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Alkaline Peptone Water Preparation, Composition, Principle, Application.

Clinical materials containing small numbers of Vibrio should be inoculated into an enrichment medium prior to plating onto a selective medium, such as TCBS Agar (M189). Alkaline Peptone Water is a suitable enrichment broth for this purpose. 

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What is alkaline Peptone Water used for?

Alkaline peptone water is a type of aqueous solution that is used as a growth medium for microorganisms. It is made by mixing peptone (a type of protein hydrolysate) and water, and adjusting the pH to 7.5-8.5 with a strong alkali such as sodium hydroxide or potassium hydroxide. The high pH and the presence of peptone provide a suitable environment for the growth of many types of bacteria. Alkaline peptone water is used in various laboratory and industrial applications, including the isolation and cultivation of microorganisms and the production of antibiotics.

In order to find Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and other Vibrio spp., alkaline peptone water is often employed as an enrichment medium prior to selective enrichment and isolation (Ex: TCBS agar).

Medium pH is between 8.6 and 9.0. In contrast to faecal coliforms and other commensals, Vibrio species thrive in alkaline environments and proliferate.

Composition of Alkaline Peptone Water 

IngredientsGms/litre
Peptone10.0
Sodium Chloride20.0

Final pH (at 25°C): 8.6 ± 0.2

Principle And Interpretation of Alkaline Peptone Water

  • There is a wide range of options for isolating Vibrio species from water, food, and human clinical samples.
  • These typically begin with a process of pre-enrichment, followed by plating on a solid medium and subsequent morphological, biochemical, and serological identification.
  • To successfully cultivate Vibrio species, use the Alkaline Peptone Water medium. This enriching broth serves the goal well.
  • This medium’s alkalinity suppresses most of the undesirable background bacteria while the 2% (w/v) sodium chloride included in this medium enhances the growth of Vibrio cholerae.
  • Peptone is a dietary supplement that includes a supply of nitrogen and carbon, as well as long-chain amino acids, vitamins, and other vital elements. Osmotic balance is preserved by sodium chloride.

Preparation of Alkaline Peptone Water

  1. To prepare 1 litre of distilled water, add 20 grammes.
  2. Combine thoroughly, transfer to storage containers, and autoclave at 121 degrees Celsius for 15 minutes to kill any bacteria.
  3. Submerge swab samples in Alkaline Peptone Water for inoculation.
  4. A sterile microbiological loop can be used to transport material into the medium if it is not being grown straight from a swab.
  5. Aseptically add 1 gramme of the faeces sample to the medium and stir until completely combined.
  6. There are two standard incubation times for the infected broths: 5-6 hours at 35-37°C, and 18-20 hours at 18-20°C.

Result Interpretation on Alkaline Peptone Water

Growth in tubes is indicated by turbidity compared to an un-inoculated tube (control).

OrganismsGrowth
Vibrio parahaemolyticus Turbid growth
Vibrio vulnificusTurbid growth
Vibrio furnissii Turbid growth
Vibrio cholerae serotype InabaTurbid growth
Vibrio cholerae serotype OgawaTurbid growth

Inoculation Procedure For Alkaline Peptone Water

Rectal swabs, faecal suspensions, and liquid stool can all be used to inoculate a patient with aphthous ulcerative colitis.

A. To be used as a source of enrichment

  1. Place a sample into the APW bottle (the inoculums from the stools should not exceed 10% of the volume of the broth).
  2. For 6-8 hours, incubate the tube at 35°C to 37°C with the top slightly ajar.
  3. Since vibrios preferentially grow in the surface and uppermost portion of the broth, subcultures to TCBS should be made with one to two loopful of APW after 6 to 8 hours of incubation.
  4. If you’re subculturing, don’t jiggle the tube.
  5. Subculture the broth at 18 hours to a new tube of APW if you are unable to do so after 6 to 8 hours of incubation.
  6. After 6 to 8 hours in the incubator, this second tube can be subcultured to a solid media.

B. As a mode of transportation

  1. Transfer approximately 1 ml of the specimen into 10 ml of sterile alkaline peptone water when cholera is suspected.
  2. The infected media must arrive at the lab within eight hours of collection (Proteus species will grow eventually in alkaline peptone water.)

In approximately 4-6 hours, APW becomes cloudy because to the rapid multiplication of Vibrio cholerae. Examine a wet preparation/perform a hanging drop motility test, create a gramme stain of the smear (which may show gram-negative curved bacilli), and/or grow the organisms in TCBS medium to verify that they are vibrios. The sensitivity and specificity of the test for detecting Vibrio cholerae lipopolysaccharide antigen in a stool sample can be improved by enriching the sample in APW first.

Storage and Shelf Life of Alkaline Peptone Water

  • Store between 10-30°C in a tightly closed container and the prepared medium at 15-25°C.
  • Use before expiry date on the label.
  • On opening, product should be properly stored dry, after tightly capping the bottle inorder to prevent lump formation due to the hygroscopic nature of the product.
  • Improper storage of the product may lead to lump formation. 
  • Store in 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 stated expiry period.

Application of Alkaline Peptone Water

  • It is used for the enrichment of Vibrio cholera and Vibrio species from food, water and clinical samples.
  • Used in hanging drop method for direct microscopic examination of samples.
  • It is also used as a transport media.
  • Used as a non-selective enrichment broth for the cultivation of Aeromonas.

Limitations of Alkaline Peptone Water

  1. Certain strains of Vibrio species requiring higher sodium chloride concentration may show poor growth.
  2. Further recovery from this enriched broth onto selective media is required.
  3. Biochemical characterization is carried out from pure isolates for complete identification.
  4. Growth from the enrichment broth is used for plating on selective media. For biochemical identification a pure culture is recommended.
  5. Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
  6. 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 clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques.

How do you make 0.1% Peptone Water?

To make 0.1% peptone water, you can follow these steps:

  1. Determine the desired volume of the solution.
  2. Weigh out the appropriate amount of peptone based on the desired volume of the solution. To make a 0.1% solution, you will need 0.1 grams of peptone for every 100 milliliters of solution.
  3. Dissolve the peptone in water. You can use a small amount of hot water to help dissolve the peptone, if necessary.
  4. Adjust the volume of the solution to the desired level by adding more water, if necessary.
  5. Autoclave the solution to sterilize it, if desired.
  6. Store the solution at the appropriate temperature until it is needed.

It is important to note that the concentration of peptone in the solution can affect the growth and behavior of bacteria, so it is important to carefully measure the amount of peptone used to prepare the solution. A concentration of 0.1% peptone is relatively low and may be suitable for cultivating bacteria that have specific nutritional requirements or that are sensitive to higher concentrations of peptone.

How do you make Peptone Water buffer?

To make a peptone water buffer, you can follow these steps:

  1. Determine the desired pH of the buffer and the desired volume of the buffer solution.
  2. Calculate the amount of acid or base needed to adjust the pH of the peptone water to the desired level. You can use a pH calculator or the Henderson-Hasselbalch equation to determine the amount of acid or base needed.
  3. Prepare the peptone water by dissolving the appropriate amount of peptone in water.
  4. Adjust the pH of the peptone water to the desired level by adding the calculated amount of acid or base. You can use a pH meter or pH indicator strips to measure the pH of the solution.
  5. If necessary, adjust the volume of the buffer to the desired level by adding more peptone water.
  6. Autoclave the buffer to sterilize it, if desired.
  7. Store the buffer at the appropriate temperature until it is needed.

It is important to note that the pH of the buffer may change over time due to the consumption of the buffer by bacteria or the evaporation of the solvent. It may be necessary to periodically adjust the pH of the buffer to maintain the desired pH level.

FAQ

How do you make alkaline Peptone Water?

Composition / preparation. To 1 litre of distilled water, add 40g (20g peptone and 20g sodium chloride). Mix thoroughly, distribute into final containers, and sterilise at 121°C for 15 minutes in an autoclave.

Is alkaline Peptone Water selective?

Yes, alkaline peptone water is a selective medium because it promotes the growth of certain types of bacteria while inhibiting the growth of others. It is selective because it contains peptone, which is a nutrient that provides a source of nitrogen and other essential nutrients for the growth of some bacteria, and it has an alkaline pH, which may inhibit the growth of certain types of bacteria that prefer an acidic pH. It is often used to selectively cultivate bacteria that are able to grow in alkaline conditions, such as certain strains of Escherichia coli.

What is the pH of alkaline Peptone Water?

The pH of alkaline peptone water is typically around 7.5 to 9.5. It is considered to be alkaline because it has a pH above 7.0. The pH of a medium can be adjusted by adding an acidic or basic substance to it. In the case of alkaline peptone water, the pH is raised by adding an alkaline substance, such as sodium hydroxide or potassium hydroxide, to the medium. The specific pH of the medium may vary depending on the recipe used to prepare it and the type of bacteria being cultivated.

What bacteria grows in Peptone Water?

Peptone water is a simple, nutritious medium that can support the growth of a wide variety of bacteria. It is often used to cultivate various strains of Escherichia coli, as well as other gram-negative bacteria. In addition, some strains of gram-positive bacteria, such as Bacillus and Staphylococcus, may also be able to grow in peptone water. It is a good choice for cultivating bacteria that do not have specific nutritional requirements, as it provides a general source of nutrients that can support the growth of a wide range of bacteria. However, it may not be suitable for cultivating bacteria that require specialized nutrients or conditions for growth.

What is the difference between peptone and Peptone Water?

Peptone is a complex protein that is produced by the partial hydrolysis of animal or plant proteins. It is used as a source of nitrogen and other nutrients in the growth media for bacteria and other microorganisms.
Peptone water is a medium that contains peptone as a nutrient source for bacteria. It is a simple, nutritious medium that is used to cultivate a wide variety of bacteria. In addition to peptone, it may also contain other ingredients, such as salts and sugars, to support the growth of bacteria. It is a liquid medium, so it is well suited for cultivating bacteria that require liquid media for growth.
In summary, peptone is a specific type of protein that is used as a nutrient in the growth of bacteria, while peptone water is a medium that contains peptone and other ingredients to support the growth of bacteria.

What is peptone made from?

Peptone is a complex protein that is produced by the partial hydrolysis of animal or plant proteins. It is made by breaking down proteins into smaller peptides and amino acids through the action of enzymes or acids. The resulting mixture of peptides and amino acids is then purified and dried to produce peptone.
Animal proteins are the most common source of peptone. Common sources of animal protein include meat, milk, and eggs. Plant proteins, such as soy, can also be used to make peptone, although animal-derived peptone is more commonly used in the laboratory. Peptone is a commonly used ingredient in the growth media for bacteria and other microorganisms because it provides a rich source of nitrogen and other essential nutrients for their growth.

What is 0.1 Peptone Water?

It is likely that “0.1 peptone water” refers to a solution of peptone water that contains 0.1% peptone. Peptone water is a medium that is used to cultivate bacteria and other microorganisms. It typically contains peptone as a source of nitrogen and other essential nutrients, as well as other ingredients to support the growth of bacteria. The concentration of peptone in the medium can vary depending on the specific recipe and the type of bacteria being cultivated. A concentration of 0.1% peptone would indicate that the solution contains a low level of peptone. It is possible that this concentration of peptone may be used to cultivate bacteria that have specific nutritional requirements or that are sensitive to higher concentrations of peptone.

How do you adjust the pH in alkaline Peptone Water?

To adjust the pH of alkaline peptone water, you can add an acidic or basic substance to the medium to lower or raise the pH, respectively. The specific amount and type of substance you use will depend on the starting pH of the medium and the desired final pH.
To lower the pH of the medium, you can add an acidic substance, such as hydrochloric acid or sulfuric acid. You can add a small amount of the acid to the medium and then measure the pH using a pH meter or pH indicator strips. If the pH is not at the desired level, you can continue to add small amounts of acid until the desired pH is reached.
To raise the pH of the medium, you can add a basic substance, such as sodium hydroxide or potassium hydroxide. You can add a small amount of the base to the medium and then measure the pH using a pH meter or pH indicator strips. If the pH is not at the desired level, you can continue to add small amounts of base until the desired pH is reached.
It is important to note that the pH of the medium can affect the growth and behavior of bacteria, so it is important to carefully adjust the pH to the appropriate level for the specific type of bacteria you are cultivating.

How long can you store Peptone Water?

Peptone water can be stored for a relatively long time if it is properly prepared and stored. It is generally recommended to prepare the medium fresh before use, as it can lose its potency over time. If you need to prepare the medium in advance, it can be stored at 4°C (39°F) for up to one week.
If you need to store the medium for longer periods of time, it can be autoclaved (sterilized) and then stored at room temperature or in the refrigerator. Autoclaved peptone water can be stored for several months. However, it is important to note that the medium may lose its potency over time, even when it is stored properly. It is generally best to prepare fresh batches of the medium as needed to ensure optimal performance.

Does E coli use peptone?

Yes, Escherichia coli (E. coli) is a type of bacteria that can use peptone as a source of nutrients. Peptone is a complex protein that is produced by the partial hydrolysis of animal or plant proteins. It is often used as a source of nitrogen and other essential nutrients in the growth media for bacteria and other microorganisms. E. coli is a gram-negative bacterium that is able to utilize a wide range of nutrients for growth, including peptone. In fact, peptone is a commonly used ingredient in the growth media for E. coli and other gram-negative bacteria. It is a rich source of nutrients that can support the growth and proliferation of these bacteria in the laboratory.

Why is peptone used in nutrient broth?

Peptone is used in nutrient broth because it is a rich source of nutrients that can support the growth of a wide variety of bacteria and other microorganisms. Nutrient broth is a liquid medium that is used to cultivate bacteria and other microorganisms in the laboratory. It typically contains a variety of nutrients, including peptone, to support the growth of a wide range of bacteria.
Peptone is a complex protein that is produced by the partial hydrolysis of animal or plant proteins. It is a good source of nitrogen and other essential nutrients, such as amino acids and vitamins, that are necessary for the growth and proliferation of bacteria. It is also relatively easy to obtain and relatively inexpensive, making it a popular choice for use in nutrient broth and other growth media.
In addition to peptone, nutrient broth may also contain other ingredients, such as salts and sugars, to support the growth of bacteria and provide a complete source of nutrients. It is a simple, versatile medium that is widely used in the laboratory for the cultivation of a wide variety of bacteria and other microorganisms.

Is Peptone Water soluble?

Yes, peptone water is a liquid medium, so it is soluble in water. Peptone water is a simple, nutritious medium that is used to cultivate a wide variety of bacteria and other microorganisms. It is made by dissolving peptone and other ingredients, such as salts and sugars, in water. The resulting solution is a clear, amber-colored liquid that is used to support the growth of bacteria in the laboratory.
Peptone is a complex protein that is produced by the partial hydrolysis of animal or plant proteins. It is a rich source of nutrients, including nitrogen and amino acids, that are necessary for the growth and proliferation of bacteria. When peptone is dissolved in water, it forms a nutrient-rich solution that can support the growth of a wide variety of bacteria.
In addition to being soluble in water, peptone is also soluble in other solvents, such as alcohol and glycerol. It is a versatile ingredient that is widely used in the growth media for bacteria and other microorganisms in the laboratory.

Why do we use Peptone Water for dilution?

Peptone water is often used for dilution in microbiological experiments because it is a simple, nutritious medium that supports the growth of a wide variety of bacteria. It is a liquid medium that contains peptone as a source of nitrogen and other essential nutrients, as well as other ingredients, such as salts and sugars, to support the growth of bacteria.
Dilution is a common technique used in microbiology to reduce the concentration of bacteria in a sample. It is often used to determine the number of bacteria present in a sample, to isolate pure cultures of bacteria, or to prepare samples for further testing. Peptone water is often used as the diluent because it is a simple, non-selective medium that does not inhibit the growth of most bacteria.
To perform a dilution, a known volume of a bacterial sample is mixed with a larger volume of peptone water to reduce the concentration of bacteria in the sample. The resulting dilution can then be plated on a solid medium or used for further testing as needed. Peptone water is a useful tool for performing dilutions because it is easy to work with and supports the growth of a wide range of bacteria.

What type of media is alkaline Peptone Water?

Alkaline peptone water is a type of liquid medium that is used to cultivate bacteria and other microorganisms in the laboratory. It is considered to be a selective medium because it promotes the growth of certain types of bacteria while inhibiting the growth of others. It is selective because it contains peptone, which is a nutrient that provides a source of nitrogen and other essential nutrients for the growth of some bacteria, and it has an alkaline pH, which may inhibit the growth of certain types of bacteria that prefer an acidic pH.
In general, liquid media are used to cultivate bacteria and other microorganisms that require a liquid medium for growth. They are suitable for cultivating bacteria that are able to grow and multiply in a liquid environment, such as many strains of gram-negative bacteria. Liquid media are often used in conjunction with solid media, which are used to cultivate bacteria that require a solid surface for growth.
Alkaline peptone water is a versatile medium that is widely used in the laboratory for the cultivation of a wide variety of bacteria, including certain strains of Escherichia coli. It is a good choice for cultivating bacteria that are able to grow in alkaline conditions and do not have specific nutritional requirements.

What is peptone saline water?

Peptone saline water is a liquid medium that is used to cultivate bacteria and other microorganisms in the laboratory. It is similar to peptone water, but it also contains salts to provide additional nutrients and to help maintain the osmotic balance of the medium. Saline solutions, such as saline water, are commonly used in the laboratory because they have a similar ionic composition to body fluids, which makes them well suited for use in biological systems.
Peptone saline water typically contains peptone as a source of nitrogen and other essential nutrients, as well as salts, such as sodium chloride, to provide additional nutrients and to help maintain the osmotic balance of the medium. It is a simple, nutritious medium that is used to cultivate a wide variety of bacteria and other microorganisms. It is a good choice for cultivating bacteria that do not have specific nutritional requirements and that are able to grow in the presence of salts.
In general, peptone saline water is a liquid medium that is used to cultivate bacteria and other microorganisms in the laboratory. It is similar to peptone water, but it contains additional ingredients, such as salts, to provide additional nutrients and to help maintain the osmotic balance of the medium.

What is buffer Peptone Water?

Buffer peptone water is a type of liquid media used in microbiological laboratories. It is a neutral pH liquid growth medium that is used to cultivate a variety of microorganisms, including bacteria, fungi, and yeast. It is made by combining peptone, a protein-rich ingredient, with water and a buffer solution, which helps to maintain a stable pH level. Buffer peptone water can be used as a base medium for preparing other types of media, or it can be used on its own for the cultivation of microorganisms. It is a useful tool for researchers studying microorganisms, as it provides a supportive environment for the growth and proliferation of a wide range of microorganisms.

References

  • https://himedialabs.com/TD/M618.pdf
  • https://microbenotes.com/alkaline-peptone-water/
  • https://microbeonline.com/alkaline-peptone-water-apw-principle-preparation-uses/
  • https://www.onlinebiologynotes.com/alkaline-peptone-water-composition-preparation-and-uses/

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MN Editors. (September 19, 2021).Alkaline Peptone Water Preparation, Composition, Principle, Application.. Retrieved from https://microbiologynote.com/alkaline-peptone-water/

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MN Editors. "Alkaline Peptone Water Preparation, Composition, Principle, Application.." Microbiology Note, Microbiologynote.com, September 19, 2021.

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