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

Summary

Here, we present a protocol to detect hydrogen sulfide producing bacteria with a modified protocol used for bismuth sulfide (BS) precipitation. The key advantages of this method are that it is easy to evaluate and does not require specialized equipment.

Abstract

Hydrogen sulfide (H₂S) is a toxic gas produced by bacteria in the proteolysis of sulfur-containing amino acids and proteins that plays an important role in human health. The H₂S production test is one of the important bacterial biochemical identification tests. The traditional methods are not only tedious and time-consuming but also prone to inhibition of bacterial growth due to the toxic effect of heavy metal salts in sulfur-containing medium, which often leads to negative results. Here, we established a simple and sensitive method to detect H₂S in bacteria. This method is a modified version of bismuth sulfide (BS) precipitation that uses 96-well transparent microtiter plates. Bacterial culture was combined with bismuth solution containing L-cysteine and cultivated for 20 min, at the end of which a black precipitate was observed.The visual detection limit for H₂S was 0.2 mM. Based on the visual color change, the simple, high-throughput, and rapid detection of H₂S producing bacteria can be achieved. In summary, this method can be used to identify H₂S production in bacteria.

Introduction

Hydrogen sulfide producing bacteria can utilize sulfur-containing amino acids and proteins to produce hydrogen sulfide (H₂S). The production of H₂S occurs usually in gram-negative Enterobacteriaceae family bacteria and also in members of Citrobacter spp., Proteus spp., Edwardsiella spp., and Shewanella spp.¹. These bacteria have the ability to reduce sulphate into hydrogen sulfide (H₂S) in order to obtain energy. Hydrogen sulfide has been implicated in the development of bacterial drug resistance. H₂S protects bacteria from the toxicity of reactive oxygen speci....

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 (Table 1). Salmonella paratyphi A, Fusobacterium nucleatum, Pseudomonas aeruginosa, and Proteus vuigaris can produce H₂S, as outlined in previous liter....

Results

Detection of hydrogen sulfide producing bacteria
The performance of the H₂S test was investigated using pure cultures of selected bacterial strains, as listed in Table 1. The results indicated that Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus vuigaris can produce H₂S with black BS precipitate, while Salmonella paratyphi A, Staphylococcus aureu.......

Discussion

The hydrogen sulfide production test is one of the conventional phenotypic tests for the identification and differentiation of bacterial strains. Many bacterial species can produce hydrogen sulfide in their natural environment, such as aquatic water. These bacterial species include Salmonella sp., Citrobacter sp., Proteus sp., Pseudomonas sp., some strains of Klebsiella sp., Escherichia coli, and some species of anaerobic Clostridia^15.......

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
Enterococcus faecalis	ATCC	19433
Fusobacterium nucleatum	ATCC	25586
Klebsiella pneumoniae	ATCC	43816
L-cysteine	Amresco	52-90-4
Proteus vuigaris	CMCC	49027
Salmonella paratyphi A	CMCC	50001
Salmonella paratyphi B	CMCC	50094
Staphylococcus aureus	ATCC	25923
Triethanolamine-HCl	Shanghai Aladdin Biochemical Technology Co., Ltd.	637-39-8