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.

Result Observations Interpretation
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 used
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

  1. Obtain a tube containing a slant with a butt.  A butt is where the agar goes straight across the bottom of the tube, when there is an agar slant above it (agar at an angle in the tube).  Only use tubes where the butt is at least half an inch or so (1.2 cm), if it is not, you may not be be able to get growth under anaerobic (or at least reduced oxygen) conditions.
  2. Observe the media in the tube and record its color and appearance in your notebook.
  3. Label the tube, include your name, the date, and either the bacteria sample or the test's name.
  4. Do NOT Use your Wire Loop, as this will soon ruin it. Use your inoculating needle for this experiment.
  5. Aseptically transfer bacteria to the tube.  (Flame an inoculating needle (not the loop), cool it well, pick up some bacteria on the needle by rolling the tip in a colony, and then transfer some to the slant by streaking the slant in an S-like pattern.)
  6. Keep the needle uncontaminated and pick up some more bacteria and then stab the needle straight down into the butt, going nearly to the tube bottom. Pull the needle straight out the way you came in.  Do not push the needle against the bottom of the tube and make sure that the needle is cooled well as heat can easily move it.
  7. Remind the instructor to set up the control tubes (uninoculated controls) which are essential for observing and comparing the results. 
  8. Place your tubes in the rack for this experiment. 
  9. Read the tubes for fermentation at 24 hours, read for Hydrogen sulfide production at 48 hours (if the tubes are black, 24 hours is sufficient).  For slow growing or slow fermenting organisms, up to twice the time may be needed to make observations.  (If your results are unclear, continue incubating for up to twice the above times).  Do not over or under incubate or your results may be off.  Over incubating may cause the tubes to turn red because when the sugars become used up, proteins may be digested.


  1. Look at what is happening aerobically (on the slant) and anaerobically (in the butt). Growth may occur in both locations.
  2. Often holding the tubes up to a light will aid your observations.  Also roll the tube, because sometimes a color change is only near the surface of the slant and has to be seen at the correct angle. 
  3. Look for the occurrence of fermentation (a yellow color), peptone digestion (red), no change (orange), H2S production (black), gas production (cracks or lifting of the agar) in both the slant and the butt, compare your tube to an uninoculated control.
  4. The black coloration may mask the color of the butt, if so, record it as yellow.  H2S is produced in an acid environment with reduced or no oxygen.
  5. Any color change is significant, no matter how slight. Also, do not confuse your inoculation stab with a crack due to gas production.
  6. Check your results against the table, the results are complex.  Make your interpretations.  Use whatever pictures are available to you as an aid.
  7. Use the thioglycollate test to establish true anaerobic growth, but use the TSI test to confirm the oxygen requirements found in the thioglycollate test.  (For example, if you had growth only in the top of a thioglycollate tube, you should only see growth in the slant of a TSI tube.)


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.