1) Triple sugar iron agar is a differential medium used to identify enteric bacteria based on their ability to ferment sugars (glucose, lactose, sucrose) and reduce sulfur. 2) Organisms that ferment sugars will cause the medium to turn yellow, while those that do not ferment will leave it red. Patterns in color change between the slant and butt indicate which sugars were fermented. 3) Sulfur reducing organisms will produce black precipitates from hydrogen sulfide reacting with iron in the medium, which can provide additional information about the organism.

1. TRIPLETRIPLE
SUGAR IRONSUGAR IRON
AGARAGAR
PREPARED. BYPREPARED. BY::
GHASSANGHASSAN
ABU HAJJAJABU HAJJAJ..
SUPERVISORSUPERVISOR::
DR. Abdelraouf A. ElmanamaDR. Abdelraouf A. Elmanama

2. INTRODUCTION:
Triple sugar iron agar (TSI) is a
differential medium that contains
lactose, sucrose, a small amount of
glucose (dextrose), ferrous sulfate,
and the pH indicator phenol red. It
is used to differentiate enterics
based on the ability to reduce
sulfur and ferment carbohydrates.

3. THE TEST:
As with the phenol red fermentation
broths, if an organism can ferment
any of the three sugars present in the
medium, the medium will turn yellow.
If an organism can only ferment
dextrose, the small amount of
dextrose in the medium is used by the
organism within the first ten hours of
incubation..

4. After that time, the reaction that
produced acid reverts in the aerobic
areas of the slant, and the medium in
those areas turns red, indicating
alkaline conditions. The anaerobic
areas of the slant, such as the butt, will
not revert to an alkaline state, and they
will remain yellow. This happens with
Salmonella and Shigella.

5. NOTE:
SIM medium should be read after an
incubation of only 24 hours because a
longer incubation time can cause a
false negative. Vigorous fermenters
such as Escherichia coli and
Entrobacter cloacae will ferment all the
available sugars and then begin using
the amino acids. This will produce
amine groups and cause the medium to
turn alkaline.

6. If an organism can reduce sulfur, the
hydrogen sulfide gas which is produced
will react with the iron to form iron
sulfide, which appears as a black
precipitate. If the precipitate is formed, it
can mask any acid/alkaline results.
Sulfur reduction requires an acidic
environment, so if the black precipitate
is present, some fermentation took
place.

7. If the butt of the slant is obscured by the
precipitate, look at the top of the slant to
determine if the organism could ferment
only dextrose (red), or if it could ferment
either lactose and/or sucrose (yellow).
If the fermentation produced gas, you
may see fissures in the medium, or the
entire slant may be raised above the
bottom of the test tube.

8. Enterobacter cloacae exhibits fermenation of glucose and gas
production but no sulfur reduction
This would be read K/A,G. Staphylococcus aureus exhibits acidic
fermentation.
This would be read A/A. Salmonella typhimurium ferments glucose
and reduces sulfur. This would be read K/A, H2S. Micrococcus luteus
uses the amino acids and does not grow in the butt of the slant. This
would be read K/NC.

9. Bacillus megaterium fermented sugars but did
not grow in the anaerobic environment of the
butt. This would be read A/NC. Enterobacter
aerogenes fermented the sugars but turned to the
amino acids. This would be read K/A.

10. Results (slant/buttResults (slant/butt(( SymbolSymbol InterpretationInterpretation
Red/yellowRed/yellow K/AK/A Glucose fermentation only;Glucose fermentation only;
Peptone catabolizedPeptone catabolized
Yellow/yellowYellow/yellow A/AA/A Glucose and lactose and/orGlucose and lactose and/or
sucrose fermentationsucrose fermentation
Red/redRed/red K/KK/K No fermentation; PeptoneNo fermentation; Peptone
catabolizedcatabolized
Red/no color changeRed/no color change K/NCK/NC No fermentation; Peptone usedNo fermentation; Peptone used
aerobicallyaerobically
Yellow/yellow with bubblesYellow/yellow with bubbles A/A,GA/A,G Glucose and lactose and/orGlucose and lactose and/or
sucrose fermentation; Gassucrose fermentation; Gas
producedproduced
Red/yellow with bubblesRed/yellow with bubbles K/A,GK/A,G Glucose fermentation only; GasGlucose fermentation only; Gas
producedproduced
Red/yellow with bubbles andRed/yellow with bubbles and
black precipitateblack precipitate
K/A,G, H2SK/A,G, H2S Glucose fermentation only; GasGlucose fermentation only; Gas
produced; H2S producedproduced; H2S produced
Red/yellow with black precipitateRed/yellow with black precipitate K/A, H2SK/A, H2S Glucose fermentation only; H2SGlucose fermentation only; H2S
producedproduced
Yellow/yellow with blackYellow/yellow with black
precipitateprecipitate
A/A, H2SA/A, H2S Glucose and lactose and/orGlucose and lactose and/or
sucrose fermentation; H2Ssucrose fermentation; H2S
producedproduced
A=acid production; K=alkaline reaction; G=gas

11. A/AA/A E. coli, Yersinia, Aeromonas, VibrioE. coli, Yersinia, Aeromonas, Vibrio
K/AK/A Salmonella, Edwardsiella, ShigellaSalmonella, Edwardsiella, Shigella
K/KK/K nonfermenters such asnonfermenters such as PseudomonasPseudomonas
and othersand others
A/KA/K erroneously inoculated a Gram + or someerroneously inoculated a Gram + or some
other weird thingother weird thing
SOME COMMON SUGAR REACTIONS IN TSIA