Hydrogen Sulfide (H2S) Production

Introduction Results Methods Photos


Triple Sugar Iron (TSI) agar slants differentiate bacteria on their ability to ferment glucose, lactose, and/or sucrose and on their ability to reduce sulfur to hydrogen sulfide.

TSI slants contain glucose (0.1%), lactose (1.0%) and sucrose (1.0%), ferrous (iron) sulfate and peptone (this is an alternative energy source that is not fermentable).

If there is no fermentation, then there should be no change (except for growth on the slanted surface).

If ONLY GLUCOSE is fermented, then the bottom of the slant (called the “butt”) will turn yellow.  However, there is not enough glucose in the medium to turn the top of the slant yellow.  When the glucose is used up, peptone (which is also present in the medium) is be utilized (aerobically) forming an alkaline by-product.  So the top of the slant (where oxygen is present) may turn deeper red.

If Glucose and Lactose or Glucose and Sucrose are fermented, then the entire slant will turn yellow.

If gas is produced, the agar may be lifted off the bottom of the test tube and the agar may also break apart.

Hydrogen sulfide (H2S) may be produced from the metabolism of ferrous sulfate. H2S forms a black precipitate.



Slant/Butt/Gas Glucose Lactose Sucrose H2S
Red/Red/None - - - -
Red/Yellow/None A - - -
Red/Yellow/Gas AG - - -
Yellow/Yellow/Gas AG AG ? -
Yellow/Black/Gas AG - AG +
Red/Black/Gas AG - - +



1. Obtain two TSI slants from the back shelf.

2. Use an inoculating pick to make a stab inoculation into one of the TSI slants.

3. Use an inoculating loop to inoculate the slanted portion of the same TSI slant.

4. Incubate at appropriate temperature (whatever temperature your organism grows well at). Incubate for 24 to 48 hours (do not exceed 48 to 72 hours for this test).

5. Obtain your slants from the incubator and observe the color and gas production.