H2S Production Test – Its Principle, Methods, and Interpretation


Several bacteria and archaea can use their metabolism to convert sulfur-containing compounds to hydrogen sulfide and gain energy in the process. These sulfur-containing molecules might be inorganic, such as sulfate, sulfite, thiosulfate, or tetrathionates, organic, such as sulfur amino acids and proteins, or even elemental sulfur itself.

The sulfur in these compounds is reduced enzymatically and released as hydrogen sulfide (H2S) gas. Hydrogen sulfide-producing bacteria are a diverse group of aerobic, facultative, and anaerobic bacterial species that have the ability to create H2S gas.

H2S gas is a colorless, poisonous, acidic, and combustible reduced gas form of sulfur with a distinctive rotten egg odor. Microbial H2S generation is identified by allowing it to react with ferric ions or lead acetate to form black-colored ferrous sulfide or lead sulfide. Sodium thiosulfate is the primary sulfur source in the H2S test.


H2S producing bacteria can metabolize the sulfur-containing chemicals present in the culture media (mainly sodium thiosulfate) and decrease them, producing H2S gas. The generated H2S gas subsequently combines with ferric ions or lead acetate to make water-insoluble black ferrous sulfide or lead sulfide. This insoluble black-colored substance renders the culture media black, suggesting that the H2S production test was successful.

Chemical reactions involved in H2S production

The production of H2S by microorganisms involves specific chemical reactions. One of the primary pathways for H2S production is the reduction of inorganic sulfur compounds, such as sulfates (SO42-) or thiosulfates (S2O32-), by microbial enzymes. This reduction process results in the release of H2S gas.

The overall reaction can be represented as follows:

SO42- or S2O32- + Organic matter + Microbial enzymes → H2S + Byproducts

It’s important to note that the exact reactions and pathways involved may vary depending on the specific microorganism and environmental conditions.

Microorganisms that produce H2S

Several types of microorganisms have the ability to produce H2S. This includes various species of bacteria, such as Salmonella, Proteus, Citrobacter, and Enterobacter. These bacteria typically inhabit environments rich in organic matter, such as soil, water, and the intestinal tracts of animals. Additionally, certain yeasts and fungi can also produce H2S under specific conditions.

Methods of H2S Production test

  • SIM (Sulfide Indole Motility) test
  • Kligler Iron Agar (KIA) test
  • Triple Sugar Iron (TSI) test
  • Lead Acetate paper test


Media: There are numerous culture media available for detecting hydrogen sulfide formation. Sulphide Indole Motility medium, Kligler’s Iron Agar, TSI (Triple Sugar Iron) Agar Medium, and Lead Acetate (LA) Agar have often been used for tube H2S tests.

Salmonella-Shigella Agar, Deoxycholate citrate agar, BS (Bismuth Sulfite) medium, XLD (Xylose-lysine deoxycholate) agar medium, and HE (Hektoen Enteric) Agar are often used for the petri plate method.

Equipment: petri plates, test tubes, weighing machine, Bunsen burner, incubator, inoculating wire and loop, autoclave, lead acetate paper

Test organisms: positive control- Proteus, negative control-shigella


SIM (Sulfide Indole Motility) test

  1. Touch a well-isolated colony from a fresh culture (18 to 24 hours old) of the test bacteria with a sterile inoculating wire.
  2. Inoculate the SIM medium tube by stabbing it more than halfway (up to 3 to 5 mm above the tube’s base) with the inoculating wire.
  3. Incubate the tube aerobically (with a loose cap) at 3t°C for roughly 24 hours.
  4. Examine the production of black-colored precipitate in the medium. 

Kligler Iron Agar (KIA) test or Triple Sugar Iron (TSI) test

  1. Using a sterile inoculating loop, inoculate a well-isolated colony from a fresh culture of the test bacterium.
  2. Spread the culture over the agar plate to obtain well-isolated colonies.
  3. Incubate aerobically at 37°C for roughly 24 hours.
  4. Examine the color of the developed colonies.

Lead acetate paper method

  1. Inoculate the nutrient broth tube with a well-isolated colony from a fresh culture of the test bacteria using a sterile inoculating touch.
  2. Place a lead acetate paper strip so that one end is just above the medium and the other end is fastened at the tube’s neck by the screw cap or cotton plug.
  3. After 24 hours, incubate the tube aerobically at 37°C and look for blackening of the paper strip.


SIM (Sulfide Indole Motility) test

Positive results show culture media blackening. (The entire media or the junction of slant and butt, or any part of butt or slant, may turn black)

There is no blackening of media in any place in case of negative results.

Figure 1: tube 1 shows the negative results while tube 2 shows the positive results (blackening) (McBenedict et al., 2018)

Kligler Iron Agar (KIA) test or Triple Sugar Iron (TSI) test

Positive results show colorless or colored colonies with a black center, as well as black colonies.

There are no black colonies and/or black-centered colonies in negative results.

Figure 2: figure A represents the negative results and figure B represents the blackening on media (positive results) (Hasan, 2021)

Lead acetate paper method

Lead acetate paper blackening (brownish-black color development) represents positive results and no blackening shows negative results.

Figure 3: figure represents the negative results on left lead acetate paper and positive results on right lead acetate paper (Toyotome et al., 2021)

Example of H2S Production Results

Bacteria that are positive for H2S production testBacteria that are negative for H2S production test
Salmonella typhiStaphylococcus aureus
Salmonella Para typhiStreptococcus pyogenes
Proteus vulgarisStreptococcus pneumoniae
Proteus mirabilisEnterococcus faecalis
Citrobacter freundiiPseudomonas aeruginosa
Escherichia coliAcinetobacter baumannii
Klebsiella pneumoniaeHaemophilus influenzae
Edwardsiella tardaNeisseria meningitidis
Shigella dysenteriaeNeisseria gonorrhoeae
Salmonella typhiVibrio cholerae


This test can be used :

  • As a presumptive test for bacterial identification.
  • Salmonella spp. (H2S test positive) and Shigella spp. (H2S test negative) are distinguished.
  • For Enterobacterales Characterization and Identification
  • To do fecal culture and pathogen characterization.
  • Rapid detection of fecal coliforms in water.


  • Because it is not a confirmatory test, other biochemical test results are required for complete identification of the medium.
  • Sucrose in the medium has the potential to suppress H2S generation.
  • SIM, NB, DCA, and other basic mediums may not support the growth of some fastidious organisms.
  • Lead acetate is the most sensitive to H2S detection, although it is hazardous to microorganisms.
  • Some bacteria require prolonged incubation, such as 3 days for Campylobacter spp. and even longer for other slow-growing bacteria.
  • Confusion while selecting test media.

Quality control

  • Calibration is required to assure the accuracy and dependability of the H2S concentration monitoring equipment. To guarantee that the equipment is working properly, it should be calibrated before and after each usage.
  • To ensure the accuracy and precision of the measurements, quality control samples should be included in the test. To check that the measurement is accurate and the instrument is working properly, a control sample with a known H2S content might be utilised.
  • To achieve consistency and accuracy in the H2S production test, standard operating procedures (SOPs) should be established and closely followed. through avoid errors, the SOP should include all phases, from sample collection through data analysis.
  • Personnel involved in the H2S production test should be appropriately trained and monitored to ensure proper testing. Personnel should be familiar with the test processes and carefully adhere to the SOPs.


  • HASAN, T. O. 2021. Genomic Diversity Analysis of Salmonella spp. from Broiler and Layer Flocks and their Feed and Water in Karbala, Iraq. University of Baghdad.
  • MCBENEDICT, B., WANKE, H., HANG’OMBE, B. & CHIMWAMUROMBE, P. J. A. I. M. 2018. Detection of Escherichia coli and harmful enteric bacterial pathogens in domestic hand-dug wells in the Cuvelai Etosha Basin of Namibia. 8, 297-313.
  • TOYOTOME, T., TAKINO, M., TAKAYA, M., YAHIRO, M. & KAMEI, K. J. J. O. F. 2021. Identification of volatile sulfur compounds produced by Schizophyllum commune. 7, 465.
  • Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
Ujala Shabbir
Ujala Shabbir

Ujala Shabbir is a microbiologist pursuing her MPhil in Microbiology at the University of Veterinary and Animal Sciences in Lahore. Her research is focused on the "preparation of calcium-conjugated FMDV vaccine and its comparative immunogenicity with other vaccine delivery systems".
She completed her Bachelor of Science degree in Applied Microbiology from the same university in 2021 with a CGPA of 3.72/4.
During her undergraduate studies, she took various microbiology-related courses and gained practical experience through internships.

Articles: 13

Newsletter Updates

Enter your email address below and subscribe to our newsletter

Leave a Reply

Your email address will not be published. Required fields are marked *