Triple Sugar Iron (TSI) Agar
TSI is a differential media that can detect both fermentation and hydrogen sulfide production. It is also a rich medium allowing the growth of fastidious species. It contains a pH indicator (phenol red), four protein sources or extracts, three sugars (glucose, lactose, and sucrose to test for fermentation) and iron and sulfur compounds (to test for the production of hydrogen sulfide gas). TSI tubes are poured to have both a slant (on the top) and a butt (on the bottom). The slant is to allow for aerobic growth, the butt to allow for anaerobic growth (or at least reduced oxygen). It is a fairly complex test, having a number of combination results that are possible. When fermentation occurs, acid products are made which will change the color of the media from orange to yellow. If fermentation occurs with the production of gas, there will be cracks, breaks, or lifting of the agar in the tube. If fermentation of the sugars does not occur, the bacteria may digest the peptones, releasing alkaline end products. This will lower the pH and turn the medium red. If the sulfur compound is reduced, hydrogen sulfide will form and interact with the iron compound to form a black precipitate, which is especially visible in the butt. If nothing happens (no change) the medium will stay orange.
One complication is that three sugars are present, and that glucose is present in limiting amounts (0.1%). For this reason, it is usually not possible to tell which sugar has been fermented, unless only glucose is fermented. If glucose is the only sugar that is fermented, then the result will be a red slant/yellow butt after 1 day of growth because all the glucose will likely be used up in the slant, so that peptones will be digested which will make alkaline end products turning the slant red. Because the butt ferments glucose slower, it will remain yellow from the acid products of fermentation.
Purpose: this test aids in the identification and differentiation of members of Enterobacteriaceae (enterics) from other Gram negative bacilli. It can also be used for other purposes, such as aiding in the identification of hydrogen sulfide producers, sugar fermentation, and confirming oxygen requirements.
|yellow slant/yellow butt||aerobic and anaerobic fermentation of sucrose and/or lactose (and glucose)|
|red slant/yellow butt||aerobic: glucose fermented till it
ran out then peptones were digested
anaerobic: fermentation of glucose (butt ferments glucose slower)
|red slant/red butt||aerobic and anaerobic: no fermentation, peptones were metabolized (not an enteric)|
|red slant/butt unchanged||aerobic: no fermentation, peptones were
anaerobic: little or no growth (nothing utilized, not an enteric)
|no change in slant & butt||aerobic and anaerobic: nothing was metabolized, bacteria may not be growing (not an enteric)|
|black precipitate, especially in butt||sulfur reduction has occurred (producing H2S gas)|
|cracks, breaks or lifting of agar||gas production during fermentation|
TSI is very similar to Kligler Iron Agar with the main difference that TSI has sucrose and Kligler Iron Agar does not. TSI appears to be less sensitive in detecting the production of hydrogen sulfide gas than other similar tests (SIM media, Kligler's Iron Agar, etc.). So, it is possible to observe the production of hydrogen sulfide in another test and not observe it in TSI. The sucrose in TSI may suppress the formation of hydrogen sulfide. Both TSI and Kligler's Iron Agar also use ferrous sulfate to detect the production of H2S which is less sensitive than other hydrogen sulfide detectors. Also the presence of sugars in the media decreases the production of hydrogen sulfide and the higher agar content reduces the sensitivity of the test. TSI has an added sulfur source (sodium thiosulfate), but sulfur may also be obtained from certain amino acids present in the protein in the media. Gas production may not always be observed as this test is not as sensitive to detecting gas as an inverted Duham tube. However, if H2S is produced, then gas is produced even if there is no other signs of gas. If possible, it is best to use freshly made TSI tubes as oxygen will slowly diffuse into the agar. If the TSI tubes are not relatively new, then they may be melted and reformed to reduce the oxygen.
Difco Laboratories. 1998. Difco Manual, 11th ed. Difco Laboratories, Sparks, MD, USA.
Harley J.P. and Prescott L.M. 2002. Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.
MacFaddin J.F. 2000. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
Otero R.B. 1973. Laboratory Exercises in Microbiology, 1st edition. J.B. Lippincott Company, Philadelphia, PA, USA.