Bio Basic, Inc. Markham, Ontario, Canada
Power supply	China tech Co., New Taipei City, Taiwan	YP30-3-2
Riboflavin 5′-phosphate	Sigma-Aldrich, St. Louis, MO	R7774
Solar power meter	Tenmars Electronics Co., Taipei, Taiwan	TM-207
Staphylococcus aureus subsp. aureus	Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan	10451
UV-Vis optical spectrometer	Ocean Optics, Dunedin, FL	USB4000
UV-Vis spectrophotometer	Hitachi High-Tech Science Corporation,Tokyo, Japan	U-2900
Violet LED	Long-hui Electronic Co., LTD, Dongguan, China