VRBA- Composition, Principle, Preparation, Results, Uses

Violet Red Bile Agar (VRBA) is a selective and differential medium that is used to isolate and identify coliform bacteria from various sources. Coliform bacteria are Gram-negative, facultatively anaerobic, rod-shaped bacteria that can ferment lactose to produce acid and gas. They are commonly found in the intestinal tract of humans and animals, as well as in soil, water, and food. Coliform bacteria are indicators of fecal contamination and potential pathogens in water and food samples.

VRBA contains ingredients that selectively inhibit the growth of Gram-positive bacteria and other unwanted organisms, while allowing the growth of coliform bacteria. It also contains ingredients that differentiate coliform bacteria from other Gram-negative bacteria based on their ability to ferment lactose. Coliform bacteria produce purple-red colonies with a bile precipitate around them, while non-coliform bacteria produce colorless or pale colonies with no bile precipitate.

VRBA is widely used in microbiology laboratories for the isolation, detection, and enumeration of coliform bacteria in water, milk, dairy products, and clinical samples. It is also used as a confirmatory test for presumptive coliforms obtained from other media, such as MacConkey Agar or Lauryl Sulfate Broth. VRBA can help to identify the presence of Escherichia coli (E. coli), which is the most common and pathogenic member of the coliform group.

In this article, we will discuss the composition, principle, preparation, results, uses, and limitations of VRBA in detail. We will also provide some examples and images of VRBA plates with different types of bacterial growth. We hope that this article will help you to understand the basics and applications of VRBA in microbiology.😊

Violet Red Bile Agar (VRBA) is a selective and differential medium for the isolation and enumeration of coliform bacteria from various sources. The medium contains the following ingredients:

• Peptic digest of animal tissue and yeast extract: These provide essential nutrients, such as carbon, nitrogen, vitamins, and minerals, for the growth of bacteria.

• Lactose: This is the fermentable carbohydrate that serves as the energy source for the bacteria. Lactose-fermenting bacteria produce acid and gas, which can be detected by the color change of the indicator and the formation of bubbles in the medium.

• Neutral red: This is the pH indicator that turns red in acidic conditions. Lactose-fermenting bacteria lower the pH of the medium by producing acid, which results in red or purple colonies with or without a halo.

• Crystal violet and bile salts: These are the selective agents that inhibit the growth of most Gram-positive bacteria and some Gram-negative bacteria that are not coliforms. Crystal violet interferes with the cell wall synthesis of Gram-positive bacteria, while bile salts disrupt the cell membrane of bile-sensitive bacteria.

• Sodium chloride: This maintains the osmotic balance of the medium and prevents plasmolysis of bacterial cells.

• Agar: This is the solidifying agent that provides a firm surface for bacterial growth and colony formation.

The final pH of VRBA is 7.4±0.2 at 25°C. The medium appears reddish-purple in color before inoculation. After incubation, lactose-fermenting coliforms produce red or purple colonies with or without a halo, while non-lactose-fermenting bacteria produce colorless or pale colonies with greenish zones. Some enterococci may also grow on VRBA and produce pink pinpoint colonies.

Violet Red Bile Agar (VRBA) is a selective and differential medium that allows the isolation and enumeration of coliform bacteria from various sources, such as water, milk, dairy products, and clinical specimens. Coliform bacteria are Gram-negative, facultatively anaerobic, rod-shaped bacteria that can ferment lactose to produce acid and gas.

The principle of VRBA is based on the following components:

• Crystal violet and bile salts: These are the selective agents that inhibit the growth of most Gram-positive bacteria and some Gram-negative bacteria that are not coliforms. Crystal violet interferes with the cell wall synthesis of Gram-positive bacteria, while bile salts disrupt their membrane integrity. Some Gram-negative bacteria, such as Pseudomonas and Proteus, are also sensitive to these agents and are suppressed on VRBA.

• Lactose: This is the differential carbohydrate that distinguishes between lactose-fermenting and non-lactose-fermenting bacteria. Lactose-fermenting bacteria produce acid and gas from lactose, which lowers the pH of the medium and causes the precipitation of bile salts. Non-lactose-fermenting bacteria do not produce acid or gas and do not affect the pH or bile salts.

• Neutral red: This is the pH indicator that changes color according to the acidity of the medium. Neutral red is colorless at neutral or alkaline pH but turns red at acidic pH. Therefore, lactose-fermenting bacteria appear as red or purple colonies with a red halo on VRBA, while non-lactose-fermenting bacteria appear as colorless or pale colonies with a greenish halo or no halo.

By using VRBA, one can easily identify and count coliform bacteria based on their characteristic appearance on the medium. However, further confirmation tests are required to identify the specific species of coliforms or other Enterobacteriaceae that may grow on VRBA.

Violet Red Bile Agar (VRBA) is a selective and differential medium for the isolation and enumeration of coliform bacteria from water, milk, dairy products, and clinical samples. It can also be used to detect other members of the Enterobacteriaceae family, such as Salmonella and Shigella. The medium contains lactose as the fermentable carbohydrate, crystal violet, and bile salts as the selective agents, and neutral red as the pH indicator.

To prepare for VRBA, follow these steps:

• Suspend 41.53 grams of the dehydrated medium in 1000 ml of distilled water. Mix well to dissolve the powder completely.

• Heat the solution to boiling with frequent stirring. Do not autoclave or overheat the medium, as this may affect its performance.

• Cool the medium to 45°C and pour it into sterile Petri plates containing the inoculum. Alternatively, you can inoculate the medium after pouring by using a sterile loop or swab.

• Transfer a 1 ml aliquot of the test sample to a Petri dish. Add 10 ml of VRBA (at 48°C) and swirl to mix. This is called the pour plate method, which allows for uniform distribution of bacteria in the medium.

• Allow the medium to solidify before incubating at 35°C for 18 to 24 hours. For dairy products, use 32°C as the incubation temperature.

• Examine the plates for typical colonies of coliform bacteria and other Enterobacteriaceae.

To use VRBA, follow these steps:

• Observe the color and size of the colonies on VRBA. Coliform bacteria produce purple-red colonies with a diameter of 0.5 mm or larger, surrounded by a zone of precipitated bile acids. Non-coliform bacteria produce colorless or pale colonies with no bile precipitation.

• Count the number of coliform colonies on each plate and calculate the coliform count per ml or gram of the test sample. For example, if you have 10 coliform colonies on a plate inoculated with 1 ml of water sample, the coliform count is 10 per ml of water.

• Select some typical coliform colonies and perform confirmatory tests, such as Gram stain, indole test, methyl red test, Voges-Proskauer test, and citrate test. These tests help to differentiate between Escherichia coli and other coliforms, such as Enterobacter, Klebsiella, and Citrobacter.

• Report the results of VRBA and confirmatory tests according to the standard methods or guidelines for your specific application.

Here is an example of how VRBA looks like after incubation:

Image Source: Microbe Notes

The appearance of colonies on VRBA can provide some clues about the identity of the bacteria present in the sample. The following table summarizes the common results and their interpretations:

| Colony Color | Colony Size | Zone of Precipitation | Possible Organisms |

|--------------|-------------|-----------------------|--------------------|

| Purple-red | 0.5 mm or larger | Present | Lactose-positive Enterobacteriaceae (coliforms), such as E. coli |

| Pink | Pinpoint | Absent or faint | Enterococci, possibly Klebsiella |

| Colorless | Variable | Absent | Lactose-negative Enterobacteriaceae, such as Salmonella and Shigella |

The colonies can be further examined by Gram staining and biochemical tests to confirm their identity. For example, E. coli colonies can be distinguished from other coliforms by their indole production and gas formation from lactose fermentation.

VRBA is a selective and differential medium that allows the isolation and enumeration of coliform bacteria from various samples. The medium contains lactose as the carbohydrate source, neutral red as the pH indicator, and crystal violet and bile salts as the selective agents. The lactose-fermenting bacteria produce acid and gas, which lower the pH and cause the neutral red to turn red. The acid also precipitates the bile salts, forming a zone around the colonies. The non-lactose-fermenting bacteria do not produce acid or gas and remain colorless or pale on the medium.

The image below shows an example of VRBA with different types of colonies:

Violet Red Bile Agar (VRBA) is a selective and differential medium that is widely used for the isolation, detection, and enumeration of coliform bacteria in water, milk, and other dairy products. Coliform bacteria are Gram-negative, lactose-fermenting, rod-shaped bacteria that are commonly found in the intestinal tract of humans and animals. They are used as indicators of fecal contamination and hygiene quality of food and water.

VRBA can also be used to isolate and identify other members of the Enterobacteriaceae family, such as Escherichia coli, Klebsiella, Enterobacter, Citrobacter, and Proteus. These bacteria are responsible for various infections in humans and animals, such as urinary tract infections, septicemia, wound infections, and diarrhea. VRBA can help to differentiate these bacteria based on their lactose fermentation ability and colony morphology.

VRBA can also be used for the detection of enterococci, which are Gram-positive, facultative anaerobic cocci that are part of the normal flora of the gastrointestinal tract. Enterococci can cause serious infections such as endocarditis, meningitis, bacteremia, and urinary tract infections. VRBA can distinguish enterococci from other Gram-positive bacteria by their ability to grow in the presence of bile salts and crystal violet. Enterococci usually produce small, pink colonies on VRBA.

VRBA is a simple, inexpensive, and reliable medium that can be used for routine screening of food and water samples for the presence of coliforms and other enteric bacteria. It can also be used for clinical specimens such as stool, urine, blood, and wound swabs. VRBA can provide preliminary identification of the isolates based on their color and size on the medium. However, further confirmation tests are required for the definitive identification of the organisms.

• VRBA is not completely specific for enterics; other accompanying bacteria may give the same reaction.

• Due to varying nutritional requirements, some strains may be encountered that grow poorly or fail to grow on this medium.

• VRBA may not be completely inhibitory to Gram-positive organisms and will grow Gram-negative bacilli other than members of Enterobacteriaceae. Perform a Gram stain and biochemical tests to identify isolates.

• Boiling the medium for longer than 2 minutes can decrease the ability to support growth.

• It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

• Enterococci may grow, and if so, usually appear to be pinpoint in size and rose-colored.

• Violet Red Bile Agar is not intended for use in the diagnosis of a disease or other conditions in humans.

Some additional points that could be added are:

• VRBA does not differentiate between E. coli and other coliforms. A confirmatory test such as indole production or IMViC is needed to identify E. coli.

• VRBA may produce false-negative results for some lactose-fermenting bacteria that are sensitive to bile salts or crystal violet. A selective medium without these inhibitors may be used as a supplement to VRBA.

• VRBA may produce false-positive results for some non-lactose-fermenting bacteria that produce red pigments or acidify the medium by other means. A differential medium such as MacConkey Agar may be used to distinguish between lactose and non-lactose fermenters.

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