Triple Sugar Iron Agar
Triple Sugar Iron Agar M021S
Triple Sugar Iron Agar is used for the identification of gram-negative enteric bacilli on the basis of dextrose, lactose and
sucrose fermentation and hydrogen sulphide production.
Composition**
Ingredients Gms / Litre
Peptone 20.000
Meat extract 3.000
Lactose 10.000
Sucrose 10.000
Dextrose 1.000
Sodium chloride 5.000
Ferrous sulphate, heptahydrate 0.200
Sodium thiosulphate, pentahydrate 0.300
Phenol red 0.024
Agar 12.000
Final pH ( at 25°C) 7.4±0.2
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 64.32 grams (the equivalent weight of dehydrated media per litre) in 1000 ml distilled water. Heat to boiling to
dissolve the medium completely. Mix well and distribute into test tubes. Sterilize by autoclaving at 15 lbs pressure (121°C) for
15 minutes. Allow the medium to set in sloped form with a butt about 1 inch long.
Principle And Interpretation
Triple Sugar Iron Agar was originally proposed by Sulkin and Willett (1) and modified by Hajna (2) for identifying
Enterobacteriaceae . This medium complies with the recommendation of APHA, for the examination of meat and food
products (3), for the examination of milk and dairy products (4) and for microbial limit test for confirming the presence of
Salmonellae (5, 6) and in the identification of gram-negative bacilli (6, 7).
ISO Committee (8) and BIS (11) has recommended a slight modification for the identification of Salmonellae. BIS has
recommended the medium (M021S) for detection of Escherichia coli (9) and Vibrio s (10).
Peptone, yeast extract and meat extract provide nitrogenous compounds, sulphur, trace elements and vitamin B complex etc.
Sodium chloride maintains osmotic equilibrium. Lactose, sucrose and glucose are the fermentable carbohydrates. Sodium
thiosulphate and ferric or ferrous ions make H2S indicator system. Phenol red is the pH indicator. Organisms that ferment
glucose produce a variety of acids, turning the colour of the medium from red to yellow. More amount of acids are liberated
in butt (fermentation) than in the slant (respiration). Growing bacteria also form alkaline products from the oxidative
decarboxylation of peptone and these alkaline products neutralize the large amounts of acid present in the butt. Thus the
appearance of an alkaline (red) slant and an acid (yellow) butt after incubation indicates that the organism is a glucose
fermenter but is unable to ferment lactose and/or sucrose. Bacteria that ferment lactose or sucrose (or both), in addition to
glucose, produce large amounts of acid. Thus no reversion of pH in that region is possible and thus bacteria exhibit an acid slant
and acid butt. Gas production (CO2) is detected by the presence of cracks or bubbles in the medium, when the accumulated
gas escapes. Thiosulphate is reduced to hydrogen sulphide by several species of bacteria and H2S combines with ferric ions of
ferric salts to produce the insoluble black precipitate of ferrous sulphide. Reduction of thiosulphate proceeds only in an acid
environment and blackening usually occurs in the butt of the tube. Triple Sugar Iron Agar should be used in parallel with Urea
Agar / Broth (M112/M111) to distinguish between Salmonella and Proteus species. The reactions can be summarized
as follows:
Alkaline slant / acid butt- Only glucose fermented
Acid slant / acid butt- Glucose and sucrose fermented or glucose and lactose fermented or all the three sugars, glucose, lactose
and sucrose fermented.
Bubbles or cracks present- Gas production
Black precipitate present- H2S gas production
Some members of the Enterobacteriaceae and some H2S producing Salmonella may not be H2S positive on TSI Agar.
Some bacteria may show H2S production on Kligler Iron Agar but not on TSI Agar. This can happen because utilization of
sucrose in TSI Agar suppresses the enzymic pathway that result in H2S production.
Quality Control
Appearance
Light yellow to pink coloured homogeneous free flowing powder
Gelling
Firm, comparable with 1.2% Agar gel.
Colour and Clarity of prepared medium
Pinkish red coloured clear to slightly opalescent gel forms in tubes as slants
Reaction
Reaction of 6.43%w/v aqueous solution at 25ºC. pH : 7.4±0.2
Cultural Response
M021S: Cultural characteristics observed after an incubation at 35 - 37°C for 18-24 hours.
Organism Inoculum
(CFU)
Growth Slant Butt Gas H2S
Cultural Response
Citrobacter freundii ATCC
8090
50-100 luxuriant acidic
reaction,yellowing
of the medium
acidic
reaction,yellowing
of the medium
positive
reaction
positive
reaction,blackening
of medium
Enterobacter aerogenes
ATCC 13048
50-100 luxuriant acidic
reaction,yellowing
of the medium
acidic
reaction,yellowing
of the medium
positive
reaction
negative
reaction,no
blackening of
medium
Escherichia coli ATCC
25922
50-100 luxuriant acidic
reaction,yellowing
of the medium
acidic
reaction,yellowing
of the medium
positive
reaction
negative
reaction,no
blackening of
medium
Klebsiella pneumoniae
ATCC 13883
50-100 luxuriant acidic
reaction,yellowing
of the medium
acidic
reaction,yellowing
of the medium
positive
reaction
negative
reaction,no
blackening of
medium
Proteus vulgaris ATCC
13315
50-100 luxuriant alkaline
reaction,red
colour of the
medium
acidic
reaction,yellowing
of the medium
negative
reaction
positive
reaction,blackening
of medium
Salmonella Paratyphi A
ATCC 9150
50-100 luxuriant alkaline
reaction,red
colour of the
medium
acidic
reaction,yellowing
of the medium
positive
reaction
negative
reaction,no
blackening of
medium
Salmonella Typhi ATCC
6539
50-100 luxuriant alkaline
reaction,red
colour of the
medium
acidic
reaction,yellowing
of the medium
negative
reaction
positive
reaction,blackening
of medium
Salmonella Typhimurium
ATCC 14028
50-100 luxuriant alkaline
reaction,red
acidic
reaction,yellowing
of the medium
positive
reaction
positive
reaction,blackening
of medium
colour of the
medium
Shigella flexneri ATCC
12022
50-100 luxuriant alkaline
reaction,red
colour of the
medium
acidic
reaction,yellowing
of the medium
negative
reaction
negative
reaction,no
blackening of
medium
Vibrio cholerae ATCC
15748
50-100 luxuriant alkaline
reaction,red
colour of the
medium
acidic
reaction,yellowing
of the medium
negative
reaction
negative
reaction,no
blackening of
medium
Storage and Shelf Life
Store below 30°C in tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label.
