- Coliforms refer to all aerobic and facultative anaerobic, gram-negative, non-spore-forming rod-shaped bacteria that ferment lactose with gas and acid formation within 48 hours at 35°C.
- Procedures to detect, enumerate, and presumptively identify coliforms are used in testing foods and dairy products.
- One method for performing the presumptive test for coliforms uses Violet Red Bile Agar, (VRBA).
- Violet Red Bile Agar is a selective medium used to detect and enumerate lactose-fermenting coliform microorganisms.
Composition of Violet Red Bile Agar (VRBA)
|Peptic digest of animal tissue||7.000|
|Bile salts mixture||1.500|
Final pH (at 25°C): 7.4±0.2
Principle of Violet Red Bile Agar (VRBA)
- It relies on the use of the selective inhibitory components crystal violet and bile salts and the indicator system lactose and neutral red.
- Crystal violet and bile salts inhibit growth primarily of the Gram-positive accompanying bacterial flora.
- Degradation of lactose to acid is indicated by the pH indicator neutral red, which changes its color to red, and by precipitation of bile acids.
- Thus, the growth of many unwanted organisms is suppressed, while tentative identification of sought bacteria can be made.
- Organisms that rapidly attack lactose produce purple colonies surrounded by purple haloes. Non-fermenters or late lactose fermenters produce pale colonies with greenish zones.
Red, surrounded by reddish precipitation zones, diameter 1-2 mm : Lactose-positive Enterobacteriaceae: coliform bacteria, E. coli.
Pink pin-point colonies: Enterococci, possibly Klebsiella.
Colorless: Lactose-negative Enterobacteriaceae
- Peptic digest of animal tissue and yeast extract serve as sources of carbon, nitrogen, vitamins and other essential growth nutrients.
- Lactose is the fermentable carbohydrate, utilization of which leads to the production of acids.
- Neutral red indicator detects the acidity so formed.
- Crystal violet and bile salts mixture help to inhibit the accompanying gram-positive and unrelated flora. Sodium chloride maintains the osmotic equilibrium.
Preparation and Method of Use of Violet Red Bile Agar (VRBA)
- Suspend 41.53 grams in 1000 ml distilled water.
- Heat with stirring to boiling to dissolve the medium completely.
Note: DO NOT AUTOCLAVE.
- Cool to 45°C and pour into sterile Petri plates containing the inoculum.
- Transfer a 1 mL aliquot of the test sample to a petri dish.
- Add 10 mL of Violet Red Bile Agar (at 48°C) and swirl to mix.
- Allow medium to solidify before incubating at 35°C for 18 – 24 hours; use 32°C for dairy products.
- Examine for purple-red colonies, 0.5 mm in diameter (or larger), surrounded by a zone of precipitated bile acids.
- Continue with confirmatory testing of typical organism’s colonies.
Result Interpretation of Violet Red Bile Agar (VRBA)
|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)
- VRBA is used for the isolation, detection, and enumeration of coli-aerogenes bacteria in water, milk, and other dairy food products and also from clinical samples.
Limitations of Violet Red Bile Agar (VRBA)
- Violet Red Bile Agar 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 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.