Triple Sugar-Iron Agar Test
Triple Sugar-Iron Agar is used for the determination
of carbohydrate fermentation and hydrogen
sulfide production in the identification of
gram-negative bacilli.
Triple Sugar-Iron Agar contains three sugars
(glucose, lactose and sucrose), phenol red
for detecting carbohydrate fermentation and
ferrous ammonium sulfate for detection of
hydrogen sulfide production.
Carbohydrate fermentation is detected by the
presence of gas and a visible color change
(from red to yellow) of the pH indicator,
phenol red. The production of hydrogen sulfide
is indicated by the presence of a precipitate
that blackens the medium in the butt of the
tube.
Requirements
24 our typticase soy broth culture
Triple sugar-iron agar slants Media
inoculating needle
and Bunsen burner
Procedure:
• The experiment is conducted in a Laminar
Air Flow
• Take a Tryptic soy broth tube from the
rack containing 24hour culture
• Take an inoculation needle and heat it
vertically in the blue flame of the Bunsen
burner till red hot and then cool it for few
seconds
• Remove the cotton with the small finger
of your right hand and flame the mouth of
the tube
• With the cooled sterile needle, take culture
of organism from the test tube.
• Flame the mouth of the tube again, replace
the cotton and keep it back on the rack.
• Take a sterile Triple Sugar Iron (TSI)Agar
Slant from the rack
• Remove the cap and flame the mouth of
the tube
• Stab the needle containing the pure culture
into the medium in the butt of the TSI tube,
and then streak back and forth along the surface
of the slant
• Flame the mouth of the tube again, replace
the cap and keep it back on the rack.
• Flame the needle again and keep it on
the rack
• Incubate the inoculated tube and control
for 24 hours at 37°C
• After incubation, we can observe yellow
slant and yellow butt with bubbles. It means
Glucose, lactose or sucrose fermented and
also gas is produced.
• Depending on the organisms results can
vary.
