Sulfur Reduction Test: Principle, Procedure, Results, Uses

Elemental sulfur and sulfur-containing compounds – both organic and inorganic compounds – can be metabolically reduced by some microorganisms to obtain metabolic energy. Such microorganisms are called sulfur-reducing microorganisms. Several Archaea and aerobic, facultative, and anaerobic bacterial species are able to reduce sulfur into hydrogen sulfide (H2S). 

Sulfur-reducing ability is not found in all species; hence, it is studied to differentiate bacteria. The biochemical test used to assess the ability of microorganisms to reduce sulfur present in the sulfur-containing compound is called the ‘Sulfur Reduction Test’. The end byproduct of sulfur reduction is hydrogen sulfide gas and this gas is detected by the indicator during the test, so this test is also known as the ‘Hydrogen Sulfide Test’. 

It is an important diagnostic tool in medical and non-medical microbiology laboratories. It is commonly used to detect coliforms in water, identify fecal pathogens, differentiate enteric pathogenic bacteria, and characterize other pathogenic or non-pathogenic bacterial isolates.   

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Objectives of Sulfur Reduction Test

  • To assess the ability of bacteria to reduce sulfur compounds.
  • To detect the ability of bacteria to produce hydrogen sulfide (H2S) gas.
  • To biochemically differentiate bacteria for presumptive identification.

Principle of Sulfur Reduction Test

Sulfur-reducing bacteria can metabolize sulfur-containing compounds present in the culture medium and reduce the sulfur to hydrogen sulfide. The released hydrogen sulfide reacts with the indicator (mostly ferric ions or lead acetate) to form black-colored insoluble components (ferrous sulfide or lead sulfide) which turn the whole medium black. Thus, the development of black color in the medium after the incubation period indicates that the test organism is capable of reducing sulfur.

Requirements for Sulfur Reduction Test

1. Culture Medium

A wide range of sulfur compounds containing culture media are available and most of them are suitable for performing sulfur reduction tests. However, SIM (Sulfide Indole Motility) medium, KIA (Kligler’s Iron Agar), TSI (Triple Sugar Iron) Agar Medium, and Lead Acetate (LA) Agar are commonly used. This entire medium contains sodium thiosulfate as the sulfur source. 

Among these, the SIM medium is the commonly preferred medium to perform this test. SIM medium contains sodium thiosulfate as a sulfur source and peptonized iron as an indicator. 

Composition of SIM medium per 1000 mL

HM Peptone B (Beef Extract) – 3.00 grams

Peptone – 30.0 grams

Peptonized Iron – 0.020 grams

Sodium thiosulfate – 0.025 grams

Agar – 3.00 grams

Final pH – 7.3 ±0.2 at 25°C

(Reference: SIM Medium (

Preparation of SIM Medium

  • Measure the appropriate amount of SIM agar powder (36.23 grams per 1000 mL) and mix it with the appropriate volume of distilled water in a conical flask or glass bottle. 
  • Stir well using a magnetic stirrer or manually and heat to boiling so that the agar dissolves completely in water. 
  • Dispense about 5 mL of the medium in each test tube and loosely put on the cap or cotton plug the opening.  
  • Autoclave the tubes at 121°C and 15 lbs pressure for 15 minutes.
  •  Let it cool and solidify at an upright position (forming butt only).

2. Reagents

  • No additional reagents are required while using SIM or other sulfur-containing agar. 
  • Lead acetate paper is needed while performing the lead acetate paper method. 

3. Equipment

Test Tubes
Weighing Machine
Inoculating wire
Bunsen burnerAutoclave

4. Test Organism (Sample Bacteria)

5. Control Organisms

Proteus mirabilis ATCC 29906

Shigella flexneri ATCC 12022

Procedure of Sulfur Reduction Test

1. SIM Agar Method (Tube Method)

  • Using a sterile inoculating wire, touch several colonies of sample bacteria from fresh (18 to 24 hours) old culture. 
  • Stab the SIM medium in the tube halfway (up to 3 to 5 mm above the base of the tube). 
  • Incubate the tube aerobically (with a loose cap) at 35±2°C for about 24 hours.
  • Observe the development of black color on the medium.

2. Lead Acetate Paper Method

  • Using a sterile inoculating loop/wire, touch several colonies of sample bacteria from fresh (18 to 24 hours) old culture and inoculate the nutrient broth or peptone water medium. 
  • Position a lead acetate paper strip so that one end hangs just over the medium and the other end is secured at the tube’s neck with a cotton plug or screw cap.
  • Incubate the tube aerobically at 35±2°C and observe for blackening of the paper strip after 24 hours.

Result and Interpretation of Sulfur Reduction Test

  • A positive result is indicated by blackening the medium or turning the lead acetate paper into a black color. 
  • A negative result is indicated by no blackening of the medium or no change in the color of the lead acetate paper.
Sulfur Reduction Test
Sulfur Reduction Test

Quality Control

  • Proteus mirabilis ATCC 29906 gives a positive result and hence turns the SIM medium and lead acetate paper to a black color.
  • Shigella flexneri ATCC 12022 gives a negative result and hence doesn’t turn the SIM medium and lead acetate paper into black color. 

Sulfur Reduction Test Results of Some Common Bacteria 

Sulfur Reducing BacteriaSulfur Non-reducing Bacteria
Proteus spp.,
Citrobacter spp.,
Salmonella spp.,
Staphylococcus saprophyticus,
Campylobacter spp.,
Klebsiella pneumoniae,
Shigella spp.,
Staphylococcus aureus,
E. coli,
Pseudomonas aeruginosa,
Neisseria gonorrhoeae,
Vibrio cholerae,
Yersinia pestis,
Morganela morgannii


  • Don’t use an inoculating loop to inoculate the SIM tube. 
  • Look for cracking of the medium in test tubes before inoculation.
  • Incubate aerobically – with a loose cap. 
  • The selection of an appropriate medium is necessary. 
  • Avoid lead acetate paper touching the medium as lead acetate show an inhibitory effect. 

Applications of Sulfur Reduction Test

  • To differentiate and presumptively identify the member of Enterobacteriaceae
  • To differentiate Salmonella spp. (sulfur reducing) from Shigella spp. (sulfur non-reducing) and Erysipelothrix spp. (sulfur reducing) from Lactobacillus spp.  (sulfur non-reducing)
  • To rapidly detect the presence of fecal coliforms in water.

Limitations of Sulfur Reduction Test

  • It is not a confirmatory test and requires other biochemical test results to confirm bacterial identification. 
  • Sucrose in the medium may inhibit H2S production. 
  • The use of inoculum from a liquid medium may require a longer incubation period. 


  1. Leber, Amy L., editor in chief. (2016). Clinical microbiology procedures handbook (Fourth edition) . Washington, DC : ASM Press 1752 N St., N.W., [2016] doi:10.1128/9781555818814.ch3.17.25
  2. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.) P. 183 – 185. St. Louis, Missouri: Elsevier
  3. Thakur, Swagata & Anokhe, Archana & Kalia, Vinay. (2021). Biochemical Test for Detecting Hydrogen Sulphide (H2S) Producing Bacteria. 2. 53-56. (PDF) Biochemical Test for Detecting Hydrogen Sulphide (H2S) Producing Bacteria (
  9. Hydrogen sulfide (H2S) production test – Virtual Microbiology Lab Simulator Software (
  10. Hydrogen Sulfide Test – Principle, Procedure, Uses and Interpretation (
  11. Hydrogen Sulphide (H2S) Production Test – BiochemGems
  12. Hydrogen Sulfide Test – Procedure, Uses and Interpretation –
  13. Hydrogen Sulfide (H2S) Test Principle, Procedure, Result (
  14. Common Biochemical Tests in Microbiology: Hydrogen Sulphide Production Test – Labmonk
  15. Hydrogen Sulfide Test: Principle, Procedure, Uses and Interpretation (
  16. Hydrogen Sulfide Test: Principle, Procedure And Results Interpretation – BIOCHEMINSIDER

About Author

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Prashant Dahal

Prashant Dahal completed his bachelor’s degree (B.Sc.) Microbiology from Sunsari Technical College, affiliated with Tribhuvan University. He is interested in topics related to Antimicrobial resistance, the mechanism of resistance development, Infectious diseases (Pneumonia, tuberculosis, HIV, malaria, dengue), Host-pathogen interaction, Actinomycetes, fungal metabolites, and phytochemicals as novel sources of antimicrobials and Vaccines.

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