Triple Sugar Iron Agar (TSI): Principle, Procedure and Interpretation

Whenever you see the name of this test i.e. Triple Sugar Iron Agar ,you have to
remember that it’s a test which has three sugar (Lactose, Sucrose, and Glucose)
and also iron; and it contains Agar Agar as solidifying agent (TSI is a semi solid
media having slant and butt).
CLUE: You might have (or not) realized the rationale behind the use of three
different sugar and adding iron. Lets start with very basic information and we will
proceed towards principle and interpretations.

Composition of Triple Sugar Iron Agar (TSI)

Lactose, Sucrose and Glucose in the concentration of 10:10:1 (i.e. 10 part Lactose
(1%), 10 part Sucrose (1%) and 1 part Glucose (0.1%)). TSI is similar to Kligler’s
iron agar (KIA),except that Kligler’s iron agar contains only two carbohydrates:
glucose (0.1%) and lactose (1%).
 0.1% Glucose: 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: a large amount of acid turns both butt and
slant yellow, thus indicating the ability of the culture to ferment either
lactose or sucrose.
 Iron: Ferrous sulfate: Indicator of H2S formation
 Phenol red: Indicator of acidification (It is yellow in acidic condition and
red under alkaline conditions).
 It also contains Peptone which acts as source of nitrogen. (Remember that
when ever peptone is utilized under
aerobic condition ammonia is
produced)

Why Sucorse is added in TSI?

Inoculation in TSI Agar
Addition of sucrose in TSI Agar
permits earlier detection of coliform
bacteria that ferment sucrose more rapidly
than lactose. Adding sucrose also aids the
identification of certain gram-negative
bacteria that could ferment sucrose but not
lactose. Other basic understanding is TSI
Tube contains butt (poorly oxygenated area
on the bottom) slant (angled well
oxygenated area on the top).
Inoculation in TSI Agar

Procedure for Triple Sugar Iron Agar (TSI) Test

1. With a sterilized straight inoculation needle touch the top of a well-isolated
colony
2. Inoculate TSI Agar by first stabbing through the center of the medium to
the bottom of the tube and then streaking on the surface of the agar slant.
 3. Leave the cap on loosely and incubate the tube at 35°C in ambient air for 18
to 24 hours.

Interpretation of Triple Sugar Iron Agar Test

1. If lactose (or sucrose) is fermented, a large amount of acid is produced,
which turns the phenol red indicator yellow both in butt and in the
slant. Some organisms generate gases, which produces bubbles/cracks on
the medium.
2. If lactose is not fermented but the small amount of glucose is, the oxygen
deficient butt will be yellow (remember that butt comparatively have more
glucose compared to slant i.e. more media more glucose), but on the slant
the acid (less acid as media in slant is very less) will be oxidized to
carbondioxide and water by the organism and the slant will be red (alkaline
or neutral pH).
3. If neither lactose/sucrose nor glucose is fermented, both the butt and the
slant will be red. The slant can become a deeper red-purple (more alkaline)
as a result of production of ammonia from the oxidative deamination of
amino acids (remember peoptone is a major constitutents of TSI Agar) .
4. if H2S is produced, the black color of ferrous sulfide is seen.

So the expected results of TSI Agar test are:

Triple Sugar Iron Agar Test Results

Image source: Clark College
1. Alkaline slant/no change in butt
(K/NC) i.e Red/Red = glucose,
lactose and sucrose non-fermenter
2. Alkaline slant/Alkaline butt (K/K)
i.e Red/Red = glucose, lactose
and sucrose non-fermenter
3. Alkaline slant/acidic butt (K/A);
Red/Yellow = glucose
fermentation only, gas (+ or -),
H2s (+ or -)
4. Acidic slant/acidic butt (A/A);
Yellow/Yellow = glucose, lactose
and/or sucrose fermenter gas (+ or
-), H2s (+ or -).
Some example of Triple Sugar Iron (TSI) Agar Reactions:

Name of the
organisms Slant Butt Gas H2S

Escherichia,
Klebsiella, Acid Acid
Enterobacter (A) (A) Pos (+) Neg (-)

Shigella, Alkaline Acid

Serratia (K) (A) Neg (-) Neg (- )

Salmonella, Alkaline Acid

Proteus (K) (A) Pos (+) Pos (+)

Alkaline Alkaline
Pseudomonas (K) (K) Neg (-) Neg (-)
Blood Agar: Composition, Preparation, Uses and Types of Hemolysis

Blood agar is an enriched, bacterial growth medium. Fastidious organisms, such

as streptococci, do not grow well on ordinary growth media. Blood agar is a type
of growth medium (trypticase soya agar enriched with 5% sheep blood) that
encourages the growth of bacteria, such as streptococci, that otherwise wouldn’t
grow.
Blood contains inhibitors for certain bacteria such
as Neisseriaand Haemophilus genera and the blood agar must be heated to
inactivate these inhibitors and to release essential growth factors (e.g., V factor) .
Heating of blood agar converts it into chocolate agar (heated blood turns a
chocolate color) and supports the growth of these bacteria.

Beta hemolysis in sheep blood agar.

Blood agar consists of a base containing a protein source (e.g. Tryptones),
soybean protein digest, sodium chloride (Nacl), agar and 5% sheep blood.
Composition of Blood Agar:
 Pancreatic digest of casein
 Papaic digest of soy meal
 NaCl
 Agar
 Distilled water
Combine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and
sterilize by autoclaving.
Procedure for the preparation of Blood Agar
1. Prepare the blood agar base as instructed by the manufacturer. Sterilize by
autoclaving at 121°C for 15 minutes.
2. Transfer thus prepared blood agar base to a 50°C water bath.
3. When the agar base is cooled to 50°C, add sterile blood agar aseptically and
mix well gently. Avoid formation of air bubbles. You must have warmed
 the blood to room temperature at the time of dispensing to molten agar base.
(Note: If you are planning to prepare a batch of blood agar plates, prepare
few blood agar plates first to ensure that blood is sterile).
4. Dispense 15 ml amounts to sterile petri plates aseptically
5. Label the medium with the date of preparation and give it a batch number (if
necessary).
6. Store the plates at 2-8°C, preferably in sealed plastic bags to prevent loss of
moisture. The shelf life of thus prepared blood agar is up to four weeks.
Quality control of Blood Agar
1. The pH of the blood agar range from 7.2 to 7.6 at room temperature.
2. Inoculate the plates with 5 hour broth cultures of Streptococcus
pyogenes and S. pneumoniae. Inoculate also a plate with H. influenzae and
streak with S. aureus (i.e. Satellitism Test).
3. Incubate the plates in a carbon dioxide enriched atmosphere at 35-
37°C overnight.
4. Check for the growth characteristics of each species
1. S. pyogenes: Beta-hemolysis
2. S. pneumoniae: Alpha-hemolysis
3. Satellitism of H. influenzae

Optochin and Bacitracin Sensitivity of the isolates in Blood Agar

Uses of Blood Agar:
Blood agar has two major uses:
1. Isolation, identification (with the use of either Optochin disc or Bacitracin
discand testing the sensitivity of the isolate) and antimicrobial susceptibility
of Streptococci.
2. Determine the type of hemolysis, if any.
Blood Agar and Hemolysis
Certain bacterial species produce extracellular enzymes that lyse red blood cells in
the blood agar (hemolysis). These hemolysin (extotoxin) radially diffuses
outwards from the colony (or colonies) causing complete or partial destruction of
the red cells (RBC) in the medium and complete denaturation of hemoglobin
within the cells to colorless products.

Four types of hemolysis are produced in Sheep blood agar by Streptococci

namely; Alpha hemolysis, Beta hemolysis, gamma hemolysis and alpha prime or
wide zone alpha hemolysis.

Hemolysis is best observed by examining colonies grown under anaerobic

conditions or inspecting sub-surface colonies.
How does one know if the colonies they are observing on a plate have
caused alpha hemolysis or beta hemolysis?
Note: To know the type of blood agar hemolysis, the blood agar plate must be
held up to a light source and observed with the light coming from behind
(transmitted light).
If either type of hemolysis is present, then one will observe a zone of
hemolysis surrounding a growing colony.
Alpha hemolysis: Partial lysis of the RBC to produce a greenish-gray or brownish
discoloration around the colony. α hemolysis is due to the reduction of RBC
hemoglobin to methemoglobin in the medium surrounding the colony. Many of
the alpha hemolytic streptococci are part of the normal body flora.
But Streptococcus pneumoniae which is also alpha hemolytic causes serious
pneumonia and other deadly infectious disease.
Various types of Hemolysis
Beta Hemolysis:Complete lysis of Red Blood Cells, causing a clearing of blood
from the medium under and surrounding the colonies e.g. Group A beta hemolytic
streptococci-Streptococcus pyogenes and Group B, beta hemolytic streptococci-
Streptococcus agalactiace. For group A streptococci maximal activity of both the
hemolysins; Oxygen labile SLO and oxygen stable SLS hemolysins is observed
only in anaerobic conditions.
Gamma or non hemolysis: No hemolysis of RBC. No change of the medium
under and surrounding the colonies.
Alpha prime or wide zone alpha hemolysis: A small zone of intact erythrocytes
immediately adjacent to bacterial colony, with a zone of complete red-cell
hemolysis surrounding the zone of intact erythrocytes. This type of hemolysis
may be confused with Beta hemolysis.
Double Zone hemolysis produced by Clostridium perfringens
Target Hemolysis: Clostridium perfringens is readily identified in the laboratory
by its characteristic “double zone” hemolysis also known has target hemolysis.