Name: a sensitive visual method for the detection of hydrogen sulfide producing bacteria
Uploaded: 2022-06-27T15:00:00
Description: Watch this Scientific Journal Video about A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria at JoVE.com

This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Teaching Reform Research Project of China Pharmaceutical University (2019XJYB18).

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, Fusobacte...

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+H2S+test+to+screen+for+the+detection+of+sulphur-+and+sulphate-reducing+bacteria+of+faecal+origin+in+water.">Modification of the H2S test to screen for the detection of sulphur- and sulphate-reducing bacteria of faecal origin in water.</a> Water Supply. 21 (1), 59-79 (2021).</li><li>Netzer, R., Ribi&#269;i&#263;, D., Aas, M., Cav&#233;, L., Dhawan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantification+of+priority+bacteria+in+aquaculture+using+digital+PCR.">Absolute quantification of priority bacteria in aquaculture using digital PCR.</a> Journal of Microbiological Methods. 183, 106171 (2021).</li></ol>

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&#160;sp., some strains of Klebsiella sp., Escherichia coli, and some species of anaerobic Clostridia15...

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&#160;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 species (ROS), thus antagonizing the antibacterial effect of antibiotics2,3. H2S also has an important physiological effect in maintaining homeostasis. At supraphysiological levels, H2S has been shown to be profoundly toxic to the body. In the human body, H2S has another role as a gas signaling molecule that is involved in a variety of physiological and pathological processes. H2S can regulate the systolic function of the heart and plays an important physiological role in relaxing blood vessels, inhibiting vascular remodeling, and protecting the myocardium4,5. H2S also plays an important role in regulating the nervous system and digestive tract6,7. It has been found that, when exposed to bactericidal antibiotics, bacteria produce lethal reactive oxygen species (ROS) leading to cell death8,9,10,11.
As a common biochemical test in microbiological laboratory courses, the hydrogen sulfide test is an important experiment in the identification of bacteria, especially bacteria of the family Enterobacteriaceae. At present, the hydrogen sulfide test is usually performed on a large number of sulfur-containing amino acids and lead acetate medium inoculated with the bacteria to be tested. After a period of incubation (2-3 days), the results are judged by observing whether the culture medium or lead acetate paper strip is blackened because of lead acetate production11. However, these 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. A bismuth-based method has been established for the detection of H2S12,13. H2S can react with bismuth, forming black bismuth sulfide precipitation. In order to conduct a reform for this biochemical test, a simple and quick method with no side effects on bacterial growth needs to be established. Here, we set up a simple method for the detection of hydrogen sulfide producing bacteria grown in an in vitro environment using bismuth sulfide as a substrate in a 96-well microtiter plate format.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bismuth (III)chloride</td>
			<td>Shanghai Macklin Biochemical Co., Ltd</td>
			<td>7787-60-2</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Nanjing Chemical Reagent Co., Ltd</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterococcus faecalis&#160;</td>
			<td>ATCC&#160;</td>
			<td>19433</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusobacterium&#160;nucleatum&#160;</td>
			<td>ATCC&#160;</td>
			<td>25586</td>
			<td></td>
		</tr>
		<tr>
			<td>Klebsiella pneumoniae&#160;</td>
			<td>ATCC&#160;</td>
			<td>43816</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Amresco</td>
			<td>52-90-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteus&#160;vuigaris&#160;</td>
			<td>CMCC&#160;</td>
			<td>49027</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella paratyphi A</td>
			<td>CMCC</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella paratyphi B</td>
			<td>CMCC</td>
			<td>50094</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus&#160;aureus&#160;</td>
			<td>ATCC&#160;</td>
			<td>25923</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine-HCl</td>
			<td>Shanghai Aladdin Biochemical Technology Co., Ltd.</td>
			<td>637-39-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

a sensitive visual method for the detection of hydrogen sulfide producing bacteria

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.

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,&#160;and Proteus vuigaris can produce H2S with black BS precipitate, while Salmonella paratyphi A, Staphylococcus <e...

Watch this Scientific Journal Video about A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria at JoVE.com

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

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.

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 ...

Hydrogen sulfide-producing bacteria can utilize sulfur-containing amino acids and proteins to produce hydrogen sulfide. Here, we established a sensitive and simple method to detect the hydrogen sulfide in bacteria. This method is a modified version of bismuth sulfide precipitation that used 96-well transparent microtiter plates.Bacteria culture was combined with bismuth solution containing L-cysteine and cultured for 20 minutes, at the end of which a black precipitate was observed. Demonstrating the procedures will be Wei Zhu, a research assistant from our laboratory. Begin by transferring the bacterial colonies from a Luria-Bertani or LB agar plate to 100 milliliters of LB medium, and culture at 37 degrees Celsius for 12 to 16 hours until the bacterial concentration is about 1 billion cells per milliliter.Transfer one bacterial colony of Fusobacterium nucleatum and Proteus vulgaris from Trypticase soy broth or TSB agar plates to 100 milliliters of TSB medium, and culture at 37 degrees Celsius anaerobically for 24 hours until the bacterial concentration is about 1 billion cells per milliliter. Mix 100 microliters of bacterial culture with 100 microliters of newly prepared bismuth solution in the 96-well microtiter plates, and culture for 20 minutes at 37 degrees Celsius. For each bacterial strain, perform the analysis in triplicate.After 20 minutes, 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 hydrogen sulfide or H2S. Repeat this measurement three times.Determine the sensitivity of the method using different concentrations of sodium hydrosulfide mixed with bismuth sulfide solution. Finally, determine the presence of bisulfide and sulfide ions by observing the formation of a black bismuth sulfide precipitate. Score the color of the wells using a visual scale from no color production to the darkest black color production.The performance of the hydrogen sulfide test was investigated using pure cultures of selected bacterial strains. The results indicated that Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus vulgaris can produce hydrogen sulfide with black bismuth sulfide precipitate. While Salmonella paratyphi A, Staphylococcus aureus, Aeromonas hydrophila, and Klebsiella pneumoniae failed to show any black precipitate.The key advantage of this method is that it is easy to replicate and does not require specialized equipment.

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