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, Fusobacterium nucleatum, Pseudomonas aeruginosa, and Proteus vuigaris can produce H2S, as outlined in previous literature1.
<ol>
	<li>Preparation of bacterial culture
	<ol>
		<li>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 &#176;C for 12-16 h until the bacterial concentration is about 1 x 109 cells/mL (as indicated by OD600 = 1).</li>
		<li>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 &#176;C anaerobically for 24 h until the bacterial concentration is about 1 x 109 cells/mL (as indicated by OD600 = 1).</li>
	</ol>
	</li>
</ol>
2. H2S detection assay
<ol>
	<li>Hydrogen sulfide production test
	<ol>
		<li>Mix 100 &#181;L of bacterial culture with 100 &#181;L 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, and 40 mM L-cysteine) in the 96-well microtiter plates and culture for 20 min at 37 &#176;C. For each bacterial strain, perform the analysis in triplicate.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Sensitivity of the method
	<ol>
		<li>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 solution 14.</li>
		<li>Determine the presence of HS&#8722;/S2&#8722; 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 (++++++).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Thompson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+group+of+hydrogen+sulphide+producing+bacteria.">The group of hydrogen sulphide producing bacteria.</a> Journal of Medical Research. 42 (184), 383-389 (1921).</li><li>Ono, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cysteine+hydropersulfide+inactivates+&#946;-lactam+antibiotics+with+formation+of+ring-opened+carbothioic+s-acids+in+bacteria.">Cysteine hydropersulfide inactivates &#946;-lactam antibiotics with formation of ring-opened carbothioic s-acids in bacteria.</a> ACS Chemical Biology. 16 (4), 731-739 (2021).</li><li>Mironov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+H2S-mediated+protection+against+oxidative+stress+in+Escherichia+coli.">Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (23), 6022-6027 (2017).</li><li>Shen, Y., Shen, Z., Luo, S., Guo, W., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cardioprotective+effects+of+hydrogen+sulfide+in+heart+diseases:+From+molecular+mechanisms+to+therapeutic+potential.">The cardioprotective effects of hydrogen sulfide in heart diseases: From molecular mechanisms to therapeutic potential.</a> Oxidative Medicine and Cellular Longevity. 2015, 925167 (2015).</li><li>Salloum, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide+and+cardioprotection-mechanistic+insights+and+clinical+translatability.">Hydrogen sulfide and cardioprotection-mechanistic insights and clinical translatability.</a> Pharmacology &amp; Therapeutics. 152, 11-17 (2015).</li><li>Wallace, J. L., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide-based+therapeutics:+Exploiting+a+unique+but+ubiquitous+gasotransmitter.">Hydrogen sulfide-based therapeutics: Exploiting a unique but ubiquitous gasotransmitter.</a> Nature Reviews. Drug Discovery. 14 (5), 329-345 (2015).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hydrogen+sulfide+in+ischemia-reperfusion+injury.">Role of hydrogen sulfide in ischemia-reperfusion injury.</a> Oxidative Medicine and Cellular Longevity. , 186908 (2015).</li><li>Truong, D. H., Eghbal, M. A., Hindmarsh, W., Roth, S. H., O'Brien, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+hydrogen+sulfide+toxicity.">Molecular mechanisms of hydrogen sulfide toxicity.</a> Drug Metabolism Reviews. 38 (4), 733-744 (2006).</li><li>Shatalin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+bacterial+H2S+biogenesis+targeting+antibiotic+resistance+and+tolerance.">Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance.</a> Science. 372 (6547), 1169-1175 (2021).</li><li>Fr&#225;vega, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+Typhimurium+exhibits+fluoroquinolone+resistance+mediated+by+the+accumulation+of+the+antioxidant+molecule+H2S+in+a+CysK-dependent+manner.">Salmonella Typhimurium exhibits fluoroquinolone resistance mediated by the accumulation of the antioxidant molecule H2S in a CysK-dependent manner.</a> The Journal of Antimicrobial Chemotherapy. 71 (12), 3409-3415 (2016).</li><li>Luhachack, L., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gasotransmitters:+An+innate+defense+against+antibiotics.">Bacterial gasotransmitters: An innate defense against antibiotics.</a> Current Opinion in Microbiology. 21, 13-17 (2014).</li><li>Yoshida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide+production+from+cysteine+and+homocysteine+by+periodontal+and+oral+bacteria.">Hydrogen sulfide production from cysteine and homocysteine by periodontal and oral bacteria.</a> Journal of Periodontology. 80 (11), 1845-1851 (2009).</li><li>Basic, A., Blomqvist, S., Carl&#233;n, A., Dahl&#233;n, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+bacterial+hydrogen+sulfide+production+in+vitro.">Estimation of bacterial hydrogen sulfide production in vitro.</a> Journal of Oral Microbiology. 7, 28166 (2015).</li><li>Rosolina, S. M., Carpenter, T. S., Xue, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bismuth-based,+disposable+sensor+for+the+detection+of+hydrogen+sulfide+gas.">Bismuth-based, disposable sensor for the detection of hydrogen sulfide gas.</a> Analytical Chemistry. 88 (3), 1553-1558 (2016).</li><li>Barton, L. L., Fauque, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry,+physiology+and+biotechnology+of+sulfate-reducing+bacteria.">Biochemistry, physiology and biotechnology of sulfate-reducing bacteria.</a> Advances in Applied Microbiology. 68, 41-98 (2009).</li><li>Shatalin, K., Shatalina, E., Mironov, A., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2S:+A+universal+defense+against+antibiotics+in+bacteria.">H2S: A universal defense against antibiotics in bacteria.</a> Science. 334 (6058), 986-990 (2011).</li><li>Schnabel, B., Caplin, J. L., Cooper, I. 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 authors declare no conflicts of interest.

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><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>&lt;em&gt;Enterococcus faecalis&amp;nbsp;&lt;/em&gt;</td><td>ATCC&amp;nbsp;</td><td>19433</td><td></td></tr><tr><td>&lt;em&gt;Fusobacterium&amp;nbsp;nucleatum&amp;nbsp;&lt;/em&gt;</td><td>ATCC&amp;nbsp;</td><td>25586</td><td></td></tr><tr><td>&lt;em&gt;Klebsiella pneumoniae&amp;nbsp;&lt;/em&gt;</td><td>ATCC&amp;nbsp;</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>&lt;em&gt;Proteus&amp;nbsp;vuigaris&amp;nbsp;&lt;/em&gt;</td><td>CMCC&amp;nbsp;</td><td>49027</td><td></td></tr><tr><td>&lt;em&gt;Salmonella paratyphi&lt;/em&gt; A</td><td>CMCC</td><td>50001</td><td></td></tr><tr><td>&lt;em&gt;Salmonella paratyphi&lt;/em&gt; B</td><td>CMCC</td><td>50094</td><td></td></tr><tr><td>Staphylococcus&amp;nbsp;aureus&amp;nbsp;</td><td>ATCC&amp;nbsp;</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...

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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 ...

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3.5K Views.  China Pharmaceutical University. 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.

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

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Chemistry

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging

Source: Terzi, A. et al., <a href="https://app.jove.com/v/62165">ROS Live Cell Imaging During Neuronal Development.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates a technique to detect real-time hydrogen peroxide levels in retinal ganglion cells using the roGFP2-Orp1 biosensor and live cell imaging techniques. It outlines the steps involved in cell preparation, fluorescence imaging, and the quantification of hydrogen peroxide levels.

1. In vitro&#160;reactive oxygen species (ROS) imaging of cultured retinal ganglion cell (RGC) neurons

	On the day of imaging (typically 6-24 h after ...

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

Summary

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Colorimetric Detection of Bacteria Using Litmus Test

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging