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Triple Sugar Iron Agar Test Introduction

Most of the biochemical tests are performed to determine the characteristics of the microorganisms and their reactions in certain mediums. Thus, it helps us to determine the nature of the organism inside the host body in different pathological conditions.

Triple sugar iron agar test is one such test, which is used the differentiate the different group of genera of the Enterobacteriaceae family, those of all gram-negative bacilli which can ferment the glucose thereby producing an acid. It helps us to distinguish them from the other species of gram-negative intestinal bacilli.

This test is generally performed in the biochemical laboratories, methods and procedures are discussed below.

Triple Sugar Iron Agar Test

This test is usually performed in a differential medium containing lactose, sucrose, and a small amount of glucose, along with the ferrous sulphate. This medium is used to differentiate the enteric based on the ability to reduce the sulfur and in fermenting carbohydrates.

Triple Sugar Iron Agar Test Objective

The main aim of the test is to whether the gram-negative bacilli have the capability to ferment the monosaccharides like glucose, lactose or sucrose which results in forming a hydrogen sulfide. To differentiate the members of the Enterobacteriaceae family from the other gram-negative rods.

Triple Sugar Iron Agar Test Principle

Triple sugar iron agar test is designed in such a way, to differentiate the different groups of the Enterobacteriaceae family, that are all gram-negative and are capable of fermenting glucose which initiates the production of acid and to differentiate them from the other species of gram-negative bacilli present in the intestine.

This differentiation is purely based on the fermentation of the monosaccharides like glucose, fructose and lactose which results in production of one of the acids, hydrogen sulfide.

This Triple Sugar Iron medium usually consists of about 10 parts of lactose, 10 parts of sucrose and 1 part of glucose and peptone. Here, phenol red and ferrous sulphate acts as an indicator which detects the presence of carbohydrates and it expresses it by the action of color change.

Usually here in the presence of carbohydrate in the medium, the colors change is from organ red to yellow, at the time of presence of acids. In case of oxidative decarboxylation of the peptone, the alkaline products in the medium built and results in increase in the ph. This change is indicated by change in color of the medium to orange red to deep red.

Whereas the sodium thiosulphate and the ferrous sulphate that are being present in the medium, detects the production of hydrogen sulphide which is indicted by formation of black color butt in the tube.

Initially glucose is utilized by a fermentative organism present in the medium, thus changes the entire medium into yellow when it becomes acidic within 8 to 12 hours. But this remains acidic even after 18 to 24 hours due to the presence of the organic acids and results in fermentation of glucose under the anaerobic conditions in the butt of the tube.

However, the slant reverts to the alkaline by change in color to red as of oxidation of the fermentation products under aerobic conditions on the slant. This change results in the formation of the carbon dioxide and water and oxidation of peptones in the medium to the alkaline amines. On addition to monosaccharides such as glucose, lactose and sucrose gets fermented.

Due to this fermentation products large amount of fermentation products are formed along the slant, thus neutralizing the alkaline amines and renders the slant acid, which is yellow in color because of 18-to-24-hour reaction. In case, if the slant and butt convert in alkaline form which shows that glucose is not yet fermented.

The organisms showing this reaction are usually called as non-fermenters and they derive their nutrition from the peptones that are present in the medium. And the presence or synthesis of carbon dioxide and hydrogen gas in the reaction is indicated by formation of bubbles or by observing the crack in the medium of the agar or in some cases it may also lead to separation of agar from the sides or from the bottom of the tube.

Whereas the production of hydrogen gas in the medium requires an acidic environment which leads to the blackening of the agar butt on reaction with ferric ammonium citrate in the tube.

Triple Sugar Iron Agar Test Reagents

Preparation of Triple Sugar Iron Agar:

Pancreatic Digest of Casein15.0 grams
Lactose10.0 grams
Sucrose10.0 grams
Sodium Chloride5.0 grams
Yeast Extract3.0 grams
Peptic Digest of Animal tissue5.0 grams
Beef extract3.0 grams
Dextrose1.0 grams
Ferric Ammonium Citrate0.5 grams
Sodium thiosulphate0.3 grams
Agar12.0 grams
Phenol red0.024 grams
Triple Sugar Iron Agar Test Procedure

Initially, a small amount of colony is collected by touching a well-isolated colony using a straight inoculating needle.

Triple Sugar Iron Agar is first inoculated by stabbing the center of the medium to the end of the tube. Further the surface of the agar slant is streaked.

The tube of the cap is closed loosely, and it is inoculated in an ambient air chamber for about 18 to 24 hours at a temperature of 35 to 37ºC.

After incubation the tube is observed and opted if there is any color change in the butt and slant or any cracks or blackening of the medium.

Triple Sugar Iron Agar Test Results
Result (Slant/butt)Interpretation
Red /Yellow colorFermentation of glucose alone and catabolisation of peptone.
Yellow/YellowFermentation of monosaccharides such as Glucose, sucrose, lactose.
Red/RedNo fermentation, Peptone is catabolized under aerobic and anaerobic conditions.
Yellow/ Yellow bubblesGlucose and lactose or sucrose fermentation, gas produced
Red/Yellow with bubblesFermentation of glucose alone, Gas produced.
Red/Yellow with bubbles and formation of black precipitate.Fermentation of glucose alone, Hydrogen sulphide gas is produced.
Yellow/Yellow with bubbles and black precipitateGlucose, lactose, sucrose fermentation, Production of hydrogen sulphide gas
Red/Yellow with black precipitateGlucose fermentation alone, production of hydrogen sulphide gas
Yellow/Yellow with black precipitateFermentation of glucose, lactose or sucrose, production of hydrogen sulphide gas.
Triple Sugar Iron Agar Test Citations

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