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Triple Sugar Iron Agar


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  1. To differentiate among members of the Enterobacteriaceae.
  2. To distinguish between the gram-negative enteric bacilli from other groups of intestinal bacilli based on carbohydrate fermentation and the production of hydrogen sulfide.




The triple sugar- iron agar test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli. This differentiation is based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production by the various groups of intestinal organisms. Carbohydrate fermentation is indicated by the presence of gas and a visible color change of the pH indicator, phenol red. The production of hydrogen sulphide in the medium is indicated by the formation of a black precipitate that will blacken the medium in the butt of the tube.


To facilitate the observation of carbohydrate utilization patterns, TSI Agar contains three fermentative sugars, lactose and sucrose in 1% concentrations and glucose in  0.1% concentration. Due to the building of acid during fermentation, the pH falls. The acid base indicator Phenol red is incorporated for detecting carbohydrate fermentation that is indicated by the change in color of the carbohydrate medium from orange red to yellow in the presence of acids.  In case of oxidative decarboxylation of peptone, alkaline products are built and the pH rises. This is indicated by the change in colour of the medium from orange red to deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide and is indicated by the black color in the butt of the tube.


Carbohydrate fermentation is indicated by the production of gas and a change in the colour of the pH indicator from red to yellow. To facilitate the detection of organisms that only ferment glucose, the glucose concentration is one-tenth the concentration of lactose or sucrose. The meagre amount of acid production in the slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to remain orange red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube since it is under lower oxygen tension.


After depletion of the limited glucose, organisms able to do so will begin to utilize the lactose or sucrose. To enhance the alkaline condition of the slant, free exchange of air must be permitted by closing the tube cap loosely. If the tube is tightly closed, an acid reaction (caused solely by glucose fermentation) will also involve the slant.




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