Sulfide Indole Motility (SIM) Medium: Principle, Procedure, and Results 

Sulfide Indole Motility (SIM) medium is a differential bacterial growth medium for detecting three different characteristics of an organism; 

  1. Sulfur reduction and (H2S) sulfide production 
  2. Indole production and
  3. Motility of a bacteria 
sulfur-indole-motility (SIM) agar
sulfur-indole-motility (SIM) agar

Sulfur reduction test is useful in differentiating enteric organisms. SIM agar is more sensitive than either TSI or KIA  in detecting H2S production. The indole test is one of the IMViC series of tests.  The motility test is useful for testing a wide variety of organisms.  SIM test is useful for differentiating members of Enterobacteriaceae, primarily for differentiating Salmonella and Shigella.

Left: Kligler’s iron agar (KIA) and Right: Sulfide indole motility (SIM) inoculated with weak H2S-producing organism (Salmonella Typhi). In KIA, blackening is seen in the middle of the tube at the interface of the slant and butt, but in SIM, diffuse delicate blackening is seen across the entire media. 


SIM medium contains a pancreatic digest of casein (casein peptone), a peptic digest of animal tissue (meat peptone), ferrous ammonium sulfate as an iron source, and sodium thiosulfate. Peptone contains amino acids, including tryptophan.

Ingredients Amount (gram/liter)
Casein peptone  20 g
Meat peptone  6 g
Sodium thiosulfate  0.3 g
Ferric ammonium citrate  0.2 g
Agar  3.5 g
Demineralized water  1000 mL
pH 7.3 ± 0.2 at 25°C.   

Principle of Sulfide Indole Motility (SIM) medium

Test organism is inoculated onto semisolid medium sulfide-indole-motility agar and is tested for the production of hydrogen sulfide, indole, and motility of the organism. 

Hydrogen sulfide-producing organisms produce H2S gas that reacts with iron salts, ferrous sulfate, and ferric ammonium citrate in SIM, producing a black precipitate. 

Indole-producing organisms break down tryptophan present in the SIM agar using the tryptophanase enzyme and produce indole. Indole production is detected by Kovac’s or Ehrlich’s reagent, which contains 4 (p)-dimethylamino-benzaldehyde. This reacts with the indole to produce rosindole dye, a red-colored compound. 

Nonmotile organisms grow only on the stab line, and the surrounding medium remains clear. Motile organisms move out of the stab line and make the entire tube appear turbid. (taylorsmithconsulting)  

Procedure of Sulfide Indole Motility Medium

Stab line in SIM agar
  1. Label SIM medium with the name of the organisms or lab identification number (whichever is applicable).
  2. Using a sterile inoculating needle, touch the center of a well-isolated colony. The inoculum should be substantial but does not need to be particularly heavy.
  3. Aseptically stab once to a depth of 1/3 to 1/4 inch in the middle of the corresponding tube.
  4. Withdraw the needle and sterilize it by incinerating it in a bunsen burner or micro incinerator.
  5. Aseptically inoculate the remaining tubes in the same way.
  6. Incubate aerobically at 35 to 37°C for 18 to 24 hours. 


Observe for motility and formation of black precipitate before adding a reagent to check for indole production. 


Check for the radiating (fuzzy) growth from the stab line or hold a paper with a print behind the tubes and try to read it through the tubes.

  • Motile organisms will spread out into the medium from the site of inoculation.
  • Nonmotile organisms remain at the site of inoculation 

Find more about motility test medium

Sulfide Production 

Result of the indole test using sulfur-indole-motility medium and Proteus vulgaris after a 24-hour incubation at 37
Result of the indole test using sulfur-indole-motility medium and Proteus vulgaris after a 24-hour incubation at 37°C

For sulfur reduction, observe the location of any black color. Blackening usually begins at the inoculation line, and black color may appear throughout the medium or as a black precipitate in the butt.

Indole Test 

Result of the indole test using sulfur-indole-motility medium. A-positive, B-negative
Result of the indole test using sulfur-indole-motility medium. The positive indole result is given by Escherichia coli (left)

Add a few drops of Kovac’s or Ehrlich’s reagent. 

  • Positive indole test: Appearance of the red ring after addition of reagent 
  • Negative indole test: No color change after addition of reagent 

Read more about Indole Test

SIM test results of selected bacteria 

Name of the Organism Sulfide  Indole Motility 
Escherichia coli No H2S Positive  Motile (Few strains are nonmotile) 
Vibrio cholerae  No H2S Positive  Motile
Citrobacter freundii Variable Positive (minority strains give negative results) Motile
Vibrio parahaemolyticus  No H2S Positive Motile
Enterobacter spp No H2S Negative Motile
Proteus vulgaris H2S  producer  +ve Motile
Proteus mirabilis H2S  producer  -ve  Motile
Klebsiella pneumoniae No H2S Negative  Nonmotile
Yersinia enterocolitica No H2S Variable  Motile
Morganella morganii No H2S Positive Motile
Providencia spp. No H2S Variable  Motile
Shigella dysenteriae No H2S Variable Nonmotile
Shigella flexneri No H2S Variable Nonmotile
Shigella boydii No H2S Variable Nonmotile
Shigella sonnei No H2S Negative  Nonmotile
Salmonella Paratyphi A Negative (<13% isolates are H2S producers) Negative  Motile
Salmonella Paratyphi B Positive  Negative  Motile
Salmonella Paratyphi C Positive (minority strains are H2S nonproducers)  Negative  Motile
Salmonella Typhi Positive (weak) Negative  Motile
Other Salmonella serovars  Positive (minority strains are H2S nonproducers)  Negative  Motile

*Shigella sonnei is indole negative, and other shigellae give variable indole reactions. Salmonella serovars are indole negative. Klebsiella oxytoca is indole positive. 

Similar Types of Biochemical Media 

  • Motility-indole-ornithine agar
  • Motility indole urease medium 

References and further readings

  1. Alternative Methods for Determining Indole Production using SIM medium images. ASM Education
  2. Sulfur, Indole, Motility (SIM) Media. University of Wyoming. Retrieved 8 October 2022, from
  3. SIM Medium. Microbugz. Retrieved 8 October 2022, from

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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