Triple Sugar Iron Agar

Originally proposed by Sulkin and Willett and modified by Hajna for identifying Enterobacteriaceae . This medium complies with the recommendation of APHA, for the examination of meat and food products , for the examination of milk and dairy products and for microbial limit test for confirming the presence of Salmonellae and in the identification of gram-negative bacilli. The reactions can be interpreted as follows: Alkaline slant / acid butt- only glucose fermented, Acid slant / acid butt- dextrose and sucrose fermented or dextrose and lactose fermented or all the three sugars, dextrose,lactose and sucrose fermented, Bubbles or cracks present-gas production Black precipitate present-H2S gas production.

Intended Use:

Recommended for the identification of gram-negative enteric bacilli on the basis of dextrose, lactose and sucrose fermentation and hydrogen sulphide production.

Composition**

**Formula adjusted, standardized to suit performance parameters
# Equivalent to Beef extract

Ingredients	g / L
Peptone	10.000
Tryptone	10.000
Yeast extract	3.000
HM Peptone B#	3.000
Lactose	10.000
Sucrose	10.000
Dextrose (Glucose)	1.000
Sodium chloride	5.000
Ferrous sulphate	0.200
Sodium thiosulphate	0.300
Phenol red	0.024
Agar	12.000
Final pH (at 25 °C)	7.4 ± 0.2

Directions

Suspend 64.52 grams in 1000 ml purified/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), for microbial limit tests for confirming the presence of Salmonellae (5,6), and for the identification of gram-negative bacilli (5,7).

Tryptone, peptone, yeast extract and HM peptone B provide nitrogenous compounds, sulphur, trace elements and vitamin B complex. Sodium chloride maintains osmotic equilibrium. Lactose, sucrose and dextrose are the fermentable carbohydrates. Sodium thiosulphate and ferrous ions make the H₂S indicator system. Phenol red is the pH indicator.

Organisms that ferment glucose produce acids, turning the medium from red to yellow. More acid is liberated in the butt (fermentation) than in the slant (respiration). Oxidative decarboxylation of peptone forms alkaline products that may neutralize acid in the slant. Thus an alkaline (red) slant and an acid (yellow) butt after incubation indicates a glucose fermenter that cannot ferment lactose and/or sucrose. Fermentation of lactose or sucrose (or both) in addition to glucose produces large amounts of acid, preventing reversion in the slant and yielding an acid slant and acid butt.

Gas production (CO₂) is detected by cracks or bubbles in the medium. Thiosulphate is reduced to H₂S by several species; H₂S combines with ferric ions to form an insoluble black precipitate (ferrous sulphide), usually in the butt. TSI should be used in parallel with Urea Agar/Broth (M112/M111) to distinguish between Salmonella and Proteus spp.

Reactions summary

• Alkaline slant / acid butt — only glucose fermented.
• Acid slant / acid butt — glucose and sucrose fermented or glucose and lactose fermented, or all three sugars fermented.
• Bubbles or cracks — gas production.
• Black precipitate — H₂S production.

Type of specimen

Pure bacterial isolate from water, food, or clinical sample.

Specimen Collection and Handling:

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (8,9). For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (3,4). For water samples, follow appropriate techniques for sample collection and processing as per guidelines and local standards (5). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic Use. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological laboratory practices while handling specimens and cultures. Standard precautions as per established guidelines should be followed when handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations :

1. Some members of the Enterobacteriaceae and H₂S-producing Salmonella may not be H₂S positive on TSI Agar.
2. Some bacteria may show H₂S production on Kligler Iron Agar but not on TSI Agar because utilization of sucrose in TSI Agar can suppress the enzymic pathway that results in H₂S production.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label, within the expiry period and when stored at the recommended temperature.

Quality Control

Appearance: Light yellow to pink 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 6.45% w/v aqueous solution at 25 °C, pH 7.4 ± 0.2
pH: 7.20–7.60

Cultural Response

Cultural characteristics observed after incubation at 35–37 °C for 18–24 hours.

Organism (ATCC)	Growth	Slant	Butt	Gas	H2S
Citrobacter freundii ATCC 8090	luxuriant	acidic reaction, yellowing of the medium	acidic reaction, yellowing of the medium	positive reaction	positive, blackening of medium
Klebsiella aerogenes ATCC 13048	luxuriant	acidic reaction, yellowing of the medium	acidic reaction, yellowing of the medium	positive reaction	negative, no blackening of medium
Escherichia coli ATCC 25922	luxuriant	acidic reaction, yellowing of the medium	acidic reaction, yellowing of the medium	positive reaction	negative, no blackening of medium
Klebsiella pneumoniae ATCC 13883	luxuriant	acidic reaction, yellowing of the medium	acidic reaction, yellowing of the medium	positive reaction	negative, no blackening of medium
Salmonella Typhimurium ATCC 14028	luxuriant	alkaline reaction, red colour of the medium	acidic reaction, yellowing of the medium	positive reaction	positive, blackening of medium
Shigella flexneri ATCC 12022	luxuriant	alkaline reaction, red colour of the medium	acidic reaction, yellowing of the medium	negative reaction	negative, no blackening of medium
Salmonella Paratyphi A ATCC 9150	luxuriant	alkaline reaction, red colour of the medium	acidic reaction, yellowing of the medium	positive reaction	negative, no blackening of medium
Salmonella Typhi ATCC 6539	luxuriant	alkaline reaction, red colour of the medium	acidic reaction, yellowing of the medium	negative reaction	positive, blackening of medium
Proteus hauseri ATCC 13315	luxuriant	alkaline reaction, red colour of the medium	acidic reaction, yellowing of the medium	negative reaction	positive, blackening of medium

Key: (*) Corresponding WDCM numbers. (#) Formerly known as Enterobacter aerogenes. ($) Formerly known as Proteus vulgaris.

Storage and Shelf Life

Store between 10–30 °C in a tightly closed container and the prepared medium at 2–8 °C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in a dry, ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within the stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials. Material that comes into contact with clinical samples must be decontaminated and disposed of in accordance with current laboratory techniques (8,9).