Microbiology
Triple Sugar Iron (TSI)
p1/5
Name:_________________________________
TRIPLE SUGAR IRON AGAR AND ITS
USE
©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103.
Prep of TSI slants
Variety of TSI
reactions
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 H2S.
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 areformed 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 H2S, this chemical will react with
the iron to form ferrous sulfide, which is seen as a black precipitate in the butt (a black butt).
PREPARATION OF MEDIA: (for 40 tubes)(NOTE: prepare at beginning of class.)
1. Weigh out medium, dissolve: 65 g dry medium, add to 1000 mL water, heat to boiling to
dissolve.
2. Deliver aliquots to tubes: 6 mL to16 x 150 mm tubes with a pipet. Cover with closures.
3. Autoclave capped tubes.
4. Creating the slant: Position the rack so that the tubes (with liquid medium in them) have
a 3 cm slant with a 2-3 cm butt. Let cool until solid.
Microbiology
Triple Sugar Iron (TSI)
p2/5
Name:_________________________________
INOCULATION:
1. Inoculate the slants using a pure culture with a stab by streaking over the entire surface of
the slant (zig-zag to cover surface)and then stabbing deep into the butt.
1. Inoculate 3 TSI tubes one each of:
1. Escherichia coli
2. Pseudomonas aeruginosa
3. Salmonella typhimurium
2. Incubate at 37ºC for 24 hours (48 hr may be necessary to show all H2S reactions).
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
does not ferment either lactose or sucrose Example red slant
R
YELLOW
Y
ferments lactose and/or sucrose
Example yellow slant
SCORING THE BUTT COLOR AND CONDITION:
BUTT
Code
Interpretation
Example
COLOR/CONDITION Letter
no fermentation, the bacterium is
RED
Example red butt
R
an obligate aerobe
Y
some fermentation has occurred,
acid has been produced, it is a
Example yellow butt
facultative anaerobe.
GAS FORMED
YG
Seen as cracks in the agar,
bubbles, or the entire slant may
be pushed out of the tube.
Example yellow butt with gas
(Caution: these gassy fermenters
may have bacteria close to the
opening.)
BLACK
"+"
H2S has been produced
YELLOW
Example black butt
Microbiology
Triple Sugar Iron (TSI)
p3/5
Name:_________________________________
After you have scored your TSI Agar slants, you should suggest a species of bacterium which
matches those metabolic traits discovered. While by no means definitive, the following are TSI
Agar reactions typical of a number of prominent species which can be distinguished by this
medium:
Score your cultures
BACTERIUM
SLANT BUTT H2S COMMENTS
Salmonella typhimurium
Escherichia coli
Pseudomonas aeruginosa
ACTION
indicate with +/Ferments Glucose
Ferments Lactose
Produces Gas
Produces H2S
Salmonella
typhimurium
Escherichia. coli
Pseudomonas
aeuginosa
Microbiology
Triple Sugar Iron (TSI)
p4/5
Name:_________________________________
TYPICAL REACTIONS OF SELECTED ENTERICBACTERIA:
BACTERIUM
SLANT BUTT
H2S 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
+
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
one of "paracolon" group (non-pathogenic)
Uninoculated
R/R Obligate aerobe
R/Y +;Glucose fermented H2S gas formed
R/Y; Only Glucose fermented
Y/Y +; Lactose + / or Sucrose fermented & H2S
Y/Y G; Lactose + / or Sucrose fermented & Gas
Y/Y G; Lactose + / or Sucrose fermented & Gas
Y/Y ; Lactose + / or Sucrose fermented & No Gas
Microbiology
Example of Yellow
Butt Y
Triple Sugar Iron (TSI)
p5/5
Name:_________________________________
Yellow Butt with
Gas YG
Black Butt H2S Gas
Yellow Slant
Red Butt/ Red Slant
Red Slant