Triple Sugar Iron Agar Principles,composition and colony characteristics

Triple Sugar Iron Agar  was originally proposed by Sulkin and Willett and modified by Hajna for the identification of  Enterobacteriaceae.It is selective growth media recommended for identification of gram-negative enteric bacilli on the basis of  sugars such as dextrose, lactose and sucrose fermentation and hydrogen sulphide production.

Principle of Triple Sugar Iron Agar

  • The presence of peptone, yeast extract and beef extract provide nitrogenous compounds, sulphur, trace elements and vitamin B complex etc.
  • The sodium chloride maintains osmotic equilibrium.
  • Lactose, sucrose and dextrose monohydrate are the fermentable carbohydrates sugars.
  • Sodium thiosulphate and ferric or ferrous ions make Hydrogen sulphide (H2S) indicator system.
  • Sodium thiosulphate is also an inactivator of halogen and can minimize its toxicity in the testing sample, if any during microbial limit tests.
  • Phenol red is the pH indicator.
  • Those organisms that ferment the sugar dextrose monohydrate produce a variety of acids, varying the colour of the medium from red to yellow.

More amounts of acids are liberated in butt region (fermentation) than in the slant (respiration). Growing bacteria also form alkaline products from the oxidative decarboxylation of peptone and these alkaline products neutralize the large amounts of acid present in the butt.

  • Therefore, the appearance of an alkaline (red) slant and an acid (yellow) butt after incubation indicates that the organism is a dextrose fermenter but is unable to ferment lactose and/or sucrose.
  • Bacteria that ferment lactose or sucrose or both, in addition to dextrose, produces large amount of acid enables no reversion of pH in that region and thus bacteria exhibit an acid slant and acid butt.
  • Carbon dioxide gas production is detected by the presence of cracks or bubbles in the medium, when the accumulated gas escapes.
  • Thiosulphate is reduced to hydrogen sulphide by several species of bacteria and hydrogen sulphide (H2S) combines with ferric ions of ferric salts to produce the insoluble black precipitate of ferrous sulphide.
  • Reduction of thiosulphate proceeds only in an acid environment and blackening usually occurs in the butt of the tube.
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Note that triple Sugar Iron Agar should be used in parallel with other test like  Urea Agar or Broth to distinguish between Salmonella and Proteus species. These reactions is summarised below :

ObservationReaction
Alkaline slant / acid butt only glucose fermented
Presence of bubbles or cracks Positive gas production
Presence of black precipitate Hydrogen sulphide (H2S) gas production

Also some members of the Enterobacteriaceae and Hydrogen sulphide producing Salmonella species may not be H2S positive on TSI Agar.

Some bacteria may show H2S production on Kligler Iron Agar but not on TSI Agar. This can happen because utilization of sucrose in Triple Sugar Iron Agar suppresses the enzymic pathway that result in the production of Hydrogen sulphide

Composition of Triple Sugar Iron Agar

IngredientsGrams /Litres
Beef extract3.000
Peptone20.000
Yeast extract3.000
Lactose10.000
Sucrose10.000
Dextrose monohydrate1.000
Ferrous sulphate0.200
Sodium chloride5.000
Sodium thiosulphate0.300
Phenol red0.024
Agar12.000

How to Prepare Triple Sugar Iron Agar

  • Start by suspending 64.42 grams(the equivalent weight of dehydrated medium per litre)in 1000 ml purified/ distilled water.
  • Then heat to boiling to dissolve the medium completely.
  • Mix well and distribute into test tubes and Sterilize by maintaining at 10lbs pressure (115°C) for 30 minutes.
  • Allow the medium to set in sloped form with a butt about 2.5cm long

Appearance of  Triple Sugar Iron Agar

After preparation of the growth media,the expected appearance should be pinkish red coloured clear to slightly opalescent gel forms in Petri plates or tubes

Triple Sugar Iron Agar
Triple Sugar Iron Agar

Type of Inoculums

Use pure isolate from clinical and non clinical samples such as Urine,stools,blood etc when using this media

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Cultural characteristics after Incubation at 35-37°C for 18-24 hours.

OrganismsSlantButtGasHydrogen Sulphide
Enterobacter aerogenes ATCC 13048 Acidic reaction, yellowing of the mediumAcidic reaction, yellowing of the mediumPositive reactionNo blackening of medium
Citrobacter freundii ATCC 8090 Acidic reaction, yellowing of the mediumAcidic reaction, yellowing of the mediumPositive reactionBlackening of medium
Klebsiella pneumoniae ATCC 13883 Acidic reaction, yellowing of the mediumAcidic reaction, yellowing of the medium Positive reactionNo blackening of medium
Proteus vulgaris ATCC 13315 Alkaline reaction, red colour of the mediumAcidic reaction, yellowing of the mediumNegative reactionBlackening of medium
Salmonella Paratyphi A ATCC 9150Alkaline reaction, red colour of the mediumAcidic reaction, yellowing of the mediumPositive reactionNo blackening of medium
Salmonella Typhi ATCC 6539 Alkaline reaction, red colour of the mediumAcidic reaction, yellowing of the mediumNegative reactionBlackening of medium
Salmonella Typhi ATCC 6539Alkaline reaction, red colour of the mediumAcidic reaction, yellowing of the mediumNegative reactionBlackening of medium
Salmonella Typhimurium ATCC 14028Alkaline reaction, red colour of the medium Acidic reaction, yellowing of the mediumPositive reactionBlackening of medium
Shigella flexneri ATCC 12022 Alkaline reaction, red colour of the mediumAcidic reaction, yellowing of the mediumNegative reactionNo blackening of medium
Escherichia coli ATCC 8739Acidic reaction, yellowing of the mediumAcidic reaction, yellowing of the mediumPositive reactionNegative reaction
Klebsiella pneumoniae ATCC 10031 Acidic reaction, yellowing of the mediumAcidic reaction, yellowing of the mediumPositive reactionNegative reaction
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Triple Sugar Iron agar slant characteristics
Triple Sugar Iron agar slant characteristics
1-Uninoculated -2-Shigella species-3-Providencia species-4-Pseudomonas species on Triple sugar iron agar slant
1-Uninoculated -2-Shigella species-3-Providencia species-4-Pseudomonas species on Triple sugar iron agar slant

How to store or conserve Triple Sugar Iron agar

  • Store the unprepare media below 30°C in tightly closed container and the prepared medium at a temperature of 2 – 8°C.


Reference

  • Sulkin E.S. and Willett J.C., 1940, J. Lab. Clin. Med., 25:649.
  • MacFaddin J., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
  • Hajna A.A., 1945, J. Bacteriol, 49:516.
  • Eaton A. D., Clesceri L. S. and Greenberg A W.,(Eds.), 2005, Standard Methods for the Examination of Water and Wastewater, 21st ed., APHA, Washington, D.C.
  • The Indian Pharmacopoeia 1996, Govt. of India, 1996. The Controller of Publication, Delhi.

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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