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Short answer: SIM medium is a semisolid, multitest agar in a deep tube that shows three reactions from one stab inoculation: hydrogen sulfide production, indole production and motility. Read motility and hydrogen sulfide first, then add Kovac’s reagent to test for indole. This page is general laboratory information, not medical advice or a diagnostic procedure.
What SIM medium is
ASM’s Microbe Library describes SIM medium as “a multitest agar used to test for indole production while simultaneously determining other characteristics of the bacterium,” available both as a premixed powder and as premade deep tubes. Neogen’s technical specification sheet for SIM Medium (record FS-TSS-0336, rev 1.0, effective 2023-08-28) gives the intended use as differentiating microorganisms on the basis of hydrogen sulfide production, indole production and motility “in a laboratory setting”, and states it is not intended for diagnosing disease in humans.
The same sheet says these three tests are useful in identifying Enterobacteriaceae, especially Salmonella and Shigella. It traces the medium’s lineage to semi-solid agar for motility (Tittsler and Sandholzer, 1936), to the combination of motility, hydrogen sulfide and fermentation tests for Salmonella and Shigella (Sulkin and Willett, 1940) and to Greene and colleagues (1951).
The name is easy to confuse: this is a bacteriology culture medium, unrelated to SIMS mass spectrometry or the Sims uterine curette.
How the three reactions are read
The order matters. Neogen instructs: “Add Kovac’s Reagent after determining motility and H2S production”. Both sources describe inoculating from an isolated colony by stabbing about two-thirds of the way into the medium.
1. Motility (the motility test medium reaction)
Motility shows as turbidity of the medium or growth extending away from the stab line. For a stand-alone semisolid medium used only for this reaction, see motility test medium.
2. Hydrogen sulfide (the hydrogen sulfide production test)
Hydrogen sulfide production shows as blackening along the stab line. The same reaction is read on iron-containing slants; see triple sugar iron agar results and lysine iron agar for how it appears there.
3. Indole
Indole is produced from the tryptophan in the medium. ASM gives the chemistry: the enzyme tryptophanase converts tryptophan and water to indole, pyruvic acid and ammonia, and indole reacts with p-dimethylaminobenzaldehyde under acid conditions to form a red dye (rosindole). After the first two reactions are recorded, Kovac’s reagent is added to the top of the tube: a red color in the reagent layer is positive. ASM reports a positive within seconds and describes yellow or slightly cloudy as negative; Neogen lists 3-4 drops of reagent and ASM lists 5.
Expected pattern with quality control strains
Neogen’s quality control table uses Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028 and Shigella flexneri ATCC 12022, incubated at 35 ± 2 °C for 18-24 hours. Per that sheet, E. coli is hydrogen sulfide negative and indole positive, S. typhimurium is hydrogen sulfide positive, and motility reads positive, positive and negative for E. coli, Salmonella and Shigella respectively.
SIM is not one fixed recipe: two published formulations
The practical difference for a lab is that two named sources specify the medium differently, so the incubation window and the formulation belong to the specification you buy or prepare.
| Item (per litre) | Neogen SIM Medium (FS-TSS-0336) | ASM Microbe Library protocol |
|---|---|---|
| Protein source | Enzymatic digest of casein 20.0 g; enzymatic digest of animal tissue 6.1 g | Peptone 30.0 g; beef extract 3.0 g |
| Iron source | Ferric ammonium citrate 0.2 g | Ferrous ammonium sulfate 0.2 g |
| Sodium thiosulfate | 0.2 g | 0.025 g |
| Agar | 3.5 g | 3.0 g |
| Final pH | 7.3 ± 0.2 at 25 °C | 7.3 |
| Incubation | 35 ± 2 °C, 18-24 hours | 35 ± 2 °C, 24-48 hours or until growth is evident |
| Kovac’s reagent | 3-4 drops | 5 drops |
Both sources autoclave at 121 °C for 15 minutes. Neogen’s dehydrated medium is prepared at 30 g per litre; ASM fills 4.0-5.0 mL per 16 mm tube, cools the tubes upright to form a deep, and keeps prepared tubes at 4-8 °C “for several months”.
SIM compared with related multitest media
ASM lists SIM alongside MIO (motility-indole-ornithine) and MIL as multitest agars. SIM reports hydrogen sulfide and indole as well as motility. For the plates that supply isolated colonies, see what an agar plate is.
What to check on a SIM medium specification sheet
The Neogen sheet shows what a supplier specification states. When reviewing any SIM medium, whether dehydrated powder or prepared tubes, check that the document gives:
- The intended use, including whether it is laboratory-only rather than diagnostic.
- The typical formulation, including agar, iron source and thiosulfate, so you can compare it with the protocol your lab follows.
- The incubation temperature and time the quality control results were set for.
- Quality control strains and their expected hydrogen sulfide, indole and motility results.
- Storage conditions: Neogen states dehydrated medium at 2-30 °C, away from direct sunlight, container tightly closed.
- Discard criteria: Neogen says to discard dehydrated medium that is not free flowing or has changed color. It describes dehydrated medium as homogeneous, free flowing and beige, and prepared medium as light amber, clear to slightly hazy.
- Stated limitations (below).
Common misunderstandings
- “The three results identify the organism.” Neogen states the reactions are not sufficient to speciate organisms; additional biochemical and serological tests are required for confirmation.
- “Add the reagent first, or at any time.” Kovac’s reagent goes in after motility and hydrogen sulfide are read.
- “Any inoculum works.” Neogen advises avoiding inoculum from a liquid or broth suspension because growth initiation will be delayed, and notes some strains may grow poorly or fail to grow.
- “Ehrlich’s and Kovac’s reagents are interchangeable.” ASM says Ehrlich’s reagent is more sensitive and is recommended for groups that make little indole, such as nonfermentative bacilli and anaerobes.
FAQ
What is SIM medium used for?
It differentiates bacteria by hydrogen sulfide production, indole production and motility in a single deep tube, in a laboratory setting. It is part of the work-up of Enterobacteriaceae.
Which reaction is read first?
Motility and hydrogen sulfide are read first. Kovac’s reagent for indole is added afterward.
What does a red ring mean?
After Kovac’s reagent is added, red in the reagent layer means the organism produced indole from tryptophan (indole positive).
Is SIM medium a motility test medium?
It includes a motility reading, shown by turbidity or growth spreading from the stab line, but it also reports hydrogen sulfide and indole. A medium that reports motility alone is a different product.
Which reaction is the hydrogen sulfide production test?
Blackening along the stab line. It is read before the indole reagent is added.
Sources
- Neogen, Technical Specification Sheet “SIM Medium”, FS-TSS-0336 rev 1.0, effective 2023-08-28. https://media.neogen.com/m/5cd79ab7cac7d786. Checked 2026-11-01.
- MacWilliams MP. “Indole Test Protocol”, ASM Microbe Library, created 8 December 2009. https://asm.org/getcontentasset/e2c55ea1-d5c0-43e1-bfaf-66d2715c1e21/dfc3d011-8f63-43f6-9ed8-4b444333a1d0/indole-test-protocol.pdf?language=en-US. Checked 2026-11-01.
