Triple Sugar Iron (TSI) Agar (Atlas pgs.78-79)
TSI is a differential media that can detect fermentation and hydrogen sulfide production. It is a rich medium containing a pH indicator, four protein sources or extracts, three sugars (testing for fermentation) and iron and sulfur compounds (testing 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 especially is visible in the butt. If the tube becomes largely black, it may be difficult to read the tube for fermentation. 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. 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 may be a red slant/yellow butt because when all the glucose is used up, peptones may be digested which will turn the slant red, but the butt ferments glucose slower, so it remains yellow.
Purpose: this test aids in the identification and differentiation of members of Enterobacteriaceae (enterics) from other Gram- bacilli. It can also be used for other purposes, such as aiding in the identification of hydrogen sulfide producers 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 used (not an enteric)|
|red slant/butt unchanged||aerobic: no fermentation, peptones
anaerobic: little or no growth (nothing used, not an enteric)
|no change in slant & butt||aerobic and anaerobic: nothing was used, bacteria is growing slowly if at all (not an enteric)|
|black precipitate, especially in butt||sulfur reduction has occurred (producing H2S)|
|cracks, breaks or lifting in agar||gas production during fermentation|
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, and this detector is less sensitive than other hydrogen sulfide detectors. 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.