Triple Sugar Iron Agar (TSIA) Test

Objectives of TSIA Test

  • To determine whether a gram negative bacilli ferments glucose and lactose or sucrose and forms hydrogen sulfide (H2S).
  • To differentiate members of the Enterobacteriaceae family from other gram-negative rods.

Principle of TSIA Test

The Triple Sugar Iron agar (TSIA) test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli. The differentiation is based on fermentation of glucose and lactose or sucrose and hydrogen sulfide (H2S) production. TSIA medium contains 10 parts of lactose: 10 parts of sucrose: 1 part of glucose and peptone. Phenol red and ferrous sulfate serve as indicators of acidification and H2S formation, respectively. The acid base indicator phenol red incorporated for detecting carbohydrate fermentation is indicated by the change in color of the carbohydrate medium from orange red to yellow in the presence of acids.  In case of oxidative decarboxylation of peptone, alkaline products are built and the pH rises. This is indicated by the change in colour of the medium from orange red to deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide and is indicated by the black color in the butt of the tube.

Glucose is utilized first by a fermentative organism and the entire medium becomes acidic (yellow) in 8 to 12 hours. Butt remains acidic even after 18 to 24 hours incubation period because of the presence of organic acids resulting from the fermentation of glucose under anaerobic conditions in the butt of the tube.  The slant, however, reverts to the alkaline (red) state because of oxidation of the fermentation products under aerobic conditions on the slant. This change is a result of the formation of CO2 and H2O and the oxidation of peptones in the medium to alkaline amines. When, in addition to glucose, lactose and/or sucrose are fermented, the large amount of fermentation products formed on the slant neutralizes the alkaline amines and renders the slant acidic (yellow), provided the reaction is read in 18 to 24 hours. If the slant and butt become alkaline, glucose has not been fermented. Organisms showing this reaction are defined as non-fermenters, and derive their nutrients from the peptones present in the medium. The formation of CO2 and hydrogen gas (H2) is indicated by the presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube. The production of H2S (sodium thiosulfate reduced to H2S) requires an acidic environment, and reaction with the ferric ammonium citrate produces a blackening of the agar butt in the tube.

Media Used in TSIA Test

Triple sugar iron agar

Pancreatic Digest of Casein 15.0gm
Lactose 10.0gm
Sucrose 10.0gm
Sodium Chloride 5.0gm
Peptic Digest of Animal Tissue 5.0gm
Yeast Extract 3.0gm
Beef Extract 3.0gm
Dextrose 1.0gm
Ferric Ammonium Citrate 0.5gm
Sodium Thiosulfate 0.3gm
Phenol Red 0.024gm
Agar 12.0gm

Final pH 7.3 +/- 0.2 at 25ºC.

Procedure of TSIA Test

  • With a straight inoculating needle, touch the top of a well-isolated colony.
  • Inoculate TSI agar by first stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant.
  • Leave the cap on loosely and incubate the tube at 35°-37°C in ambient air for 18 to 24 hours.
  • Following incubation, examine for color change in slant and butt, blackening and cracks in the medium.

Result Interpretation of TSIA Test

Result Interpretation of TSIA Test

S.N. Result (slant/butt) Symbol


1 Red/Yellow K/A Glucose fermentation only, peptone catabolized.
2 Yellow/Yellow A/A Glucose and lactose and/or sucrose fermentation.
3 Red/Red K/K No fermentation, Peptone catabolized under aerobic and/or anaerobic conditions.
4 Yellow/Yellow with bubbles A/A,G Glucose and lactose and/or sucrose fermentation, Gas produced.
5 Red/Yellow with bubbles K/A,G Glucose fermentation only, Gas produced.
6 Red/Yellow with bubbles and black precipitate K/A,G,H2S Glucose fermentation only, Gas produced, H2S produced.
7 Yellow/Yellow with bubbles and black precipitate A/A,G,H2S Glucose and lactose and/or sucrose fermentation, Gas produced, H2S produced.
8 Red/Yellow with black precipitate K/A,H2S Glucose fermentation only, H2S produced.
9 Yellow/Yellow with black precipitate A/A,H2S Glucose and lactose and/or sucrose fermentation, H2S produced.

Results of TSIA Test

Limitations of TSIA Test

  • It is important to stab the butt of the medium. Failure to stab the butt invalidates this test. The integrity of the agar must be maintained when stabbing.
  • Caps must be loosened during this test or erroneous results will occur.
  • Reactions in TSI should not be read beyond 24 hours of incubation, because aerobic oxidation of the fermentation products from lactose and/or sucrose proceeds, and the slant eventually reverts to the alkaline state.
  • An organism that produces hydrogen sulfide may mask acid production in the butt of the medium. However, hydrogen sulfide production requires an acid environment, thus the butt portion should be considered acid.
  • TSI is not as sensitive in detecting hydrogen sulfide in comparison to other iron containing mediums, such as Sulfide Indole Motility (SIM) Medium. Thus, organisms that have weak hydrogen sulfide production may show only trace hydrogen sulfide activity, or none at all.
  • Certain species or strains may give delayed reactions or completely fail to ferment the carbohydrate in the stated manner. However, if the organism fails to ferment glucose within 48 hours, it most likely is not in the Enterobacteriaceae family.
  • A pure culture is essential when inoculating Triple Sugar Iron Agar. If inoculated with a mixed culture, irregular observations may occur.
  • Further biochemical tests and serological typing must be performed for definite identification and confirmation of organisms.

Quality Control of TSIA Test

Test organism Slant Butt Gas production H2S production
Escherichia coli ATCC25922 Yellow Yellow +
Pseudomonas aeruginosa ATCC27853 Red Red
Salmonella enterica  ATCC14028 Red Yellow + +
Shigella sonnei ATCC9290 Red Yellow


  1. Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen edition, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
  2. Aneja K.R (2003), Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.

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