Triple Sugar Iron (TSI) Test – Principle, Procedure, Uses and Interpretation

By Prof Daniel Asrat


The Triple Sugar Iron (TSI) test is a microbiological test roughly named for its ability to test a microorganism’s ability to ferment sugars and to produce hydrogen sulfide.[1] It is often used to differentiate enteric bacteria including Salmonella and Shigella.

The TSI slant is a test tube that contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, and sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate.

All of these ingredients are mixed together, heated to sterility, and allowed to solidify in the test tube at a slanted angle. The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (an aerobic environment) or not exposed to air (an anaerobic environment).

TSI agar medium was developed based on Kligler’s iron agar, which had been used for the determination of lactose-fermentative bacteria, by addition of sucrose to be able to detect sucrose-fermentative bacteria, also.


Triple Sugar Iron Agar (TSIA) contains 3 sugar glucose (dextrose) sucrose, lactose in the ratio of1: 1:10 as the source of carbon as its name suggest. Besides this the media also contains yeast and beef extract, peptic digest of animal tissue and casein as the source of protein.The presence of fetrous sulphate and sodium thiosulphate fulfills the demand of sulphur and there is the indicator phenol red for the indication of change in the invironment of the media due to the production of acid or alkali. The media is prepared in the form of slant containing, a slant and butt region.

This test is used to differentiate gram negative enteric. Enter bacteria and other glucose fermenter first begin to metabolize glucose as glucose utilizing enzymes are present constitutively and bacteria can gain the most energy by using simplest sugar. All other sugar must be converted to glucose before they enter the EMP. Glucose utilization occur both aerobically on the slant where O2 is available and on the butt where  there is anaerobic condition.  Once the glucose fermenting bacterium has reduced all of the available glucose to pyruvate, it will further metabolize pyruvate via TCA cycle to produce acid end products. The acid in the medium cause the ph indicator phenol red, to change its colour to yellow. Thus, after 6 hours of incubation both the slant and butt of TSIA that has been inoculated with a glucose fermenter will appear yellow.

After the depletion of limited glucose in the medium, the organism that has enzymes to degrade lactose or sucrose starts to utilize them as they are present 10 times more concentration than glucose and the organism continue to make acid end products.Hence, the butt and slant of TSIA will still remain yellow for lactose and sucrose fermentors (degrades) after 18 hours of incubation and the reaction is called acid/acid.If the organism being tasted cannot use the lactose or sucrose in the medium, it must shift to protein utilization as there is no other source or energy in the media.

As the protein breakdown occurs only in aerobic condition, the byproduct of protein and amino acid metabolism i.e NH3 changes the pH environment of the medium from the neutral to alkaline  in the slant region where the condition is aerobic. Hence, the colour of slant appears red and the reaction is called alkali/acid while the colour of the butt is still yellow due to anaerobic glucose breakdown.

Gas production can be detected when holes are formed or the medium is broken into several fragments. H2S production by organism results in the blackening of the medium. This colour is due to the production of H2S from an ingredient of the medium, sodium thiosulphate, which then combines with another component of the medium, ferrous ammonium sulphate resulting in the formation of black, insoluble compound, ferrous sulphide.

FeSO4  +  H2S ——————> H2SO4  +  FeS (Black ppt)


Reagents And Materials Required

  • Test tubes
  • Conical flask
  • Cotton plugs
  • Inoculating needle
  • Autoclave
  • Bunsen burner
  • Laminar flow chamber
  • Dispose jar
  • Incubator
  • Triple sugar iron (TSI) agar
  • Isolated colonies or pure cultures of bacteria.

Composition of Triple Sugar Iron Agar (TSI)

Beef extract3.0 g
Yeast extract3.0 g
Peptone20.0 g
Glucose1.0 g
Lactose10.0 g
Sucrose10.0 g
Ferrous sulfate or ferrous ammonium sulfate0.2 g
NaCl5.0 g
Sodium thiosulfate0.3 g
Phenol red0.024 g
Agar13.0 g
Distilled water1,000 mL


  1. The ingredients of TSI agar medium (containing the 3 sugars and iron as the main components) or its ready-made powder required for 100 ml of the medium is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
  2. Its pH is determined using a pH paper or pH meter and adjusted to 7.4 using 0.1N HCI if it is more or using 0.1N NaOH if it is less.
  3. The flask is heated to dissolve the agar in the medium completely.
  4. Before it solidifies, the medium in warm molten condition is distributed into 5 test tubes (approximately 20 ml each).
  5. The test tubes are cotton-plugged, covered with craft paper and tied with thread or rubber band.
  6. They are sterilised at 121°C (15 psi pressure) for 15 minutes in an autoclave.
  7. After sterilisation, they are removed from the autoclave and kept in a slanting position to cool and solidify the medium, so as to get TSI agar slants.
  8. The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the slants by stabbing into the butt and streaking on the surface of the slants with the help of a flame- sterilised needle. The needle is sterilised after each inoculation.
  9. The inoculated slants are incubated at 37°C for 24 hours in an incubator.

Observations And Results Interpretation

1.Yellow butt and red slant with or without gas production (breaks in the agar butt)

Acid butt and alkaline slant has been formed. Here only glucose has been used anaerobically (fermentatively) making the butt acidic (yellow). No other sugar has been utilized. Since concentration of glucose is less (0.1%), the small amount of acid produced on the slant surface is oxidized rapidly making it alkaline (red).

Further, oxidative domination of peptone present in the medium produces NHY which makes the slant alkaline (red). However, in the butt, the acid condition is maintained due to reduced availability of oxygen and slower growth of the bacteria. Thus, the bacteria is glucose positive.

2.Yellow butt and yellow slant with or without gas production

Acid butt and acid slant has been formed. Here, lactose and/or sucrose have been fermented. Since their concentration in the medium is high, they produce large quantity of acids resulting in acidic slant and acidic butt and maintain the acidic condition. Thus, the bacteria is sucrose/lactose positive.

3.Red butt and red slant

None of the three sugars has been fermented. Instead, peptones have been catabolised under anaerobic and/or aerobic conditions, resulting in an alkaline condition due to production of ammonia.

If only aerobic degradation of peptones occurs, only the slant surface becomes alkaline (red). If there is aerobic and anaerobic degradation of peptone, both the slant as well as the butt becomes alkaline (red). Thus, the bacteria is sugar negative.

4.Blackening of the butt

In addition to any one of the above conditions, if blackening of butt occurs, it indicates that, the bacteria is capable of producing H2S utilising the inorganic sulphur (sodium thiosulphate) present in the medium.

The H2S combines with the ferrous sulphate in the medium to form black precipitates of ferrous sulphide resulting in a change in the colour of the medium to black. Thus, the bacteria are H2S positive.

5.No blackening of butt:

The bacteria is not capable of producing H2S utilising the inorganic sulphur (sodium thiosulphate) present in the medium. Thus, the bacteria is H2S negative.