Tryptone Water: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Tryptone Water: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Tryptone Water

Tryptone water is used for the detection of indole production by coliforms ( E. coli, Klebsiella oxytoca).

Principle of Tryptone Water

Water is the source of hydrogen and oxygen. Sodium chloride is the source of electrolytes. Casein enzymic hydrolysate is a good substrate for indole production due to its high tryptophan content. Certain bacteria break down the amino acid tryptophan with the help of enzymes that mediate the production of indole by hydrolytic activity. The indole formation can be detected by either Kovacs or Ehrlich’s reagent. Indole combines with the aldehyde present in the reagent to give red color to the alcoholic layer. The alcohol layer extracts and concentrates the red color complex.

Composition of Tryptone Water

Ingredients   Gms / Litre

  • Casein enzymic hydrolysate 20.0
  • Sodium chloride 5.0
  • Distilled water: 1000 ml
  • Final pH ( at 25°C) 7.5±0.2

 Preparation of Tryptone Water

  1. Suspend 25.0 grams of the dehydrated medium in 1000 ml purified/distilled water.
  2. Heat to boiling to dissolve the medium completely.
  3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes i.e. validated cycle.

Storage and Shelf life of Tryptone Water

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration ( discoloration), contamination (turbidity).
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for Tryptone Water

Procedure of Tryptone Water

  1. Allow the medium to warm at 37°C or to room temperature before inoculating.
  2. Inoculate the specimen/ test organism.
  3. Incubate it aerobically at  35-37°C for 18-24 hours.
  4. Examine for turbidity.
  5. Add 0.5 ml of indole reagent directly to the tube and agitate.
  6. Allow the tubes to stand for 5-10 minutes. The formation of a red ring at the top of the tube indicates indole production ( as shown above picture).

Result Interpretation 

Growth: Turbidity

No growth: Lacking turbidity

Control strain

Enterobacter aerogenes ATCC 13048: Luxuriant growth without color or ring

Escherichia coli ATCC 25922: Luxuriant growth with a red ring at the interface of the medium on the addition of Kovac’s reagent

Uninoculated tube: No growth

Visible growth of various organisms inTryptone Water

  1. Turbidity – Aerobic gram-negative bacilli
  2. Pellicle formation – Bacillus species, Pseudomonas species, Yeast cells
  3. Clotting  Staphylococcus aureus
  4. Visible colonies ( puffballs) -Staphylococci

Uses of Tryptone Water

Tryptone water is initially used for the cultivation and maintenance of fastidious aerobic and facultative bacteria such as E. coli and Pseudomonas species. A liquid medium for the detection of indole-forming bacteria because of its high content of tryptophan, it is more reliable than peptone water for this purpose.

Keynotes on Tryptone Water

  • Coliform bacteria are facultatively anaerobic, Gram-negative, non-spore-forming rods that ferment lactose vigorously to acid and gas at 35 ± 2 °C within 24 or 48 hours. They generally belong to four genera of the Enterobacteriaceae:  E. coli, and Klebsiella, Enterobacter, and Citrobacter.
  • Modified Tryptone Water with Brilliant Green Bile Broth 2 % is used to determine the most probable number (MPN) of E. coli in the food sample.
  • Indole testing is recommended as an aid in the differentiation of organisms based on indole production. For confirmation of the organisms, further biochemical tests are mandatory.

Limitations of Tryptone Water

  • Subcultures on a solid medium may be necessary to ensure the purity of the inoculant. Mixed or contaminated growth will give false reactions.
  • Some strains may show poor growth because of nutritional variations.
  • The organism cannot be confirmed by this medium and thus biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

Further Readings on Tryptone Water

  • http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?
  • https://www.himedialabs.com/TD/M463I.pdf
  • https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/coliform-bacteria
  • https://www.sigmaaldrich.com
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