Triple Sugar Iron (TSI) Agar Composition, Principle, Preparation, Results, Uses

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The majority of bacteria are able to ferment carbohydrates, especially sugars. Within them, every bacteria is able to ferment just a few of the sugars, whereas it is unable to ferment all the other sugars. So, the sugars that a bacterium is able to ferment, as well as the sugars that it can’t, is the signature of the bacterium and is an important factor in its determination. It is the Triple Sugar Iron (TSI) Agar is a type of culture medium known for its capacity to determine a microorganism’s capacity to produce sugars and generate hydrogen sulfur.

Principle of Triple Sugar Iron (TSI) Agar

The TSI Agar is a unique medium made up of multiple sugars that comprise an acid-sensitive colour (phenol red) with 11% lactose sucrose , 0.1% glucose, along with ferrous sulfate, sodium thiosulfate or ferrous ammonium sulfurate. All of these ingredients are combined and allowed to solidify at an angle, resulting within an Agar Test Tube that is an incline.

The slanted design of this medium gives an assortment of surfaces that are either exposed to oxygen-rich air in different degrees (aerobic environments) or not exposed air (an anaerobic setting) where the patterns of fermentation of the organisms are analyzed. The triple sugar-iron agar is designed to distinguish between organisms based on variations in the patterns of carbohydrate fermentation and the production of hydrogen sulfide.


Carbohydrate fermentation can be detected by the release of gas and the change in the color the indicator for pH from yellow to red. Because of the production of acid in the process of fermenting, pH decreases. The acid-base indicator Phenol Red can be used to detect the fermentation of carbohydrate, which is evident by the changing the color of the carbohydrate medium , which changes from orange-red to yellow when in presence of acid.

When there is an the decarboxylation process, oxidative Alkaline products are formed and the pH increases. This is evident by the shift in color of the medium, which ranges from orange-red into deep red. Ferrous ammonium and sodium thiosulfate Sulfate that is present in the medium are able to detect that the formation of hydrogen sulfur dioxide and is evident in the black hue in the tube’s butt. In order to make it easier to identify organisms that can only ferment glucose, the concentration of glucose is one-tenth of the amount of sucrose or lactose.

The tiny amount of acid that is produced in the tube’s slant when glucose fermentation occurs, it oxidizes quickly which causes it to be in the orange-red or return back to alkaline pH. However this acid process (yellow) remains at the tube’s butt because it’s under less oxygen tension. Following the depletion of the limiting glucose, the organisms that are able to use it will be able to make use of lactose and sucrose.

  • An alkaline/acid (red slant/yellow butt) reaction: It is indicative of dextrose fermentation only.
  • An acid/acid (yellow slant/yellow butt) reaction: It indicates the fermentation of dextrose, lactose and/or sucrose.
  • An alkaline/alkaline (red slant, red butt) reaction: Absence of carbohydrate fermentation results.
  • Blackening of the medium: Occurs in the presence of H2 
  • Gas production: Bubbles or cracks in the agar indicate the production of gas ( formation of CO2and H2)

Composition of Triple Sugar Iron (TSI) Agar

Pancreatic Digest of Casein15.0
Sodium Chloride5.0
Peptic Digest of Animal Tissue5.0
Yeast Extract3.0
Beef Extract3.0
Ferric Ammonium Citrate0.5
Sodium Thiosulfate0.3
Phenol Red0.024

Final pH (at 25°C): 7.3 +/- 0.2

Preparation and Method of Use

  1. Submerge 64.42 grams (the equivalent weight of dehydrated medium per Liter) in 1000ml purified distillate water.
  2. Bring the temperature to a boil until the medium is completely dissolving.
  3. Mix well, then distribute it to test tubes.
  4. Sterilize by ensuring that the pressure is maintained the pressure at 10lbs (115degC) until 30 min. or according to confirmed cycle.
  5. Allow the medium to settle in the sloped design by securing it with a nut about 2.5cm long.
  6. Utilizing a straight needle for inoculation make contact with the top of an isolated colony.
  7. Innoculate TSI by first stabbing the middle of the medium until the tube’s bottom before streaking over the top of the agar’s in a slant.
  8. The cap should be left loose and let the tube incubate at 35deg to 37degC in air for 18 to 24 hrs.
  9. Study your medium’s reaction.

Result Interpretation on Triple Sugar Iron (TSI) Agar

Salmonella enterica Growth; red slant, yellow butt, gas positive, black-butt (H2S produced)
Escherichia coli Growth; yellow slant, yellow butt, gas positive, no H2S produced
Pseudomonas aeruginosa Growth; red slant, red butt, no gas, no H2S produced
Shigella sonnei Growth; red slant, yellow butt, no gas, no H2S produced
Citrobacter freundiiYellow slant, yellow butt, gas production; positive reaction for H2S Blackening of medium
Enterobacter aerogenesYellow slant, yellow butt, gas production; no H2S produced
Klebsiella pneumoniaeyellow slant, yellow butt, gas positive, no H2S produced
Proteus vulgarisRed slant, yellow butt, no gas production; H2S produced
Salmonella Paratyphi ARed slant, yellow butt, gas production; no H2S produced
Salmonella TyphiRed slant, yellow butt, no gas production; H2S produced
Salmonella TyphimuriumRed slant, yellow butt, gas production; H2S produced
Shigella flexneriRed slant, yellow butt, gas negative, H2S not produced

Uses of Triple Sugar Iron (TSI) Agar

  • The test is designed to evaluate the capacity of an organism to produce lactose, glucose and sucrose, as well as their capability to create hydrogen sulfide.
  • It is used to distinguish members of Enterobacteriaceae class from rods with gram-negative chemistry.
  • It also aids in the classification of Enterobacteriaceae by their patterns of sugar fermentation.

Limitations of Triple Sugar Iron (TSI) Agar

  • It is suggested that immunological, biochemical, mass spectrometry, or molecular tests be carried out on the colonies of pure culture to confirm the identity of the colony.
  • It is essential to stab the butt of the middle. In the event that you fail to do this, it is a violation of the test. The quality of the agar needs to be maintained while stabbing. Caps must be removed in this test, otherwise false results can occur.
  • TSI Agar must be read within the stated 18-24-hour incubation time. False-positive reactions can be observed if the test is read too early. False-negative reactions can be observed if the book is read more that 24 hours.
  • The organism that produces hydrogen sulfide can cause acid to be produced in the middle. But, the production of hydrogen sulfur requires an acidic environment, therefore the portion that is located in the butt should be considered to be acid.
  • TSI isn’t as effective in detecting hydrogen sulfide contrast to other mediums with iron in like Sulfide Indole Motility (SIM) Medium.
  • Some species and strains can produce delayed reactions or do not produce the sugar in the specified way.
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