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. It is often used to differentiate enteric bacteria including Salmonella and Shigella.
If glucose is the only sugar that is fermented, then the result will be a red slant/yellow butt after 1 day of growth because all the glucose will likely be used up in the slant, so that peptones will be digested which will make alkaline end products turning the slant red.
Preparation of TSI agar medium
- Combine the ingredients, and adjust the pH to 7.3.
- Boil to dissolve the agar and dispense into tubes.
- Sterilize by autoclaving at 121°C for 15 minutes.
- Cool in a slanted position to give a 2.5 cm butt and a 3.8 cm slant.
Sodium thiosulfate and ferrous sulfate are responsible for hydrogen sulfide production detection. Sodium thiosulfate acts as the substrate for enzymatic reduction and the resultant colorless hydrogen sulfide gas reacts with ferrous sulfate to produce ferrous sulfide, an insoluble black precipitate.
Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate.
This test thus determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism. The detection of hydrogen sulphide (H2S) gas produced by an organism. is used mainly to assist in the identification of that particular organism.
The TSI agar is a special medium with multiple sugars constituting a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate.
Preparation of Carbohydrate Fermentation Broth. Weigh and dissolve trypticase, Sodium chloride, and Phenol red in 100 ml distilled water and transfer into conical flasks. Add 0.5% to 1% of desired carbohydrate into all flasks.
The black precipitate indicates that the bacteria were able to produce hydrogen sulfide (H2S) from sodium thiosulfate. Because H2S is colorless, ferric ammonium citrate is used as an indicator resulting in the formation of insoluble ferrous sulfide.
Biochemical tests are the tests used for the identification of bacterial species based on the differences in the biochemical activities of different bacteria. The ability of bacteria to form organic compounds by metabolizing certain carbohydrates and related compounds is a widely used method for the …
2. Explain why the tsi medium contains a lower concentration of glucose than of lactoseand sucrose. Because the lower [glucose] allows for the detection of utilization and since glucose is amonosaccharide it will be used up first.
What would the consequence of adding 1% glucose instead of the 0.1% when preparing the media? You would not see a reversion after 12 hours so when reading the tubes at 24 hours, you would see a false positive for sucrose and/or lactose fermentation.
Phenol red is yellow at a pH < 6.8 and red at a pH of > 7.4, therefore if a bacterium ferments a sugar to acid a yellow color will develop. Additionally, phenol red tubes contain an inverted Durham tube.
What creates the black precipitate that is found in the "butt" of a TSI agar slant inoculated with Salmonella? The triple sugar-iron agar medium contains lactose, sucrose, glucose, sodium thiosulfate, and ferrous sulfate. True. A specimen was inoculated onto the Triple Sugar Iron (TSI) medium pictured here.
The carbohydrate fermentation test is used to determine whether or not a bacteria can utilize a certain carbohydrate. It tests for the presence of acid or gas produced from carbohydrate fermentation. A bubble in the Durham tube, no matter how small, is a positive result for gas production.
The TSI test is designed for the rapid separation and presumptive identification of enteric organisms. The Triple sugar-iron medium contains a lower concentration of glucose than of lactose and sucrose to allow for detection of the utlization of this substrate only.
Why is Phenol Red Broth made without adding significant amounts of general nutrient extracts ? it is used to determine the ability of an organism to utilize a specific sugar. One of the fermentation tubes turned red and had plenty of bacterial growth in the tube. What are the functions of phenol red ?
This is a differential medium. It tests for organisms' abilities to ferment glucose and lactose to acid and acid plus gas end products. It also allows for identification of sulfur reducers.
Detection is possible because the bacterium ferments dextrose (D-glucose) by producing mixed acids (e.g. lactic, acetic and formic acids) that can then be made visible with the addition of the indicator methyl red. There are many other methods of detection to indicate the presence of E. coli.
