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Short answer
Triple sugar iron agar results are read from four things in a tube: the colour of the slant, the colour of the butt, any gas, and any black precipitate. Yellow means acid, red means alkaline, bubbles or cracking mean gas, and blackening means hydrogen sulfide (H2S). Read together, they show which of dextrose, lactose and sucrose an organism fermented and whether it produced H2S.
What triple sugar iron agar measures
Triple sugar iron (TSI) agar is a tubed differential medium used to determine carbohydrate fermentation and H2S production, and gas from carbohydrate metabolism can also be detected (American Society for Microbiology, TSI agar protocols page). Thermo Fisher’s Remel instructions for use (IFU 454982, revision of December 2011) describe it as a solid medium for qualitative differentiation of microorganisms on the basis of dextrose, lactose and sucrose fermentation and hydrogen sulfide production. The same IFU is labelled for laboratory use only, not for diagnosis.
The medium took shape in stages. According to the IFU, Russell described a dextrose and lactose medium in 1911, Krumwiede and Kohn added sucrose, Sulkin and Willett added ferrous sulfate in 1940, and Hajna developed the recent formulation by adding sucrose to Kligler iron agar.
How the slant and butt colours arise
The key to reading a tube is a ratio built into the formula: the dextrose concentration is one-tenth that of lactose and sucrose. The classical formulation listed in the Remel IFU contains, per litre, casein peptone 10.0 g, meat peptone 10.0 g, lactose 10.0 g, sucrose 10.0 g, dextrose 1.0 g, sodium chloride 5.0 g, sodium thiosulfate 0.2 g, ferric ammonium citrate 0.2 g, phenol red 25.0 mg and agar 13.0 g, at a final pH of 7.3 plus or minus 0.2 at 25 C. The IFU notes the formula is adjusted as required to meet performance standards.
Phenol red is the pH indicator, red when alkaline and yellow when acid. Ferric ammonium citrate and sodium thiosulfate are the H2S indicators, producing a black precipitate. The tubes are cooled on a slant so the medium forms a deep butt.
Because dextrose is scarce, an organism that ferments only dextrose makes a small amount of acid. In the slant, which is exposed to air, that acid oxidizes rapidly and the slant reverts to alkaline (red). In the butt, the absence of oxygen keeps the yellow acid reaction. If lactose or sucrose is also fermented, enough acid forms to keep both slant and butt yellow. This is why a tube can be yellow in the butt and red on the slant at the same time.
Reading the result: slant, butt, gas and H2S
Triple sugar iron agar test results at a glance
| Reported pattern (slant/butt) | What the Remel IFU says it indicates |
|---|---|
| ALK/A (red slant, yellow butt) | Dextrose only fermented |
| A/A (yellow slant, yellow butt) | Dextrose and/or sucrose and/or lactose fermented |
| ALK/ALK (red slant, red butt) | No sugar fermented |
Notation differs between sources. The Remel IFU writes acid as A and alkaline as ALK, while the Ohio State University microbiology lab manual on LibreTexts uses A for acid and K for alkaline, writes results as slant/butt, and adds H2S and gas afterwards only if observed. The meaning is the same: K/A and ALK/A describe the same tube.
Gas
The IFU counts gas (CO2 and H2) as positive when there are bubbles, cracking and displacement of the medium, or separation of the medium from the side or bottom of the tube.
Hydrogen sulfide
H2S is positive when the butt blackens throughout, when a black ring forms at the slant and butt junction, or when a black precipitate is visible. Heavy blackening can mask the butt colour, so a fully black butt does not by itself tell you whether the butt was acid.
Quality-control organisms and expected results
The Remel IFU gives expected results for three reference strains after 18 to 24 hours of incubation at ambient atmosphere and 33 to 37 C. They work as a worked example of how the three patterns above look in practice.
| Reference strain | Gas | Slant | Butt | H2S |
|---|---|---|---|---|
| Escherichia coli ATCC 25922 | Positive | A | A | Negative |
| Salmonella enterica serovar Typhimurium ATCC 14028 | Positive | ALK | A | Positive |
| Pseudomonas aeruginosa ATCC 27853 | Negative | ALK | ALK | Negative |
The IFU states that if aberrant quality-control results are noted, sample results should not be reported. A laboratory that runs TSI can therefore treat these three tubes as a check on the medium before trusting any patient or research isolate result.
Where results mislead: a checklist from the manufacturer’s limitations
- Loosened caps. Tubes need loosened caps. If tightly capped, acid from dextrose alone also involves the slant, so a dextrose-only organism can look like A/A.
- Blackening can hide acidity. A black butt may mask the colour of the butt, as noted above.
- Sucrose can suppress H2S. The IFU says sucrose utilization may suppress H2S enzyme mechanisms, so a negative H2S reading is not absolute.
- SIM agar is more sensitive for H2S. The IFU reports that SIM agar is more sensitive than TSI for detecting H2S.
- Look-alikes. Proteus species may give reactions like Salmonella and Shigella, so additional biochemical testing is required for definitive identification.
- Aberrant QC. Do not report sample results when the control strains do not give their expected reactions.
The IFU does not itself give inoculation or reading steps and refers users to current editions of appropriate references, which is why this page describes how results are interpreted rather than a bench procedure.
Common misunderstandings
- Red does not always mean “nothing happened”. A red slant over a yellow butt is a positive dextrose fermentation that has reverted at the slant because of oxygen and the low dextrose level.
- TSI alone does not identify an organism. It differentiates on sugar fermentation and H2S, and the IFU requires additional biochemical testing for definitive identification.
- Black is not the same as acid. The black precipitate reports H2S, not pH.
- It is not a diagnostic device on its own. The manufacturer labels the medium for laboratory use only, not for diagnosis.
How TSI sits alongside other plate media
TSI is a tubed medium, so it is usually discussed alongside plated differential and selective media. For related media pages, see MacConkey agar and xylose lysine deoxycholate (XLD) agar. For the underlying vessels and background, see what an agar plate is, what bacteriology covers, and the Gram stain procedure, reagents and results guide.
FAQ
What does triple sugar iron agar show?
It shows whether an organism ferments dextrose, lactose and sucrose, whether it produces gas, and whether it produces hydrogen sulfide, using phenol red as the acid and alkaline indicator and iron salts to reveal H2S as a black precipitate.
What are normal triple sugar iron agar test results?
There is no single normal result; the pattern depends on the organism. The Remel IFU lists expected results for three control strains: E. coli is acid over acid with gas and no H2S, S. Typhimurium is alkaline over acid with gas and H2S, and P. aeruginosa is alkaline over alkaline with no gas and no H2S.
What does a red slant with a yellow butt mean?
It is reported as ALK/A (or K/A) and indicates that only dextrose was fermented, according to the Remel IFU.
What does a black butt mean in TSI?
It means H2S was produced. The IFU notes that blackening throughout the butt may mask acidity.
Sources
- Thermo Fisher Scientific (Remel), Triple Sugar Iron (TSI) Agar, Instructions for Use IFU 454982, revision December 2011. Date checked: 2026-10-29.
- American Society for Microbiology, Triple Sugar Iron Agar Protocols, introductory description. Date checked: 2026-10-29.
- Ohio State University, Microbiology Lab SP25, 4.07 Triple Sugar Iron Medium (LibreTexts), for the A and K reporting convention. Date checked: 2026-10-29.
