Cat. no. L16 Triple Sugar Iron (TSI) Agar, 16x100mm Tube, 6ml Slant 20 tubes/box
Cat. no. L50 Triple Sugar Iron (TSI) Agar, 16x125mm Tube, 8ml Slant 20 tubes/box
Cat. no. R32 Triple Sugar Iron (TSI) Agar, 13x100mm Tube, 4ml Slant 20 tubes/box


Hardy Diagnostics Triple Sugar Iron (TSI) Agar is recommended for use in the differentiation of Enterobacteriaceae by their ability to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide.


In 1917 Sulkin and Willett described a medium containing the carbohydrates glucose, lactose, and sucrose, and iron salts. (8) The medium showed fermentation of these carbohydrates, as well as hydrogen sulfide production. Hajna modified the medium in 1945 to contain phenol red as the pH indicator, and is the formulation still in use today. (3)

Glucose is added to the medium since most enteric pathogens uniformly ferment this carbohydrate. Lactose and sucrose are added in ten times the amount of glucose, as most enteric pathogens do not ferment these sugars. As a result, non-pathogenic enterics which do ferment these sugars produce acid in the slant. Pathogenic enterics produce an initially acid slant from the low concentration of glucose, but as growth continues it changes to the alkaline reaction. Sodium thiosulfate is incorporated into the medium as a source of hydrogen sulfide. Ferrous ammonium sulfate serves as the indicator, which turns the butt black in the presence of free hydrogen sulfide gas. Enteric organisms that are capable of fermenting glucose will produce acid (a yellow butt and a red slant), a positive hydrogen sulfide result. Gas production may result and is seen as cracks and bubbles in the medium. 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.

TSI Agar is contained in a tube and is slanted to form a deep butt and short slant. Inoculation is performed with a straight needle by stabbing to the base of the butt, and streaking the slant when the needle is removed. The cap is replaced loosely to facilitate an aerobic atmosphere.


Ingredients per liter of deionized water:*

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.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Method of Use: Allow the TSI Agar to warm to room temperature before use. Using one isolated, pure colony inoculate, stab, and streak the specimen on the agar as soon as possible after collection. Incubate tubes aerobically at 35-37ºC. for 18-24 hours. Examine reaction of medium.


An alkaline/acid (red slant/yellow butt) reaction is indicative of dextrose fermentation only. An acid/acid (yellow slant/yellow butt) reaction indicates the fermentation of dextrose, lactose and/or sucrose. The absence of carbohydrate fermentation results in an alkaline/alkaline (red slant, red butt) reaction. A blackening of the medium occurs in the presence of H 2 S. Bubbles or cracks in the agar indicate the production of gas.

Consult listed references for the identification of colony morphology and further biochemical tests required for identification. (1,2,4,6)


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.

TSI Agar must be read within the 18-24 hour stated incubation period. A false-positive reaction may be observed if read too early. A false-negative reaction may be observed if read later than 24 hours.

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 (Cat. no. Q30). 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.


Standard microbiological supplies and equipment such as loops, slides, staining supplies, other culture media, microscope, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC ® 14028
C 18-24hr 35°C Aerobic Growth; red slant, yellow butt, gas positive, black butt (H 2 S produced)
Escherichia coli
ATCC ® 25922
C 18-24hr 35°C Aerobic Growth; yellow slant, yellow butt, gas positive, no H 2 S produced
Pseudomonas aeruginosa
ATCC ® 27853
C 18-24hr 35°C Aerobic Growth; red slant, red butt, no gas, no H 2 S produced
Shigella sonnei
ATCC ® 9290
C 18-24hr 35°C Aerobic Growth; red slant, yellow butt, no gas, no H 2 S produced

User Quality Control


Triple Sugar Iron (TSI) Agar should appear slightly opalescent, with a possible slight precipitate, and red in color.

S. typhimurium growing on TSI

Salmonella enterica (ATCC ® 14028) growing on TSI Agar (Cat. no. L50). Incubated aerobically for 24 hours at 35ºC.

E. coli growing on TSI

Escherichia coli (ATCC ® 25922) growing on TSI Agar (Cat. no. L50). Incubated aerobically for 24 hours at 35ºC.

P. aeruginosa growing on TSI

Pseudomonas aeruginosa (ATCC ® 27853) growing on TSI Agar (Cat. no. L50). Incubated aerobically for 24 hours at 35ºC.


Uninoculated tube of TSI Agar (Cat. no. L50).


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Hajna. 1945. J. Bact.; 49:516.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Sulkin and Willett. 1917. J. Med. Research; 37:225.