Plot No. C40, Road No. 21Y, MIDC, Wagle Industrial Estate,
Thane(W)-400604, MH, India.
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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
| Ingredients | Gms/Litre |
|---|---|
| HM Peptone## | 20.000 |
| Yeast extract | 3.000 |
| HM extract# | 3.000 |
| Lactose | 10.000 |
| Sucrose | 10.000 |
| Dextrose (Glucose) | 1.000 |
| Sodium chloride | 5.000 |
| Ferrous sulphate | 0.300 |
| Sodium thiosulphate | 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
# Equivalent to Meat extract
## Equivalent to Meat peptone
Directions
Streak the test inoculum aseptically into the slant and incubate at appropriate conditions. Incubate the slants at 30-35°C for 18-24 hours.
Principle And Interpretation
Triple Sugar Iron Agar was 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 HM peptone, yeast extract and HM extract provide nitrogenous compounds, sulphur, trace elements and vitamin B complex etc. Sodium chloride maintains osmotic equilibrium. Lactose, sucrose and dextrose are the fermentable carbohydrates. Sodium thiosulphate and 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 enables no reversion of pH in that region 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. 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
Type of specimen
Isolate microorganism from water, food
Specimen Collection and Handling
For food samples, follow appropriate techniques for sample collection and processing as per guidelines. For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards. After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- Some members of the Enterobacteriaceae and 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 enzymatic pathway that result 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 when stored at recommended temperature.
Quality Control
Appearance
Sterile Triple Sugar Iron agar slant in long glass tube (screw cap tubes).
Colour of medium
Red coloured slant
Quantity of medium
14 ml of medium in glass tube
Reaction
7.20-7.60
Sterility Check
Passes release criteria
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.
| Organism | Inoculum (CFU) | Growth | Slant | Butt | Gas | H2S |
|---|---|---|---|---|---|---|
| Citrobacter freundii ATCC 8090 | 50-100 | luxuriant | acidic reaction, yellowing of the medium | acidic reaction, yellowing of the medium | positive reaction | positive, blackening of medium |
| # Klebsiella aerogenes ATCC 13048 (00175*) | 50-100 | luxuriant | acidic reaction, yellowing of the medium | acidic reaction, yellowing of the medium | positive reaction | negative, no blackening of medium |
| Escherichia coli ATCC 25922 (00013*) | 50-100 | luxuriant | acidic reaction, yellowing of the medium | acidic reaction, yellowing of the medium | positive reaction | negative, no blackening of medium |
| Klebsiella pneumoniae ATCC 13883 (00097*) | 50-100 | luxuriant | acidic reaction, yellowing of the medium | acidic reaction, yellowing of the medium | positive reaction | negative, 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, 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, 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, blackening of medium |
| Salmonella Typhimurium ATCC 14028 (00031*) | 50-100 | luxuriant | alkaline reaction, red colour of the medium | acidic reaction, positive yellowing of the medium | positive reaction | positive, blackening of medium |
| Shigella flexneri ATCC 12022 (00126*) | 50-100 | luxuriant | alkaline reaction, red colour of the medium | acidic reaction, negative yellowing of the medium | negative reaction | negative, no blackening of medium |
Key: *Corresponding WDCM numbers, # Formerly known as Enterobacter aerogenes
Storage and Shelf Life
Store between 2-8°C. Use before expiry date on the label. Product performance is best if used within 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 and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques.
| Product Name | Triple Sugar Iron Agar Slant |
|---|---|
| SKU | SL045L |
| Customized Product Available | No |
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