TRIPLE SUGAR IRON (TSI) TEST

TRIPLE SUGAR IRON (TSI) TEST

 

AIM

         To check whether Gram negative bacilli particularly Enterobacteriaceae family to utilize Dextrose (or Glucose), Lactose and Sucrose fermentatively, and produce Hydrogen sulfide.

PRINCIPLE     

        In 1917, Sulkin and Willett described a medium containing the carbohydrates, Glucose, Lactose and Sucrose, and Iron salts. The medium showed fermentation of these carbohydrates, as well as Hydrogen sulfide production. Hajna modified the medium in 1945 to contain phenol red as the pH indicator for identifying Enterobacteriaceae, and is the formulation still in use today.

      Triple sugar iron agar (TSI) is a Differential medium that contains three Carbohydrate sugars, Lactose (10 %), Sucrose (10 %), a small amount of Glucose or Dextrose (1 %), Ferrous sulfate, and the pH indicator Phenol red.  It is used to differentiate Enterobacteria based on the ability to reduce Sulfur and ferment Carbohydrates. As with the Phenol red, if an organism can ferment any of the three sugars present in the medium, the medium will turn Yellow.  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. Thus, the appearance of an Alkaline (Red/Pink) (K) slant and an Acid (Yellow) (A) butt after incubation indicates that the organism is a Dextrose fermenter but is unable to ferment Lactose and Sucrose. Sodium thiosulfate and Ferrous ammonium sulfate present in the medium detects the production of Hydrogen sulfide. Ferrous ammonium sulfate serves as the indicator, which turns the butt Black in the presence of free Hydrogen sulfide gas. The production of H₂S (sodium thiosulfate reduced to H₂S) requires an acidic environment, and reaction with the ferric ammonium citrate produces a blackening of the agar butt in the tube. Gas (CO₂) production may result and is seen as cracks and bubbles in the medium.

MATERIALS REQUIRED

1. Test bacteria
2. Triple sugar iron (TSI) agar slant
3. Inoculation loop and Stab wire
4. Incubator

PROCEDURE

a)     With a sterilized straight inoculation needle, touch the top of a well-isolated bacterial colony.

b)   Inoculate on TSI Agar by first stabbing through the center of the medium to the bottom of the tube and then streaking on the surface of the agar slant. 

c)     Incubate the tubes at 37 °C in ambient air for 18 to 24 hours.

d)   Following incubation, examine for color change in slant and butt, blackening and cracks in the medium.

e)    TSI Agar must be read within the 18-24 hour stated incubation period. A false-positive reaction may be observed if read too early. A false-negative reaction may be observed if read later than 24 hours.

OBSERVATION AND RESULTS

·       Alkaline slant / Acid butt - Only Dextrose fermented.

·     Acid slant / Acid butt - Dextrose and Sucrose fermented or Dextrose and Lactose fermented or all the three sugars, Dextrose, Lactose and Sucrose fermented.

·       Bubbles or cracks present - Gas (CO₂) production.

·       Black precipitate present - H₂S production.

S.No	Name of the Bacteria	Slant	Butt	Gas	H2S
1	Escherichia, Klebsiella, Enterobacter	Acid (A)	Acid (A)	Present	Absent
2	Shigella, Serratia	Alkaline (K)	Acid (A)	Absent	Absent
3	Salmonella, Proteus	Alkaline (K)	Acid (A)	Present	Present
4	Pseudomonas	Alkaline (K)	Alkaline (K)	Absent	Absent

 

                                                       a                 b                c                 d

a)  Acid slant (Yellow) / Acid butt (Yellow) - Dextrose and Sucrose fermented or Dextrose and Lactose fermented or all the three sugars, Dextrose, Lactose and Sucrose fermented.

b)     Alkaline slant (Pink) / Acid butt (Yellow) - Only Dextrose fermented.

c)     Black precipitate present - H₂S production

d)    Bubbles or cracks present - Gas (CO₂) production.

Figure – 31.1: Triple Sugar Iron (TSI) test