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Triple sugar iron agar (TSIA) is a differential agar medium used to different lactose-fermenting Enterobacteriaceae from non-lactose fermenting enterobacteria. The medium usually contains three sugars viz: sucrose, glucose, and lactose, and phenol red as a pH indicator. TSIA is a synonymous biochemical test to Kliger’s iron agar (KIA) test. Both TSIA and KIA are used to test the ability of an Enterobacteriaceae to ferment glucose and lactose. These agar medium also test the ability of an enteric organism to reduce sulphur and produce gas (e.g. hydrogen sulphide, H2S). TSIA/KIA is commonly used to identify E. coli, Salmonella species, Proteus species and Shigella species. A yellow butt and pink-red slope shows glucose fermentation and acid production only. Lactose fermentation (inclusive of glucose fermentation) also turns the medium yellow.

Salmonella and Shigella shows this type of reaction. Production of gas (which often cause cracks in the medium) during the fermentation of any of these sugars also leads to the lifting up of the agar off the bottom of the tube. Salmonella paratyphi and some faecal commensals are known to cause cracks in TSIA/KIA media. A yellow slope and a yellow butt indicate lactose fermentation, and this show the presence of a lactose fermenter e.g. E. coli. A pink-red slope and butt indicates that the organism is a non-fermenter, and thus cannot ferment either glucose or lactose. Production of a black precipitate or colour that covers the butt indicates the production of H2S gas. Salmonella species especially S. typhimurium is notorious in the production of H2S gas in TSIA/KIA medium. Shigella species are not known to produce H2S gas in TSIA/KIA media.


  1. Prepare TSIA/KIA media according to manufacturer’s instructions.
  2. Use a pure culture of the test isolate to perform this test.
  3. Pick a speck or colony of the test isolate using a straight inoculating loop.
  4. Stab the TSIA/KIA media with the collected test isolate (making sure the stab reaches the butt of the tube) and streak the slope upon removal.
  5. Incubate inoculated tube(s) at 37oC for 18-24 hrs.
  6. Observe the tube(s) for glucose and lactose fermentation after incubation. Also examine the tube(s) for H2S production (Figure 1).
Figure 1. Triple sugar iron agar (TSIA) test. From left to right: Tube 1 is an Uninoculated Negative Control Tube. Tube 2 shows Glucose and Lactose and/or Sucrose Fermentation with gas production at the same time, and this is indicated by the yellow slant and butt and also the lifting of the agar off the bottom of the tube. Tube 3 shows No Sugar Fermentation (i.e. a non-lactose fermenter is present) as indicated by red slant (alkaline) and red butt (alkaline). The organism cannot ferment lactose, thus it deaminates amino acid to produce NH3 which create an alkaline environment that turns the tube red. Tube 4 shows Positive Glucose Fermentation as indicated by red slant (alkaline) and yellow butt (acid). Tube 5 shows Positive H2S Production and Glucose Fermentation only. Tube 6 shows a Glucose and Lactose and/or Sucrose Fermentation as indicated by a yellow slant (acid) and yellow butt (acid). Photo courtesy:

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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