Cat. no. C7110 CRITERION™ Triple Sugar Iron (TSI) Agar 130gm
Cat. no. C7111 CRITERION™ Triple Sugar Iron (TSI) Agar 500gm
Cat. no. C7112 CRITERION™ Triple Sugar Iron (TSI) Agar 2kg
Cat. no. C7113 CRITERION™ Triple Sugar Iron (TSI) Agar 10kg
Cat. no. C7114 CRITERION™ Triple Sugar Iron (TSI) Agar 50kg


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

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.


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.


Gram weight per liter: 65.0gm/L
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.


Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original pink.

Store the prepared culture medium at 2-8ºC.



1. Suspend 65.0gm of the dehydrated culture media in 1 liter of distilled or deionized water.

2. Heat to boiling and mix to dissolve completely.

3. Sterilize in the autoclave at 121ºC. for 15 minutes.

4. Cool to 45-50ºC. and aseptically add enrichments, if desired.


For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. No. L50.


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. 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 autoclaves, incinerators, and incubators, etc., 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 (H2S produced)
Escherichia coli
ATCC ® 25922
C 18-24hr 35°C Aerobic Growth; yellow slant, yellow butt, gas positive, no H2S produced
Pseudomonas aeruginosa
ATCC ® 27853
C 18-24hr 35°C Aerobic Growth; red slant, red butt, no gas, no H 2 S produced

User Quality Control


CRITERION™ Triple Sugar Iron (TSI) Agar powder should appear homogeneous, free-flowing, and pink in color. The prepared media should appear slightly opalescent with a possible slight precipitate, and red in color.


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. Bacteriol.; 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.

ATCC is a registered trademark of the American Type Culture Collection.