A Sensitive Visual Method for the Detection of Hydrogen Sulfide-Producing Bacteria

Abstract

Source: Zhu, W., & Chu, W. Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria. J. Vis. Exp. (2022)

This video demonstrates a sensitive visual method for the rapid detection of hydrogen sulfide–producing bacteria. A color change from yellow to black indicates bacterial activity through the formation of bismuth sulfide.

Protocol

1. Bacterial strains

NOTE: For this experiment, nine standard strains were used, including Salmonella paratyphi A, Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa PAO1, Aeromonas hydrophila YJ-1, Proteus vuigaris, and Klebsiella pneumoniae. Salmonella paratyphi A, Fusobacterium nucleatum, Pseudomonas aeruginosa, and Proteus vuigaris can produce H₂S.

1. Preparation of bacterial culture
   1. Transfer one bacterial colony of Salmonella paratyphi A, Salmonella paratyphi B, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa PAO1, Aeromonas hydrophila YJ-1, and Klebsiella pneumoniae from a Luria-Bertani (LB) agar plate to 100 mL of LB medium and culture at 37 °C for 12-16 h until the bacterial concentration is about 1 x 10⁹ cells/mL (as indicated by OD₆₀₀ = 1).
   2. Transfer one bacterial colony of Fusobacterium nucleatum and Proteus vuigaris from trypticase soy broth (TSB) agar plates to 100 mL of TSB medium and culture at 37 °C anaerobically for 24 h until the bacterial concentration is about 1 x 10⁹ cells/mL (as indicated by OD₆₀₀ = 1).

2. H₂S detection assay

1. Hydrogen sulfide production test
   1. Mix 100 µL of bacterial culture with 100 µL of newly prepared bismuth solution (pH 8.0; 10 mM bismuth (III) chloride, 0.4 M triethanolamine-HCl, 20 mM pyridoxal 5-phosphate monohydrate, 20 mM EDTA (Ethylenediaminetetraacetic acid), and 40 mM L-cysteine) in the 96-well microtiter plates and culture for 20 min at 37 °C. For each bacterial strain, perform the analysis in triplicate.
   2. After 20 min, check for color change. If the color of the solution changes from light yellow to black, this indicates that the bacteria is able to produce H₂S. Repeat this measurement 3x.
2. Sensitivity of the method
   1. Determine the sensitivity of the method using different concentrations of sodium hydrosulfide (NaHS): 2 mM, 1 mM, 0.8 mM, 0.6 mM, 0.4 mM, 0.2 mM, 0.1 mM, and 0 mM, mixed with BS (Bismuth sulfide) solution.
   2. Determine the presence of HS⁻/S²⁻ by observing the formation of a black BS precipitate. Score the color of the wells using a visual scale from no color production (-) to darkest black color production (++++++).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bismuth (III) chloride	Shanghai Macklin Biochemical Co., Ltd	7787-60-2
EDTA	Nanjing Chemical Reagent Co., Ltd	60-00-4
Fusobacterium nucleatum	ATCC	25586
L-cysteine	Amresco	52-90-4
Triethanolamine-HCl	Shanghai Aladdin Biochemical Technology Co., Ltd.	637-39-8
Sodium hydrosulfide
Pipette