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

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

Introduction of Peptone Water

Peptone water is the formulation of Shread, Donovan, and Lee. It is the simplest broth medium used for the growth of the organism and a base for determining carbohydrate fermentation patterns of non-fastidious micro-organisms. It is also used for the detection of indole production by the bacteria after the addition of Kovacs or Ehlrich reagent.

Principle of Peptone Water

Water is the source of hydrogen and oxygen. Sodium chloride is the source of electrolytes. Peptone is a complex mixture of partially digested proteins. It contains proteoses, amino acids, polypeptides, phosphates, minerals (K, Mg), and accessory growth factors like nicotinic acid and riboflavin. Peptone used in this medium is rich in tryptophan content. The presence of indole can be detected using either Kovacs or Ehlrich reagent. Peptone Water is also applied as a base for carbohydrate fermentation studies with the addition of sugar and indicators such as bromothymol blue, bromocresol purple, or phenol red. Gas formation is achieved using Durham’s tube.

Composition of Peptone Water

Ingredients Gms / Litre

  • Peptone: 10.0
  • Sodium chloride: 5.0
  • Final pH (at 25°C) 7.2±0.2

 Preparation of Peptone Water

  1. Suspend 15.0 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 Peptone 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 Peptone Water

Procedure of Peptone 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 24 hours ( or depending on the nature of suspected organisms).
  4. Examine for turbidity.

Result Interpretation 

Growth: Turbidity

No growth: Lacking turbidity

Control strain

Staphylococcus aureus ATCC V25923: Luxuriant growth

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 in Peptone 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 Peptone Water

  • Peptone water is the simplest medium and it is used as a growth medium as a base for carbohydrate fermentation media.
  • It is a liquid medium and also suitable for the detection of indole.
  • It is a minimal growth medium that is used for determining carbohydrate fermentation patterns of non-fastidious organisms (E. coli. Klebsiella, Proteus, etc).
  • It is useful as a diluent or for making suspensions of non-fastidious organisms for antimicrobial sensitivity testing, motility test, microbial enumeration procedures, and so on.
  • Converting peptone water in the alkaline zone ( pH 8.4) is suitable for the cultivation and enrichment of Vibrio cholerae (etiological agent of cholera).
  • This medium is also useful for the detection of gas formation (using Durham’s tube)

Limitations of Peptone Water

  • This is the simplest medium due to its low nutrient content and this is not useful for the growth or maintenance of the fastidious organism.
  • pH determination is mandatory since some sugar solutions may affect the pH of the Peptone Water and hence checking must be done.
  • 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

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