VRBL Agar (Violet Red Bile Lactose Agar)

VRBL Agar (Violet Red Bile Lactose Agar) is a selective medium for the isolation, detection, and enumeration of lactose-fermenting bacteria and the differentiation of coliforms or Coli aerogenes group from non-lactose fermenting organisms in water, milk, and other dairy products, dairy equipment, and other foodstuffs.

Coliforms are gram-negative bacilli, non-sporulated, oxidase negative, aerobic or anaerobic, able to multiply in the presence of bile salts or other surface agents which can ferment lactose rapidly, producing acid and gas, usually within 24 hours or 48 hours at the temperature of 44 ± 0.5°C. 

According to the International Organization for Standardization (ISO), fecal coliforms are also known as thermotolerant coliforms.

It belongs to several bacterial species: Enterobacter aerogenes, Enterobacter cloacae, Klebsiella (Klebsiella pneumoniae, Klebsiella oxytoca), Citrobacter freundii, Citrobacter diversus, Citrobacter amalonaticus, and Escherichia coli. Moreover, Other organisms that can also ferment lactose are Erwinia and Serratia (slowly), Moellerella wisconsensis, Salmonella enterica subsp. III (Salmonella arizonae), Yersinia enterocolitica and Hafnia alvei.

Composition of VRBL Agar

Store the prepared media at 2°C-8°C and protect it from direct sunlight. Store the dehydrated powder in a cool, dry place in tightly sealed containers or bottles at 15°C-25°C to protect from moisture and light. After opening and replacing the cap, replacing the cap, store the container in a low-humidity area at the same storage temperature.

The expiration date is on the package or container. Discard it if the dehydrated medium is not freely flowing or its appearance has altered from its original color.

Principle of VRBL Agar

• VRBL agar, which contains bile salts and crystal violet, is based on MacConkey Agar. It is not required to use in the diagnosis of disease or other conditions in humans.
• Peptone is a source of nitrogen, vitamins, minerals, and amino acids essential for growth.
• Yeast extract is a source of vitamins, especially those in the B group. 
• Lactose is a fermentable sugar or carbohydrate that helps to provide carbon and energy.
• Bile salts and Crystal violet are inhibitors of non-target Gram-positive and some Gram-negative bacteria. 
• Neutral red is a pH indicator that alters pH, which changes neutral red to a red-purple color due to the production of acid during fermentation. 
• Sodium chloride provides essential electrolytes for transport and osmotic balance. Bacteriological agar is the solidifying agent.
• Microorganisms or Coliforms that rapidly ferment lactose produce purple or red-purple colonies with precipitation of bile salts often surrounded by a halo of the same color, red-purple (in the case of strong acidification connected to the use of lactose). Non-lactose fermenters and late lactose fermenters produce pale colonies with greenish areas. Occasionally, the cocci of the intestinal tract can form small, punctiform red colonies. The diameter of red-purple colonies is usually > 0.5mm.
• Selectivity can increase by anaerobic incubation or elevated temperature above 42°C for 18 hours, which may apply to suppress the growth of unwanted microorganisms or other gram-positive bacteria. 

Macroscopic Observation on VRBL Agar

After incubation at 37°C for 18-24 hours under anaerobic conditions, the color and growth of colonies are below:

Preparation of VRBL Media

1. Weigh 38.5 grams of VRBL agar and suspend it on 1 liter of purified/ distilled water. And wait it to absorb for 5 minutes.
2. Heat it gently with frequent agitation and Mix the medium properly until a homogenous suspension obtain by boiling it for a few seconds.
3. Note: Do not autoclave, sterilize, or overheat. Cool it to 45°C.
4. If necessary, maintain a pH of 7.4 ± 0.2.
5. Pour in sterilized Petri dishes or tubes. Medium appearance seems light purple-violet, transparent.

The medium can be used upto 4 hours after preparation. 

Procedure

1. Perform the Pour or spread plate procedure with or without overlay. (For the pour plate method, freshly prepare the medium, tempered to 47°C, and use within 3 hours.) 
2. Incubate at 37°C or 18-24 hours for coliforms; psychrotrophs at 4°C for ten days; mesotrophs at 32°C for 24-48 hours; thermotrophs at 42°C for 18 hours; For Escherichia coli, incubate at a temperature of 44 ± 0.1°C. For enumeration of thermotolerant coliforms, Incubate at 44 ± 1°C for 24 ± 2 hr.
3. Examine for purple or red/purple colonies with red/purple haloes with a diameter ≤0.5 mm.
4. Continue confirmatory testing of typical organism’s colonies according to the applicable standards.

Interpretation

• After incubation of coli-aerogens groups at 35 or 37°C for 24 h, coliform colonies on Petri plates should count.
• Coliform forms round, purple, or red-purple colonies with red-purple haloes (lactose +ve types) or pale with greenish zones (lactose -ve types) with a diameter ≤ 0.5 mm.

Uses of VRBL Media

• VRBL agar is used to examine food, animal feed, and environmental samples for the presence of coliforms included as a subgroup within the Enterobacteriaceae.
• VRBL agar is used to enumerate fecal coliforms and thermotolerant coliforms in foods.

Limitations of VRBL Media

1. Some strains may grow poorly or unable to grow on this medium because of variations in nutrition.
2. During the pour-plating procedure, the impact of elevated temperature on stressed coliforms is one of the problems, so fecal coliform agar (FCA) can also use to recover and enumerate stressed fecal coliform.

Precautions of VRBL Media

1. Wear a proper lab coat and gloves. Carefully handle chemicals and infectious live microorganisms (bacteria) while working in the lab.
2. According to local rules and regulations, media that have come into contact with food samples are considered contaminated and should be disposed of as tainted.
3. The time interval between the end of preparation of the stock solution (or the 10^-1 dilution in the case of a solid product) and the moment the dilutions come into contact with the culture medium must not exceed 15 minutes.

References

1. Leclercq A, Wanegue C, Baylac P. Comparison of fecal coliform agar and violet red bile lactose agar for fecal coliform enumeration in foods. Appl Environ Microbiol. 2002;68(4):1631-1638. doi:10.1128/AEM.68.4.1631-1638.2002
2. https://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/CDA_/1093.20131002.pdf
3. https://www.neogen.com/globalassets/pim/assets/original/10041/official_ncm0089_violet-red-bile-lactose-agar-vrbl-iso_technical-specifications_en-us.pdf
4. https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/171/699/70188dat.pdf
5. https://microbiologie-clinique.com/vrbl-agar.html
6. https://www.bio-rad.com/sites/default/files/2022-05/Bulletin_5051.pdf