Triple Sugar Iron Test: Do’s & Don’ts

The Triple Sugar Iron test is the most widely used tubed differential medium in the identification of the Enterobacteriaceae by assessing carbohydrate fermentation and H2S generation, although it is helpful for other gram-negative bacteria as well.

To help identify intestinal gram-negative bacilli, differential agar with two sugars was first used. Kligler noticed that bacteria produce hydrogen sulfide and utilized phenol red as a pH indicator while adding lead acetate and iron salts to detect hydrogen sulfide generation to create Kigler's Iron Agar. But when a third sugar was introduced, this agar underwent further modification. The addition of the third sugar, sucrose, made it possible to identify coliform bacteria earlier since sucrose is fermented more quickly than lactose. Additionally, the presence of sucrose facilitated the discovery of certain gram-negative bacteria that could ferment sucrose but not lactose. 

Three types of carbohydrates can be found in iron agar: lactose (1%), sucrose (1%), and glucose (0.1%). Tubes carrying molten agar are tilted during preparation so that medium has an aerobic slant and an anaerobic bottom. Bacteria can break down carbohydrates either fementatively (without oxygen) or aerobically (with oxygen). The medium will turn from reddish-orange (the initial hue) to yellow when any of the sugars are fermented due to the pH reduction. Alkalization of the peptones is indicated by a deep red colour. Some bacteria convert sodium thiosulfate in the media to hydrogen sulfide (H2S), an invisible gas. The medium's ferric ions and hydrogen sulfide will combine to form iron sulfide, a precipitate that is insoluble and dark in color. A TSI slant can be interpreted in a number of different ways depending on how carbohydrates are used and how much hydrogen sulfide is produced. 

Let's look at some dos and don'ts for this amazing colourful test, to help us correctly read the results.

References:

1. MacFaddin, J. Biochemical Tests for Identification of Medical Bacteria. 3rd ed. Lippincott Williams and Wilkins. Philadelphia,2000.

2. Forbes BA, Sahm DF, Weissfeld AS. Bailey and Scott’s diagnostic microbiology. 13th ed. St. Louis: Mosby, 2013.

3. Lehman D. Triple Sugar Iron Agar Protocols. American Society for Microbiology,2005.