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.

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 metabolized (not an enteric)
red slant/butt unchanged aerobic: no fermentation, peptones were metabolized
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

  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 an inch or so (2.4 cm), if it is less, 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.  (Using the loop will also split the agar falsely giving the impression that gas has bee produced.)
  5. Aseptically transfer bacteria to the tube.  (Either flame or incinerate 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 (about 3-5 mm from the bottom). Pull the needle straight out the way you came in.  Do not push the needle against the bottom of the tube as this will ruin it.
  7. Tighten the lid so it is between 1/2 to 1 turn open so that air can get in the tube.  Air must get in the tube for correct readings on the slant.
  8. Remind the instructor to set up the control tubes (uninoculated controls) which are useful for comparing results. 
  9. Place your tubes in the rack for this experiment. 
  10. Read the tubes for fermentation at 1 day, read for hydrogen sulfide production at 2 days (if the tubes are black, 1 day is sufficient).  For slow growing or slow fermenting organisms, up to twice the time may be needed to make observations.  Do not over or under incubate or your results may be unreliable.  Over incubating may cause all the sugars to be used up so that the organism starts to digest proteins and create alkaline products, causing a yellow tube to revert to red.  (If your results are unclear, you may overincubate for up to twice the normal times, but disregard any color change from yellow to red.) 


  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 which is why it is seen in the butt of a sugar fermenter.
  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.)
  8. A few organisms may produce so much gas that the medium may be forced up into the tube cap.  If this occurs, handle the tube with great care to avoid contaminating the culture, yourself, and the room.

Instructor Notes:

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.