How do culture media reveal hydrogen sulfide (H₂S)? Every one of them needs the same two things: a sulfur source and an iron (or lead) indicator. This MedTech Essentials guide covers how the system works, which media use it, and the organisms you must memorize for the MTLE and other MedTech exams.
The detection principle
A sulfur source, most commonly sodium thiosulfate or sulfur-containing amino acids such as cysteine and methionine, is reduced by bacterial enzymes under acidic conditions. This releases colorless H₂S gas. An iron salt then reacts with the gas to form insoluble, black iron sulfide (FeS).
Sulfur sources and iron indicators
- Sodium thiosulfate is the usual sulfur source in TSI, KIA, LIA, SS, HE, XLD, TCBS and SIM. Bacteria reduce it to release H₂S.
- Ferrous sulfate (Fe²⁺) reacts directly with H₂S to form FeS.
- Ferrous ammonium sulfate is a double salt that also supplies Fe²⁺. It is used in SIM, where peptonized iron is the alternative indicator.
- Ferric sulfate compounds supply Fe³⁺, which must be reduced to Fe²⁺ before FeS forms.
- Ferric citrate and ferric ammonium citrate are soluble Fe³⁺ sources used in SS, HE, XLD, TCBS, LIA and TSI.
H₂S detection media at a glance
| Culture medium | Sulfur source | H₂S indicator | End product / visual result |
|---|---|---|---|
| Triple Sugar Iron Agar (TSI) | Sodium thiosulfate | Ferrous sulfate / ferric ammonium citrate | FeS, blackening of the agar butt |
| Kligler’s Iron Agar (KIA) | Sodium thiosulfate | Ferrous sulfate / ferric ammonium sulfate | FeS, blackening of the agar butt |
| Lysine Iron Agar (LIA) | Sodium thiosulfate | Ferric ammonium citrate | FeS, black precipitate in the deep/butt |
| Salmonella-Shigella Agar (SS) | Sodium thiosulfate | Ferric citrate / ferric ammonium citrate | FeS*, black-centered colonies |
| Hektoen Enteric Agar (HE) | Sodium thiosulfate | Ferric ammonium citrate | FeS*, black precipitate / black-centered colonies |
| Xylose-Lysine-Deoxycholate Agar (XLD) | Sodium thiosulfate | Ferric ammonium citrate | FeS, black-centered colonies |
| Bismuth Sulfite Agar (BS) | Peptones (sulfur-containing amino acids) | Ferrous sulfate | FeS, brown-to-black colonies with a metallic sheen |
| Sulfide-Indole-Motility Medium (SIM) | Sodium thiosulfate (plus sulfur-containing amino acids in peptones) | Ferrous ammonium sulfate or peptonized iron | FeS, black precipitate along the inoculation line |
| TCBS Agar | Sodium thiosulfate | Ferric citrate | FeS*, black-centered colonies (rare H₂S-positive vibrios) |
| Lead Acetate Paper Strip | Sodium thiosulfate / cysteine / peptone (in KIA or TSI) | Lead acetate | Lead sulfide (PbS), blackening or browning of the paper strip |
*The black product is FeS, which is ferrous sulfide. Ferric iron (Fe³⁺) from ferric citrate or ferric ammonium citrate is reduced to Fe²⁺ before FeS forms.
TSI versus KIA
Both detect H₂S with sodium thiosulfate and an iron salt. The key difference for exams is the sugars: TSI = glucose + lactose + sucrose, while KIA = glucose + lactose only.
Bismuth sulfite agar
BS agar contains ferrous sulfate as the H₂S indicator. Bismuth sulfite and brilliant green are the selective agents that suppress most other bacteria while letting Salmonella grow. The sulfite here belongs to the selective bismuth system, not to a thiosulfate-type sulfur source. Salmonella colonies turn brown to black with a metallic sheen.
SIM medium
SIM tests sulfide production, indole production and motility in one tube. Sodium thiosulfate supplies the sulfur and an iron salt (ferrous ammonium sulfate or peptonized iron) acts as the indicator. A black precipitate along the stab line means H₂S is positive.
Lead acetate and other methods
Lead acetate paper strips are suspended over TSI or KIA to detect tiny amounts of H₂S gas, forming black or brown lead sulfide (PbS). They are not a plated agar medium. For board review, remember the sensitivity order:
Deoxycholate and citrate act mainly as selective agents in media such as XLD and SS, not as H₂S indicators.
The TSI black-butt rule
H₂S production needs an acidic environment. So when the butt is black, the blackening hides the yellow underneath, but you still record it as an acid (yellow) butt (A) and report H₂S as positive.
H₂S-positive organisms: SPACEd
- S: Salmonella spp. (except S. Paratyphi A)
- P: Proteus spp. (P. mirabilis, P. vulgaris)
- A: Arizona, now Salmonella enterica subsp. arizonae
- C: Citrobacter freundii
- E: Edwardsiella tarda
Gram-positive exception: Erysipelothrix rhusiopathiae is a catalase-negative, non-spore-forming, Gram-positive bacillus that produces H₂S in TSI.
References
Bailey & Scott’s Diagnostic Microbiology, 15th and 16th eds.
Delost, Introduction to Diagnostic Microbiology for the Laboratory Sciences, 2nd ed.
Mahon, Textbook of Diagnostic Microbiology, 6th ed.
FDA Bacteriological Analytical Manual (BAM) media index and manufacturer media instructions.
Supplemental review material only. Always verify against your updated textbooks and official references.
Quick check: 6 questions
Try answering in your head first, then tap Show answer.
1. What is the most common sulfur source in TSI, KIA, LIA, SS, HE, XLD, TCBS and SIM?
- Cysteine only
- Sodium thiosulfate
- Ferrous sulfate
- Bismuth sulfite
Show answer
2. Which sugar is in TSI but NOT in KIA?
- Glucose
- Lactose
- Sucrose
- Xylose
Show answer
3. The butt of a TSI tube is completely black. How do you record the butt reaction?
- Alkaline (red) butt, H₂S negative
- Acid (yellow) butt, H₂S positive
- No change, H₂S negative
- Cannot be interpreted
Show answer
4. Which method is the MOST sensitive for detecting H₂S?
- LIA
- TSI
- SIM
- Lead acetate paper strip
Show answer
5. Which Salmonella is the exception to the H₂S-positive rule in the SPACEd mnemonic?
- S. Typhimurium
- S. Paratyphi A
- S. enterica subsp. arizonae
- S. Enteritidis
Show answer
6. Which Gram-positive bacillus produces H₂S in TSI?
- Listeria monocytogenes
- Erysipelothrix rhusiopathiae
- Bacillus cereus
- Corynebacterium diphtheriae
Show answer
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