Triple sugar iron (TSI) agar

Principle

• TSI contains glucose (0.1%), lactose (1%) and sucrose (1%), with phenol red indicator and ferrous salt plus thiosulfate for H2S detection.
• Glucose fermentation alone first acidifies the whole tube; once the small amount of glucose is used, the aerobic slant reverts to alkaline from peptone breakdown, while the anaerobic butt stays acid.
• Lactose and/or sucrose fermentation produces enough acid to keep the slant acid.

Specimen

• Pure colony of a Gram-negative rod.

Reagents and equipment

• TSI agar slant with a deep butt; straight inoculating needle.

Procedure

• Stab the butt to within about 3–5 mm of the bottom, withdraw, then streak the slant surface.
• Loosen the cap and incubate at 35–37 °C.
• Read at 18–24 h.

Results

• K/A (red slant/yellow butt): glucose fermented only, e.g. Shigella; Salmonella usually K/A with H2S.
• A/A (yellow/yellow): glucose plus lactose and/or sucrose fermented, e.g. E. coli, Klebsiella, Enterobacter.
• K/K or K/NC (red/red or unchanged): non-fermenter, e.g. Pseudomonas aeruginosa.
• Gas: bubbles, cracks or agar lifted from the tube base.
• H2S: black precipitate (ferrous sulfide) in the butt; the butt is then considered acid, e.g. Salmonella, Proteus, Citrobacter freundii.

Quality control and pitfalls

• Controls: E. coli (A/A, gas), Salmonella or Proteus (K/A, H2S), P. aeruginosa (K/K).
• Reading before 18 h may give false A/A; after 24 h slants may revert to alkaline.
• A tight cap or no stab alters results; use a needle, not a loop.
• Kligler iron agar (KIA) is similar but lacks sucrose.

Clinical use

• Initial differentiation of Enterobacterales from non-fermenters and screening of stool isolates for Salmonella and Shigella.