Alkaline Peptone Water – Preparation, Composition, Principle, Application.

What is alkaline Peptone Water?

• Alkaline Peptone Water is a commonly used enrichment medium in microbiology for the detection and cultivation of Vibrio species, including Vibrio cholera, V. parahaemolyticus, V. vulnificus, and other Vibrio spp. It is typically employed before selective enrichment and isolation techniques, such as the use of TCBS (Thiosulfate Citrate Bile Salt Sucrose) agar.
• The pH of Alkaline Peptone Water ranges from 8.6 to 9.0, creating an alkaline environment that is conducive to the growth and multiplication of Vibrio species. At the same time, this alkalinity inhibits the growth of faecal coliforms and other commensal bacteria.
• Originally formulated by Shread et al. as a nonselective enrichment broth for Enterobacteriaceae, Alkaline Peptone Water has been modified for the specific purpose of enriching Vibrio species. The pH of the medium is adjusted to 8.4, which enables the growth of Vibrio organisms while suppressing the growth of commensal intestinal bacteria. Enriching samples in Alkaline Peptone Water prior to plating on selective media, such as TCBS agar, has been shown to enhance the recovery rate of Vibrio spp.
• When working with clinical materials that contain low numbers of Vibrio bacteria, it is recommended to inoculate them into an enrichment medium before plating on selective media. Alkaline Peptone Water serves as a suitable enrichment broth for this purpose. Its relatively high pH of approximately 8.4 provides a favorable environment for the growth of Vibrio species.
• Alkaline Peptone Water is recommended by the American Public Health Association (APHA) for the enrichment of Vibrio species from various sources, including seafood, infectious materials, and clinical specimens such as feces. The medium contains peptone, which provides nitrogen and carbon sources, long-chain amino acids, vitamins, and other essential nutrients. Sodium chloride is also included to maintain osmotic equilibrium.
• To use Alkaline Peptone Water, 10 grams of seafood or other specimen is added to 90 ml of the medium, and the mixture is incubated for 18-20 hours at 37°C. Prolonged incubation can lead to the growth of suppressed contaminating organisms. Turbidity in the tubes, compared to an uninoculated control tube, indicates growth. The enriched broth is then used for plating on selective media, and for further biochemical identification, it is recommended to obtain a pure culture.

Composition of Alkaline Peptone Water 

Ingredients	Gms/litre
Peptone	10.0
Sodium Chloride	20.0

Final pH (at 25°C): 8.6 ± 0.2

Principle of Alkaline Peptone Water

• The principle of Alkaline Peptone Water revolves around its ability to selectively cultivate Vibrio species while inhibiting the growth of unwanted background flora during the pre-enrichment step in the isolation process.
• When isolating Vibrio species from environmental, food, or clinical samples, a pre-enrichment step is often employed before plating onto a solid medium for subsequent identification based on morphological, biochemical, and serological characteristics. Alkaline Peptone Water serves as a suitable enrichment broth for this purpose.
• The incorporation of 2% (w/v) sodium chloride in Alkaline Peptone Water plays a crucial role in promoting the growth of Vibrio cholerae and other Vibrio species. Vibrio organisms are halophilic, meaning they require a certain level of salt concentration for optimal growth. The sodium chloride content in the medium provides the necessary osmotic equilibrium required for the growth of Vibrio species.
• Additionally, the alkalinity of the medium inhibits the growth of most unwanted background flora present in the samples. This selective effect is particularly important for isolating Vibrio species, as it helps to suppress the growth of other competing microorganisms, allowing Vibrio organisms to thrive and dominate the enrichment broth.
• The medium’s composition includes peptone, which serves as a source of nitrogen and carbon, long-chain amino acids, vitamins, and other essential nutrients necessary for the growth and metabolic activities of the Vibrio species being targeted. These components support the proliferation of Vibrio organisms during the enrichment process.
• In summary, the principle of Alkaline Peptone Water lies in its selective enrichment capability for Vibrio species. The incorporation of sodium chloride promotes the growth of Vibrio organisms, while the alkalinity of the medium inhibits unwanted background flora. The addition of peptone and other essential nutrients provides the necessary growth factors for Vibrio species, allowing for their successful cultivation in the enrichment broth.

Preparation of Alkaline Peptone Water

To prepare Alkaline Peptone Water, follow the following steps:

1. Measure 20 grams of Alkaline Peptone Water powder.
2. Dissolve the powder in 1 liter of distilled water.
3. Mix the solution thoroughly until the powder is completely dissolved.
4. Distribute the prepared medium into final containers, ensuring proper labeling.
5. Sterilize the containers with the medium by autoclaving at 121°C for 15 minutes.
6. After autoclaving, allow the medium to cool down to room temperature before further use.

To inoculate specimens into Alkaline Peptone Water, follow the appropriate methods depending on the sample type:

1. Swab specimens: Directly inoculate the swab into the Alkaline Peptone Water, ensuring that the swab contacts the medium and transfers the sample material effectively.
2. Other material (not from a swab): Using a sterile microbiological loop, aseptically transfer the specimen into the Alkaline Peptone Water medium. Make sure to transfer an appropriate amount of the material for effective enrichment.

For faecal specimens:

1. Aseptically transfer approximately 1 gram of the faecal sample into the Alkaline Peptone Water medium.
2. Mix the sample well within the medium to ensure proper distribution.

After inoculation, the prepared broths are typically incubated at the recommended temperature and duration for the specific purpose:

1. Incubate the broths at 35-37°C for 5-6 hours or,
2. Incubate the broths at 18-20°C for 18-20 hours.

These incubation conditions provide an optimal environment for the growth and enrichment of Vibrio species or other target microorganisms. The incubation time may vary depending on the specific requirements of the organisms being targeted and the desired level of enrichment.

Following incubation, the enriched broth can be further processed for isolation and identification, such as plating onto selective media or performing biochemical tests to identify the target microorganisms.

Result Interpretation on Alkaline Peptone Water

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

Organisms	Growth
Vibrio parahaemolyticus	Turbid growth
Vibrio vulnificus	Turbid growth
Vibrio furnissii	Turbid growth
Vibrio cholerae serotype Inaba	Turbid growth
Vibrio cholerae serotype Ogawa	Turbid 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.

Quality Control

The quality control of Alkaline Peptone Water involves assessing various parameters to ensure its suitability and performance. Here are some aspects of the quality control process:

1. Appearance: Alkaline Peptone Water should have a cream to yellow homogeneous free-flowing powder appearance. Any deviation from this appearance may indicate a quality issue.
2. Colour and Clarity: The prepared medium should exhibit a light yellow color and a clear solution without any precipitate. The absence of color and clarity abnormalities ensures that the medium is suitable for use.
3. Reaction: The reaction of a 2% w/v aqueous solution of Alkaline Peptone Water at 25°C should have a pH value of 8.4 ± 0.2. This pH range ensures the medium’s proper alkalinity, which is essential for the selective growth of Vibrio species.
4. pH: The acceptable pH range for Alkaline Peptone Water is 8.20-8.60. This range ensures consistency and reliability in the performance of the medium.
5. Cultural Response: The cultural response of Alkaline Peptone Water is assessed by inoculating it with specific reference strains and evaluating the growth and cultural characteristics of the organisms after incubation. Luxuriant growth of Vibrio cholerae ATCC 15748 and Vibrio parahaemolyticus ATCC 17802 is expected as an indication of proper medium performance.

The key provided denotes the corresponding WDCM (World Data Centre for Microorganisms) numbers for the reference strains used in the quality control assessment.

By evaluating these parameters, laboratories can ensure that the Alkaline Peptone Water they use meets the required quality standards and is suitable for the enrichment and isolation of Vibrio species from various samples. It helps to maintain consistent and reliable results during laboratory procedures and subsequent identification of target organisms.

Uses of Alkaline Peptone Water

Alkaline Peptone Water finds various uses in microbiology laboratories. Some of the common applications include:

1. Enrichment of Vibrio species: Alkaline Peptone Water is primarily used for the enrichment of Vibrio cholera and other Vibrio species from food, water, and clinical samples. The alkaline pH of the medium promotes the growth of Vibrio organisms while inhibiting the growth of other commensal bacteria. This selective enrichment step helps in increasing the recovery rate of Vibrio species during isolation and identification procedures.
2. Microscopic examination: Alkaline Peptone Water can be utilized for direct microscopic examination of samples using the hanging drop method. This method allows for the observation of motility, morphology, and other characteristics of microorganisms present in the sample. It aids in the preliminary identification of Vibrio species based on their motility and cellular morphology.
3. Enrichment and transport medium: Alkaline Peptone Water serves as both an enrichment and transport medium. It provides a favorable environment for the growth of Vibrio species during the enrichment process. Additionally, it can maintain the viability of microorganisms during transport from the sample collection site to the laboratory, ensuring that the organisms remain viable for subsequent analysis.
4. Cultivation of Aeromonas: Initially, Alkaline Peptone Water was used as a non-selective enrichment broth for the cultivation of Aeromonas species. Although its primary application shifted towards Vibrio species, it can still be used for the cultivation and enrichment of Aeromonas when required.

Overall, Alkaline Peptone Water is a versatile medium used for the enrichment, isolation, and transportation of Vibrio species. Its alkaline pH and nutrient composition make it suitable for the growth of these organisms, aiding in their detection and identification from various sources such as food, water, and clinical samples.

Limitations of Alkaline Peptone Water

Despite its usefulness, Alkaline Peptone Water has certain limitations that should be considered. These limitations include:

1. Overgrowth of contaminating organisms: Prolonged incubation of the Alkaline Peptone Water can lead to the growth of suppressed contaminating organisms. This can complicate the isolation and identification process by potentially overshadowing the growth of target Vibrio species.
2. Need for selective media: The growth obtained from the enrichment broth is used for plating onto selective media to isolate the target Vibrio species. This additional step is necessary to obtain pure colonies of the organisms of interest for further analysis and identification.
3. Poor growth of certain Vibrio strains: Some strains of Vibrio species, particularly those requiring higher sodium chloride concentrations, may exhibit poor growth in Alkaline Peptone Water. This can lead to decreased recovery and detection of these specific strains.
4. Further recovery required: Although Alkaline Peptone Water serves as an enrichment medium, further recovery of the organisms onto selective media is necessary for optimal isolation and identification. The enriched broth is used as a starting point for subsequent steps in the isolation process.
5. Biochemical identification from pure isolates: For complete identification and characterization, biochemical tests and other identification methods are carried out using pure isolates obtained from the enriched broth. This ensures accurate identification and differentiation of Vibrio species from other microorganisms present in the sample.

Considering these limitations, it is important to employ appropriate techniques and follow the recommended procedures to maximize the recovery and identification of target Vibrio species while minimizing the impact of potential limitations associated with Alkaline Peptone Water as an enrichment medium.

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

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/