Triple Sugar Iron Agar (TSI)
A visual guide to Triple Sugar Iron Agar (TSI), used to determine glucose/lactose/sucrose fermentation, gas production, and hydrogen sulfide (H2S) synthesis in Gram-negative rods.
TSI slants A-D
Read both the slant (aerobic) and butt (anaerobic) color reactions, gas cracks, and black H2S precipitate.
Yellow slant/yellow butt. Fermentation of glucose plus lactose and/or sucrose. Bubbles/cracks push the agar up (gas positive).
Red slant/black butt. Glucose fermentation only (slant reverts to alkaline red), H2S produced (black FeS butt), and gas bubbles present.
Red slant/red butt. Non-utilizer of glucose, lactose, and sucrose. No gas and no black precipitate.
Uninoculated reddish-orange agar slant for comparing color changes, gas production, and baseline tube integrity.
Yellow slant/yellow butt. Fermentation of glucose plus lactose and/or sucrose. Bubbles/cracks push the agar up (gas positive).
Red slant/black butt. Glucose fermentation only (slant reverts to alkaline red), H2S produced (black FeS butt), and gas bubbles present.
Red slant/red butt. Non-utilizer of glucose, lactose, and sucrose. No gas and no black precipitate.
Uninoculated reddish-orange agar slant for comparing color changes, gas production, and baseline tube integrity.
What to look for
| Visual clue | Meaning | Student shorthand |
|---|---|---|
| Yellow Slant / Yellow Butt | Acid produced from glucose and lactose/sucrose fermentation | A/A (Fermenter) |
| Red Slant / Yellow Butt | Glucose fermented only; peptones oxidized aerobically on slant back to alkaline red | K/A (Glucose-only fermenter) |
| Red Slant / Red Butt | No sugars fermented; peptones utilized aerobically and anaerobically | K/K (Non-utilizer) |
| Black Butt | H2S gas produced, reacting with iron indicator to form black ferrous sulfide precipitate | H2S Positive (usually covers K/A butt) |
| Bubbles / Cracks / Agar push-up | Gaseous end-products (CO2 or H2) produced during fermentation | Gas Positive |
TSI Interpretive Matrix
| Pattern | What to call it | How to think about it |
|---|---|---|
| Yellow/Yellow (A/A) + Gas | Glucose, lactose/sucrose fermented | Typical for Escherichia coli, Klebsiella pneumoniae. |
| Red/Black (K/A H2S+) + Gas | Glucose fermented, lactose/sucrose negative, H2S and Gas | Typical for Salmonella typhimurium, Proteus vulgaris. |
| Red/Red (K/K) | No sugars fermented; non-utilizer | Typical for Pseudomonas aeruginosa, Acinetobacter. |
| Red/Yellow (K/A) | Glucose fermented only | Typical for Shigella flexneri. |
- TSI agar differentiates enteric bacteria based on glucose, lactose, sucrose fermentation, and thiosulfate reduction.
- It contains glucose (0.1%), lactose (1%), sucrose (1%), ferrous ammonium sulfate (H2S indicator), and phenol red (pH indicator).
- The slant is aerobic, while the deep butt is anaerobic.
A = Acid (Yellow). K = Alkaline (Red). A/A = all sugars fermented. K/A = glucose only. Black = H2S. Cracks = Gas.