What is Violet Red Bile Agar (VRBA)?

• Violet Red Bile Agar (VRBA) is a modified version of MacConkey’s original formulation and is primarily used for the enumeration of lactose-fermenting coliform microorganisms. Coliforms are a group of bacteria belonging to the family Enterobacteriaceae and are characterized by their ability to ferment lactose, producing gas and acid within 48 hours at 35°C. These bacteria are aerobic or facultative anaerobic, gram-negative, non-spore-forming rod-shaped organisms.
• The detection and identification of coliforms play a crucial role in examining various food products, ingredients, and raw materials. VRBA is a selective medium that contains specific components to inhibit the growth of unwanted organisms while facilitating the tentative identification of the desired bacteria.
• The key components of VRBA include crystal violet and bile salts, which possess selective inhibitory properties against gram-positive and unrelated flora. The presence of lactose as a fermentable carbohydrate allows coliforms that rapidly attack lactose to produce distinctive purple colonies surrounded by purple halos. On the other hand, non-fermenters or late lactose-fermenters produce pale colonies with greenish zones.
• VRBA also incorporates neutral red as an indicator to detect the acidity produced during lactose fermentation. The acidic environment formed due to lactose utilization is visually indicated by the color change of neutral red. Furthermore, peptic digest of animal tissue and yeast extract serve as sources of essential growth nutrients such as carbon, nitrogen, and vitamins, enabling the bacteria to thrive.
• While VRBA provides a selective environment for coliforms, it is important to note that other accompanying bacteria may exhibit similar reactions on this medium. Therefore, further biochemical tests are required for positive identification of the coliform bacteria.
• The American Public Health Association (APHA) recommends the use of VRBA for coliform enumeration, and the selectivity of the medium can be enhanced by incubating it under anaerobic conditions or at elevated temperatures, equal to or above 42°C.
• In summary, Violet Red Bile Agar is a selective medium used in the detection and enumeration of lactose-fermenting coliform microorganisms. It suppresses the growth of unwanted organisms while facilitating the identification of coliform bacteria based on their distinctive colony characteristics. VRBA is an important tool in the testing and quality control of food and dairy products, helping to ensure their safety and suitability for consumption.

Intended Use

Violet Red Bile Agar is used to count bacteria that cause coliforms in dairy products.

Composition of Violet Red Bile Agar (VRBA)

Ingredients	Gms/liter
Peptone	7.000
Yeast extract	3.000
Sodium chloride	5.000
Bile salts mixture	1.500
Lactose	10.00
Neutral red	0.030
Crystal violet	0.002
Agar	15.00

Final pH (at 25°C): 7.4±0.2

Principle of Violet Red Bile Agar (VRBA)

The principle of Violet Red Bile Agar (VRBA) revolves around its selective inhibitory components and indicator system. The medium contains crystal violet and bile salts, which primarily inhibit the growth of accompanying Gram-positive bacterial flora. These components help suppress the growth of unwanted organisms while allowing for tentative identification of the desired bacteria.

Lactose, a fermentable carbohydrate present in VRBA, serves as a substrate for bacterial metabolism. Bacteria that rapidly attack lactose produce colonies that are purple in color, surrounded by purple haloes. This color change is a result of the degradation of lactose to acid, which is indicated by the pH indicator, neutral red. Neutral red changes its color to red in an acidic environment, facilitating the identification of lactose-fermenting organisms. Additionally, the precipitation of bile acids also indicates the acid production.

Non-fermenters or late lactose-fermenters, on the other hand, produce pale colonies with greenish zones, indicating their inability to efficiently utilize lactose for acid production.

Specific colony colors and characteristics observed on VRBA can provide further clues for the identification of certain bacterial groups. For example, lactose-positive Enterobacteriaceae, including coliform bacteria and Escherichia coli, appear as red colonies surrounded by reddish precipitation zones with a diameter of 1-2 mm. Pink pin-point colonies may indicate the presence of Enterococci, possibly Klebsiella. Colorless colonies are indicative of lactose-negative Enterobacteriaceae.

The medium also contains peptic digest of animal tissue and yeast extract, which serve as sources of carbon, nitrogen, vitamins, and other essential growth nutrients. Lactose utilization by bacteria leads to the production of acids, which can be detected by the neutral red indicator. Crystal violet and bile salts help inhibit the accompanying Gram-positive and unrelated flora, ensuring selectivity. Sodium chloride maintains the osmotic equilibrium necessary for bacterial growth on VRBA.

In summary, the principle of VRBA relies on the selective inhibitory components of crystal violet and bile salts, the fermentable carbohydrate lactose, and the indicator system of neutral red. These components work together to suppress the growth of unwanted organisms, allow for tentative identification of desired bacteria based on colony characteristics, and facilitate the detection of lactose fermentation and acid production. VRBA is a useful medium in the identification and enumeration of lactose-fermenting bacteria, particularly coliforms, in various samples.

Preparation of VRBA

To prepare Violet Red Bile Agar (VRBA), the following steps can be followed:

1. Weigh out 41.53 grams of VRBA powder and suspend it in 1000 ml of distilled water. Stir the mixture to facilitate dissolution of the medium.
2. Heat the suspension with continuous stirring until it reaches boiling point. Make sure the medium is completely dissolved. It’s important to note that VRBA should not be autoclaved.
3. Allow the medium to cool down to approximately 45°C. This temperature ensures that the medium is still in a liquid state and can be poured into Petri plates without causing harm to the agar or inhibiting bacterial growth.
4. Sterilize the Petri plates separately using the appropriate sterilization method. This can be done by autoclaving the plates at 121°C and 15 pounds of pressure for 15 minutes.
5. Once the VRBA medium has cooled to 45°C, pour it into the sterile Petri plates that contain the desired inoculum. It’s important to work in aseptic conditions during this step to prevent contamination.

After pouring the medium, allow it to solidify in the Petri plates before using them for bacterial culture. The plates can then be incubated at the appropriate temperature and conditions required for the growth and detection of lactose-fermenting coliform microorganisms.

Procedure on Violet Red Bile Agar (VRBA)

Presumptive testing for coliforms in solid medium

1. Transfer 1mL of amount of test sample to an Petri dish.
2. Add 10mL of Violet Red Bile Aggar (at 48°C) and stir to mix.
3. Allow the medium to set prior to incubating at 35°C for 18 to 24 hours. You can use 32degC for dairy products.
4. Check for colonies of purple-red, 0.5 mm in diameter (or greater) and surrounded by an area of bile acids.
5. Continue to confirm the test results of typical colonies of coliforms.

Quality Control

Quality control measures are important to ensure the reliability and performance of Violet Red Bile Agar (VRBA). Here are the quality control parameters and observations for VRBA:

1. Appearance: The VRBA powder should have a light yellow to pink homogeneous free-flowing appearance. This indicates that the medium is in the expected physical state.
2. Gelling: After preparation, VRBA should form a firm gel comparable to a 1.5% Agar gel. This ensures that the medium has achieved the desired consistency for microbial growth.
3. Colour and Clarity of Prepared Medium: VRBA should form a reddish-purple colored gel that is clear to slightly opalescent in Petri plates. This indicates that the prepared medium meets the expected color and clarity standards.
4. Reaction: A 4.15% w/v aqueous solution of VRBA should have a pH of 7.4±0.2 at 25°C. This pH range ensures optimal conditions for microbial growth and detection.
5. pH: The pH of VRBA should fall within the range of 7.20-7.60. This pH range is suitable for supporting the growth of specific bacteria and obtaining accurate results.
6. Cultural Response: VRBA undergoes cultural testing using specific strains of microorganisms to assess its performance. The following strains are commonly used for cultural response evaluation:a. Enterobacter aerogenes ATCC 13048: The expected cultural response includes luxuriant growth with pink to pinkish-red colonies.b. Escherichia coli ATCC 25922: The expected cultural response includes luxuriant growth with pinkish-red colonies and the presence of a bile precipitate.c. Salmonella Enteritidis ATCC 13076: The expected cultural response includes luxuriant growth with colonies appearing colorless to orangish-yellow.d. Staphylococcus aureus ATCC 25923: The expected cultural response includes inhibited growth, meaning no or minimal bacterial colonies are observed.

By evaluating these cultural responses, it can be determined whether VRBA supports the growth of specific strains and produces the expected colony characteristics.

These quality control measures help ensure the consistency and performance of VRBA, allowing for accurate and reliable detection and enumeration of lactose-fermenting bacteria.

Result Interpretation on Violet Red Bile Agar (VRBA)

The interpretation of results on Violet Red Bile Agar (VRBA) depends on the colony color and characteristics observed. Here are the typical interpretations for different types of organisms:

1. Lactose fermenters:
   • Appearance: Purple-red colonies
   • Description: These organisms are capable of fermenting lactose, resulting in the production of acids. The colonies on VRBA appear purple-red and may or may not have a zone of precipitate around them.
2. Lactose non-fermenters:
   • Appearance: Colorless to transparent colonies
   • Description: These organisms do not possess the ability to ferment lactose. As a result, the colonies on VRBA appear colorless or transparent.
3. Gram-positive cocci:
   • Appearance: Colorless, pinpoint colonies
   • Description: Gram-positive cocci, which may include organisms like Enterococci, produce small pinpoint colonies that are colorless on VRBA.

Here are some specific examples of organism growth and their corresponding appearances on VRBA:

• Enterobacter aerogenes:
  • Growth: Mucoid, pink to pinkish-red colonies
  • Description: Enterobacter aerogenes exhibits mucoid growth, and the colonies on VRBA appear pink to pinkish-red.
• Escherichia coli:
  • Growth: Pinkish-red with bile precipitate
  • Description: Escherichia coli shows pinkish-red colonies on VRBA, often accompanied by a zone of precipitate due to the bile salts present in the medium.
• Salmonella Enteritidis:
  • Growth: Colorless to orangish-yellow colonies
  • Description: Salmonella Enteritidis exhibits growth on VRBA, with colonies appearing colorless to orangish-yellow.
• Salmonella typhimurium:
  • Growth: Fair to good growth; colorless colonies
  • Description: Salmonella typhimurium shows fair to good growth on VRBA, and the resulting colonies are colorless.
• Staphylococcus aureus:
  • Growth: Partial to complete inhibition
  • Description: Staphylococcus aureus may exhibit either partial or complete inhibition on VRBA, meaning the growth is limited or absent.

It’s important to note that these interpretations are general guidelines, and additional confirmatory tests or biochemical tests should be performed for accurate identification of the organisms. The results obtained on VRBA provide initial indications of lactose fermentation and colony characteristics, but further analysis is often required for a definitive identification of the bacterial species.

Organisms	Growth
Enterobacter aerogenes	Mucoid, pink to pinkish-red
Escherichia coli	Pinkish red with bile precipitate
Salmonella Enteritidis	Colorless to orangish-yellow
Salmonella typhimurium	Fair to good growth; colorless colonies
Staphylococcus aureus	Partial to complete inhibition

Uses of Violet Red Bile Agar (VRBA)

Violet Red Bile Agar (VRBA) has various applications in microbiology, particularly in the isolation, detection, and enumeration of coli-aerogenes bacteria. Here are some common uses of VRBA:

1. Water Testing: VRBA is utilized in water quality analysis to detect the presence of coli-aerogenes bacteria. It helps in assessing the microbial contamination of water sources, including drinking water, swimming pools, and recreational waters.
2. Milk and Dairy Product Testing: VRBA is employed in the dairy industry to evaluate the microbial quality of milk and dairy products. It allows for the identification and enumeration of coli-aerogenes bacteria, which are indicators of fecal contamination and can be potential sources of foodborne pathogens.
3. Food Testing: VRBA is applied in the examination of various food products, including processed foods, raw ingredients, and ready-to-eat items, to determine the presence of coli-aerogenes bacteria. This aids in assessing the hygienic conditions during food processing and handling.
4. Clinical Microbiology: VRBA finds utility in clinical laboratories for the isolation and detection of coli-aerogenes bacteria in clinical samples. These bacteria can be associated with gastrointestinal infections and urinary tract infections, making VRBA a useful tool for their identification and enumeration.

Overall, VRBA is a valuable medium for the isolation, detection, and enumeration of coli-aerogenes bacteria in diverse samples, ranging from water and dairy products to clinical specimens. By utilizing VRBA, microbiologists can assess the microbial quality of various environments and identify potential sources of contamination, thus contributing to public health and safety measures.

Limitations of Violet Red Bile Agar (VRBA)

Violet Red Bile Agar (VRBA) has certain limitations that should be taken into consideration. Here are some of the limitations associated with VRBA:

1. Lack of Complete Specificity: VRBA is not entirely specific for enteric bacteria. Other accompanying bacterial species may exhibit similar reactions on the medium, leading to potential false-positive or false-negative results. Therefore, further confirmatory tests and identification methods are necessary for accurate species identification.
2. Varying Nutritional Requirements: Some strains of bacteria may have specific nutritional requirements that are not fully met by VRBA. This can result in poor or no growth of certain strains, leading to potential underestimation of their presence in the sample.
3. Limited Inhibition of Gram-Positive Organisms: While VRBA is designed to inhibit the growth of Gram-positive bacteria, it may not be completely inhibitory to all Gram-positive organisms. This can lead to the growth of Gram-positive bacilli other than members of the Enterobacteriaceae family on VRBA. To accurately identify isolates, additional tests such as Gram staining and biochemical tests are recommended.
4. Boiling Time: Prolonged boiling of the VRBA medium for more than 2 minutes can negatively affect its ability to support bacterial growth. It is important to follow the recommended protocol and not exceed the specified boiling time.
5. Supplementary Identification Tests: VRBA provides initial indications of lactose fermentation and colony characteristics. However, for complete identification, it is recommended to perform further tests such as biochemical, immunological, molecular, or mass spectrometry testing on colonies obtained from pure culture.
6. Growth of Enterococci: Enterococci, although not specifically targeted by VRBA, may still grow on the medium. They usually appear as pinpoint colonies and have a rose-colored appearance.
7. Clinical Diagnostic Limitations: VRBA is not intended for use in the diagnosis of diseases or other conditions in humans. It is primarily used for environmental and food testing purposes.

Considering these limitations, it is important to use VRBA in conjunction with other appropriate methods and tests to ensure accurate identification and characterization of bacteria in various samples.

FAQ

References

• https://exodocientifica.com.br/_technical-data/M049.pdf
• http://www.labm.com/products/violet-red-bile-agar-vrba.asp
• https://www.humeau.com/media/blfa_files/TC_211695-Violet-Red-Bile-Agar_EN_110909.pdf
• https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/VioletRedBileAgar.htm