INTRODUCTION:

Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative
enteric bacteria based on their physiological ability (or lack thereof) to:
a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H
2
S.
The medium contains 1.0% each of sucrose and lactose and 0.1% glucose. If only glucose is fermented, acid
produced in the butt will turn it yellow, but insufficient acid products are formed to affect the methyl red in the
slant. However, if either sucrose or lactose are fermented, sufficient fermentation products will be formed to
turn both the butt and the slant yellow. If gas is formed during the fermentation, it will show in the butt either as
bubbles or as cracking of the agar. If no fermentation occurs (as for an obligate aerobe), the slant and butt will
remain red. The medium also contains ferrous sulfate. If the bacterium forms H
2
S, this chemical will react with
the iron to form ferrous sulfide, which is seen as a black precipitate in the butt (a black butt). The following
table summarizes these reactions:


TYPICAL REACTIONS OF SELECTED ENTERICBACTERIA:



TRIPLE SUGAR
IRON
INGREDIENTS

FUNCTION

RESULT/INTERPRETATION
phenol red a pH indicator:
below 6.8 it is yellow
above 82., it is red
Phenol red turns yellow in an acid environment. It thus indicates
whether the acids of fermentation have been produced. Failure to
turn the butt yellow indicates that no fermentation has occured, and
that the bacterium is an obligate aerobe.
0.1 % glucose if only glucose is fermented,
only a small amount of acid
is produced
If only glucose is fermented, only enough acid is produced to turn the
butt yellow. The slant will remain red.
1.0 % lactose
1.0% sucrose
if the culture can ferment
either lactose (lac+) and/or
sucrose (suc+), a large
amount of acid is produced
a large amount of acid turns both butt and slant yellow, thus
indicating the ability of the culture to ferment either lactose or
sucrose
FeSO4
(ferrous
sulfate)
A source of iron and sulfur A few bacteria are capable of reducing the SO4= to H2S (hydrogen
sulfide).
The iron combines with the H2S to form FeS (ferrous sulfide) a black
compound. This will turn the butt black. Thus, a black butt indicates
H2S production.



TRIPLE SUGAR IRON AGAR: INTERPRETATION OF RESULTS
SCORING THE SLANTS: Examine the slant and butt, and record data using the following criteria:

SLANT COLOR: Code letter: Interpretation Example
RED R does not ferment either lactose or sucrose Example red slant
YELLOW Y ferments lactose and/or sucrose Example yellow slant
SCORING THE BUTT COLOR AND CONDITION:

BUTT
COLOR/CONDITION
Code
Letter
Interpretation Example
RED R
no fermentation, the bacterium is an obligate
aerobe
Example red butt
YELLOW Y
some fermentation has occurred, acid has
been produced, it is a facultative anaerobe.
Example yellow butt
GAS FORMED YG
Seen as cracks in the agar, bubbles, or the
entire slant may be pushed out of the tube.
(Caution: these gassy fermenters may have
bacteria close to the opening.)
Example yellow butt with gas
BLACK "+" H
2
S has been produced Example black butt

BACTERIUM SLANT BUTT H
2
S COMMENTS
Shigella dysenteriae R Y

Causes food infection dysentery
Salmonella typhimurium R YG + Causes food poisoning
Salmonella typhi R Y + Causes typhoid fever
Aerobacter aerogenes Y YG

Similar to Klebsiella,but
nonrespiratory
Escherichia coli Y YG

Most common of GIflora
Citrobacter freundii Y YG +
One of "paracolon" group (non-
pathogenic)
Proteus vulgaris Y YG + Causes GU infections, highly motile
Klebsiella pneumoniae Y
R or
YG
Pneumonia in debilitated patients
(nosocomial)
Pseudomonas
aeruginosa
R R

GI inhabitant, causes wound, GU
infections
Alcaligenes faecalis R R

GI inhabitant, opportunistic
pathogen of GU



A/A/G-/H-
- Acid slant (A)
- Acid butt (A)
- Unable to produce gas (G-)
- Unable to produce H2S (H-

A/A/G-/H+
- Acid slant (A)
- Acid butt (A)
- Unable to produce gas (G-)
- Able to produce H2S (H+)

K/K/G-/H-
- Alkaline slant (K)
- Alkaline butt (K)
- Unable to produce gas (G-)
- Unable to produce H2S (H-)

All of these ingredients are mixed together and allowed to solidify in the test tube at a slanted angle. The slanted
shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying
degrees (an aerobic environment) or not exposed to air (an anaerobic environment).
TSI agar medium was developed based on Kligler's iron agar, which had been used for the determination of
lactose fermentative bacteria, by addition of sucrose to be able to detect sucrose fermentative bacteria also.