Peptone Water – Principle, Composition, Preparation, Uses

What is Peptone Water?

  • Peptone Water is a broth medium that follows the formulation developed by Shread, Donovan, and Lee. It serves as a growth medium for various organisms and acts as a foundation for determining the carbohydrate fermentation patterns of non-fastidious organisms. Additionally, Peptone Water is utilized in the detection of indole production by the organism.
  • This medium is highly recommended as a pre-enrichment medium for Enterobacteriaceae organisms, including Salmonella and Cronobacterium species. It is commonly employed in the analysis of food and animal feeding stuffs, water, milk, milk products, and other related products.
  • Peptone Water conforms to the composition and performance criteria specified in ISO 6579-2017, ISO 21528-1:2004, and ISO 22964-2017. These standards ensure the reliability and consistency of the medium for its intended purposes.

Principle of Peptone Water

The principle of Peptone Water revolves around its two basic components: peptone and sodium chloride. Peptone serves as a source of nitrogenous and carbonaceous compounds, providing essential nutrients such as long-chain amino acids and vitamins. Sodium chloride, on the other hand, plays a crucial role in maintaining the osmotic balance of the medium and provides necessary electrolytes.

Peptone Water is particularly well-suited for studying indole production. The peptone used in this medium is abundant in tryptophan, which aids in the detection of indole using either Kovacs or Ehlrich reagent. The presence of indole can be indicative of certain bacterial species and their metabolic capabilities.


Furthermore, Peptone Water is employed as a base for carbohydrate fermentation studies. By adding specific sugars and indicators like bromocresol purple, phenol red, or bromothymol blue, it becomes possible to investigate an organism’s ability to ferment a particular carbohydrate. This aids in the differentiation of various genera and species.

During carbohydrate fermentation, acidity is produced as a byproduct. To detect this acid formation, a phenol red indicator can be added to the Peptone Water. Under acidic conditions, the medium undergoes a color change from red to yellow. Additionally, if the organism produces gas during fermentation, a Durham’s tube is inserted into the medium to capture and detect the gas production.


In summary, the principle of Peptone Water lies in its composition of peptone and sodium chloride, which provide essential nutrients and maintain osmotic balance. It is particularly useful for studying indole production and serves as a base for carbohydrate fermentation studies, aiding in the differentiation of bacterial species based on their metabolic capabilities.

Composition of Peptone Water

IngredientsGms / L
Sodium chloride5.0

Final pH should be (at 25°C) 7.2±0.2


Preparation of Peptone Water

To prepare Peptone Water, follow these steps:

  1. Measure and suspend 15.0 grams of Peptone in 1000 ml of distilled water. Ensure that the Peptone is completely dissolved in the water.
  2. Thoroughly mix the suspension to achieve a homogenous mixture.
  3. Distribute the prepared Peptone Water into the final containers. These containers can be test tubes, bottles, or any other suitable vessels, depending on the volume required for your experiments or analysis.
  4. Sterilize the Peptone Water by autoclaving. Place the containers in an autoclave chamber and subject them to a pressure of 15 lbs (pounds) per square inch at a temperature of 121°C (Celsius). Maintain these conditions for 15 minutes.
  5. After the sterilization process is complete, allow the Peptone Water to cool down before using it in your experiments or storing it for later use.

By following these steps, you can prepare Peptone Water, ensuring that it is free from any contaminants and ready to be used as a growth medium or for conducting various tests and analyses in microbiology.


Requirements for The test with Peptone Water

  • Peptone-water
  • inoculation loop or dropper as a test specimen
  • Controlled strains ( Staphylococcus aureus ATCC V25923, Escherichia coli ATCC 25922, and uninoculated medium)
  • Bunsen burner
  • Incubator
  • Garbage can

Procedure of Peptone Water

The procedure for conducting the Peptone Water test involves several steps:

  1. Warm the Peptone Water medium to a temperature of 37°C or room temperature before starting the inoculation process. This temperature is optimal for the growth of most microorganisms.
  2. Prepare an inoculum of the specimen or test organism. This can be done by obtaining a pure culture of the organism and transferring a small amount into the Peptone Water medium using a sterile loop or pipette.
  3. Inoculate the Peptone Water medium with the prepared inoculum. Carefully introduce the inoculum into the medium while minimizing the introduction of contaminants.
  4. After inoculation, incubate the Peptone Water medium aerobically at a temperature range of 35-37°C (or as per the specific requirements of the suspected organisms). Incubation time may vary depending on the nature of the microorganisms being tested.
  5. Allow the medium to incubate for approximately 24 hours. This incubation period allows sufficient time for the growth of the organisms.
  6. After the incubation period, observe the Peptone Water medium for signs of turbidity. Turbidity refers to the presence of cloudiness or opaqueness in the medium, indicating the growth of microorganisms. The formation of visible particles or sediment within the medium can also be an indication of microbial growth.

By following this procedure, the Peptone Water test allows for the detection and evaluation of microbial growth in the medium. The presence or absence of turbidity provides valuable information about the growth characteristics of the tested organism, aiding in the identification and analysis of microorganisms.


Result Interpretation on Peptone Water

The results obtained from using Peptone Water can be categorized into positive and negative outcomes.

  • A positive result is observed when the medium has been inoculated with the desired organism. In this case, growth of the organism will be evident, leading to the development of turbidity in the medium. Turbidity refers to the cloudiness or opaqueness of the liquid, indicating the presence of microbial growth. This growth can be visually assessed by observing the formation of visible particles or sediment within the medium.
  • On the other hand, a negative result is obtained when the Peptone Water is left uninoculated or not contaminated with the target organism. In such cases, no growth will be observed within the medium. The absence of microbial growth will result in a clear and transparent appearance of the medium, indicating a negative result.

The presence or absence of growth and turbidity in the Peptone Water can provide valuable information about the ability of the tested organism to thrive and proliferate in this particular medium. These results, in combination with other tests and observations, can contribute to the identification and characterization of the microorganism under investigation.


Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours

OrganismTest Result
Staphylococcus aureusnegative reaction, no red ring at the interface of the medium on addition of Kovac’s reagent
Escherichia colipositive reaction, red ring at the interface of the medium on addition of Kovac’s reagent
Salmonella Typhimuriumnegative reaction, no red ring at the interface of the medium on addition of Kovac’s reagent

Storage and Shelf life of Peptone Water

  • Keep in a cool, dark place between 2 and 8 degrees Celsius.
  • If there are any visible symptoms of damage (discoloration, contamination), the media should not be used (turbidity).
  • Because of its sensitivity to light and heat, the product can prevent damage from sun exposure, steam, dampness, and even frost.

Uses of Peptone Water

Peptone Water has a variety of uses in microbiology and laboratory settings. Some of the main applications of Peptone Water include:

  • Growth Medium: Peptone Water is commonly used as a broth medium for the growth of microorganisms. It provides essential nutrients, including nitrogenous and carbonaceous compounds, long-chain amino acids, and vitamins, to support the growth and proliferation of a wide range of microorganisms.
  • Carbohydrate Fermentation: Peptone Water serves as a base for carbohydrate fermentation media. By adding specific sugars and indicators, such as bromocresol purple, phenol red, or bromothymol blue, it can be used to study the ability of organisms to ferment specific carbohydrates. This aids in the differentiation of genera and species based on their fermentation patterns.
  • Indole Detection: Peptone Water is employed for the detection of indole production by microorganisms. The peptone used in the medium contains tryptophan, which serves as a substrate for indole production. This test helps in the identification and characterization of certain bacterial species.
  • Microbial Enumeration: Peptone Water can be used as a diluent or suspension medium for non-fastidious microorganisms in microbial enumeration procedures. It provides a suitable environment for the organisms to be easily enumerated or counted.
  • Cultivation of Vibrio Species: Peptone Water with a pH adjusted to 8.4 (alkaline condition) is specifically suitable for the cultivation and enrichment of Vibrio species. These bacteria, including pathogens like Vibrio cholerae, can thrive in this alkaline environment.

Overall, Peptone Water is a versatile medium used for various purposes in microbiology laboratories. Its applications include supporting microbial growth, detecting indole production, studying carbohydrate fermentation patterns, and facilitating microbial enumeration and cultivation of specific bacterial species.

Limitations of Peptone Water

Peptone Water, despite its usefulness, has certain limitations that should be taken into consideration. These limitations include:

  • Identification Limitation: Peptone Water is primarily a growth medium and does not provide complete identification of microorganisms. To obtain comprehensive identification, additional biochemical, immunological, molecular, or mass spectrometry testing should be performed on colonies isolated from pure cultures.
  • Unsuitability for Fastidious Microorganisms: Peptone Water has a low nutrient content, making it less suitable for the growth and maintenance of fastidious microorganisms. Fastidious organisms have specific nutritional requirements that may not be adequately met by this medium.
  • Nutritional Variations: The nutritional composition of Peptone Water can vary, which can lead to variations in growth among different strains of microorganisms. Some strains may exhibit poor growth due to inadequate nutrient availability in the medium.
  • pH Interference: Certain sugar solutions used in conjunction with Peptone Water for carbohydrate fermentation studies can affect the pH of the medium. It is important to monitor and adjust the pH accordingly to ensure accurate results.
  • Sub-cultures for Purity: To ensure the purity of the inoculant, sub-cultures may be necessary. Mixed or contaminated cultures introduced into Peptone Water can yield false reactions, leading to inaccurate interpretations of test results.

It is crucial to be aware of these limitations and consider them while using Peptone Water in microbiological studies. Adhering to appropriate testing protocols, ensuring proper sub-culturing, and considering alternative media when dealing with fastidious organisms can help overcome these limitations and enhance the accuracy and reliability of the results obtained.

Peptone water is a versatile medium that can be used to assess motility, antimicrobial sensitivity (when used as a diluent or suspension), indole production, the oxidation-fermentation (base) test, and as an enrichment media (pH sift) for Vibrio species. Different cultural media also rely on it as their foundation.


What is Peptone Water used for?

Peptone water is a nutrient medium that is used to support the growth of microorganisms. It is made by adding peptone, a mixture of hydrolyzed proteins, to water. Peptone water is often used in laboratory settings for the isolation and cultivation of microorganisms, as well as for the enrichment of samples for bacterial identification and testing. It can be stored at low temperatures (around 0-10°C) for a limited period of time before use.
In addition to its use in the laboratory, peptone water is also sometimes used in industrial applications, such as the production of antibiotics and other pharmaceuticals. It may also be used in quality control testing to ensure the safety and purity of products.
Peptone water can be adjusted to different pH levels, depending on the needs of the microorganisms being cultivated. For example, alkaline peptone water is used to cultivate certain types of Vibrio species, while neutral peptone water is used to cultivate a wider range of bacteria.

What is Peptone Water made of?

Peptone water is a microbiological growth medium made of sodium chloride and peptic digest of animal tissue. At 25 degrees Celsius, the medium has a pH of 7.20.2 and is rich in tryptophan. Peptone water is a nonselective broth medium that can be used as a main bacterial growth enrichment media.

What is the difference between peptone and Peptone Water?

Peptone is a type of partially digested protein that is used in the preparation of nutrient broth. It is typically derived from animal sources, such as beef or milk, and is broken down into smaller peptides and amino acids through the process of hydrolysis. Peptone is a key ingredient in the preparation of many types of media used in microbiology, including peptone water.
Peptone water is a type of nutrient broth that is made by adding peptones to water. It is a nutritious, neutral pH liquid that supports the growth of many types of microorganisms, including bacteria, fungi, and yeasts. Peptone water is commonly used as a base for preparing other types of media or as a growth medium on its own.
So, in summary, peptone is a type of protein used to prepare peptone water, which is a type of nutrient broth used to support the growth of microorganisms.

Why is Peptone Water used for dilution?

Peptone water is a solution of peptone, which is a mixture of hydrolyzed proteins, in water. It is commonly used as a diluent because it contains nutrients that can support the growth of microorganisms. Additionally, peptone water is generally a good source of water for the growth of microorganisms because it is free of contaminants and other materials that could interfere with microbial growth. In general, peptone water is used for dilution because it is a convenient and reliable source of nutrients and water for the growth of microorganisms.

What is called peptone?

Peptone is a mixture of hydrolyzed proteins that is produced by the digestion of proteins with enzymes or acid. It is commonly used as a source of nutrients for the growth of microorganisms in laboratory culture media. Peptone is typically made from animal or plant proteins, and it can be derived from a variety of sources, including milk, eggs, and plant seeds. In addition to its use in culture media, peptone is also used in the production of food products, cosmetics, and other consumer goods.

How do you increase the pH of Peptone Water?

There are several ways to increase the pH of peptone water:
1. Add a base: To increase the pH of peptone water, you can add a base such as sodium hydroxide (NaOH) or potassium hydroxide (KOH). You will need to use a pH meter or pH strips to determine the amount of base to add in order to achieve the desired pH.
2. Use a buffer: A buffer is a substance that can resist changes in pH. You can use a buffer solution to increase the pH of peptone water by adding a small amount of the buffer to the solution.
3. Use a pH adjuster: There are several commercial products available that are specifically designed to adjust the pH of solutions. These products can be added to peptone water to increase the pH.
It is important to note that the pH of peptone water can also be increased by adding other substances that are basic in nature, such as bicarbonate or carbonate ions. However, it is generally recommended to use a pH meter or pH strips to accurately measure the pH of the solution and ensure that the desired pH is achieved.

What does peptone smell like?

Appearance Tan powder. Molecular weight N/A. Odor Cooked meat.

Can bacteria grow in peptone water?

Yes, bacteria can grow in peptone water. Peptone water is a nutrient-rich medium that can support the growth of many types of bacteria. In fact, peptone water is commonly used in laboratory settings as a simple, nutritive medium for the cultivation and maintenance of bacteria. Peptone water is generally considered a good source of nutrients for the growth of microorganisms because it contains a mixture of hydrolyzed proteins, which can provide the amino acids and other nutrients that bacteria need to grow and multiply. However, it is important to note that the specific types of bacteria that can grow in peptone water will depend on a variety of factors, including the pH, temperature, and oxygen levels of the medium.

What is source of peptone?

Peptone is derived predominantly from bovine or porcine sources, including meat, internal organs, gelatin, and milk, in addition to plants and yeasts. Peptone is a partially digested protein that is produced through the enzymatic or acidic degradation of proteinaceous material.



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