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 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. Let’s 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: If lactose or sucrose or both sugar are fermented, a
large amount of acid will produce which turns both butt and slant yellow. So the
appearance of yellow color in both slant and butt indicates that the isolate has the
ability to ferment lactose or sucrose or both.
 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 a source of nitrogen. (Remember that
whenever peptone is utilized under aerobic condition ammonia is produced)
 Why Sucrose 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).

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 carbon
dioxide 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 constituent 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 -).
5. some example of Triple Sugar Iron (TSI) Agar Reactions:

Name of the organism Slant Butt Gas H2S

Acid (A) Acid (A) Pos (+) Neg (-)
Escherichia, Klebsiella, Enterobacter
Alkaline (K) Neg (-) Neg (-)
Shigella, Serratia Acid (A)
 Alkaline (K)
Salmonella, Proteus Acid (A) Pos (+) Pos (+)
Pseudomonas Alkaline (K) Alkaline (K) Neg (-) Neg (-)

Amies Transport Medium

 Amies medium is an improved transport medium, containing charcoal to prolong the
viability of pathogenic organisms.
 It is a semisolid media recommended for use in qualitative procedures for the
transport of clinical swab specimens to the laboratory.
 It is the modified Stuart’s Transport Medium produced by replacing glycerophosphate
with an inorganic phosphate buffer and adding charcoal to the medium.
 This modified medium gave a higher percentage of positive results than the transport
medium of Stuart.

Amies Transport Medium provides a reduced environment due to the presence of sodium
thioglycollate and the small amount of agar. Charcoal helps to neutralize materials that are toxic to
sensitive pathogens like Neisseria gonorrhoeae. Calcium magnesium, potassium and sodium salts
help the survival of gonococcal cells and also control the permeability of bacterial cells. Phosphates
buffer the medium.

Uses of Amies Transport Medium

1. Amies transport medium is used for collecting, transporting and preserving

microbiological specimens.
2. It is formulated to maintain the viability of microorganisms without a significant
increase in growth, being nonnutritive, phosphate buffered and semi-solid.
3. It is a transport medium used to preserve the viability of anaerobes such
as Neisseria gonorrhea and other pathogens from swabs.
4. It is used for preservation of microbiological specimen.
5. Amies Transport Medium is recommended for the throat, vaginal, and wound
samples.
Limitations of Amies Transport Medium

 The old medium should be freshly steamed and the charcoal resuspended before use.
 Some growth of contaminants may also occur during the long period of transport.
 After transportation, the specimen should be inoculated in the proper medium as soon
as possible. For optimum results, the time lapse between sample collection and
inoculum onto culture medium should be reduced to the minimum.
 It may not be suitable for the transport of fastidious organisms.
 Gonococci survive well in Amies Transport Medium for at least 6 to 12 hours
provided they are not exposed to temperature extremes. By 24 hours, the numbers of
gonococci decrease to an extent that may prevent their recovery if small numbers
were present initially in the specimen.

Result Interpretation on Amies Transport Medium

A fastidious organism is any organism that has a complex nutritional requirement. In other words, a
fastidious organism will only grow when specific nutrients are included in its diet. The more
restrictive term fastidious microorganism is often used in the field of microbiology to describe
microorganisms that will grow only if special nutrients are present in their culture medium.[1] Thus
fastidiousness is often practically defined as being difficult to culture, by any method yet tried. An
example of a fastidious bacterium is Neisseria gonorrhoeae, which requires blood or hemoglobin and
several amino acids and vitamins to grow.[2] Other examples include Campylobacter spp. and
Helicobacter spp., which are capnophilic – require elevated CO2 – among other requirements.