Violet Red Bile Agar
Intended use
Recommended for selective isolation, detection and enumeration of coli-aerogenes bacteria in water, milk other dairy food
products and clinical samples.
Composition
Ingredients Gms / Litre
Peptone 7.000
Yeast extract 3.000
Sodium chloride 5.000
Bile salts mixture 1.500
Lactose 10.000
Neutral red 0.030
Crystal violet 0.002
Agar 15.000
Final pH ( at 25°C) 7.4±0.2
Directions
Suspend 41.53 grams in 1000 ml purified / distilled water. Heat to boiling to dissolve the medium completely. DO NOT
AUTOCLAVE. Cool to 45-50°C and immediately pour into sterile Petri plates containing the inoculum. If desired, the
medium can be sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
Principle And Interpretation
The coliform group consists of several genera of bacteria belonging to the family Enterobacteriaceae. The historical
definition of this group has been based on the method used for detection i.e. lactose fermentation. This group is defined as all
aerobic and facultative anaerobic, gram-negative, non-spore-forming rod shaped bacteria that ferment lactose with gas and
acid formation within 48 hour at 35°C (1,2). Examination of foods, ingredients and raw materials, for the presence of
marker groups such as coliforms is the one of the common tests.
Violet Red Bile Agar, a modification of MacConkey's original formulation (1) is used for the enumeration of coliaerogenes bacterial group. It relies on the use of the selective inhibitory components crystals violet and bile salts and
the indicator system lactose, and neutral red. Thus, the growth of many unwanted organisms is suppressed, while tentative
identification of sought bacteria can be made. Organisms, which rapidly attack lactose, produce purple colonies surrounded
by purple halos. Non-fermenters or late lactose-fermenters produce pale colonies with greenish zones (3). VRBA is
recommended by APHA (4,5). Selectivity of VRBA can be increased by incubation under anaerobic conditions and/ or
at elevated temperature, i.e. equal to or above 42°C (6-8). It is also recommended by ISO (9).
Peptone 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. Violet Red Bile Agar is not completely specific for enteric; other accompanying
bacteria may give the same reaction. Further biochemical tests are necessary for positive identification (5).
Type of specimen
Clinical samples - Stool; Food and dairy samples; Water samples
Specimen Collection and Handling:
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (3,4,5).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (10).
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (11,12).
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions :
In Vitro diagnostic Use. For professional use only. Read the label before opening the container. Wear protective gloves/
protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens
and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens.
Safety guidelines may be referred in individual safety data sheets.
Limitations :
1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to
validate the medium for any specific microorganism other than mentioned in the COA based on the user’s unique
requirement.
3. Further biochemical tests must be carried out for complete identification.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at
recommended temperature.
Quality Control
Appearance
Light yellow to pink homogeneous free flowing powder
Gelling
Firm, comparable with 1.5% Agar gel.
Colour and Clarity of prepared medium
Reddish purple coloured clear to slightly opalescent gel forms in Petri plates.
Reaction
Reaction of 4.15% w/v aqueous solution at 25°C. pH : 7.4±0.2
pH
7.20-7.60
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.
Inoculum
(CFU)
Organism Growth Recovery Colour of
colony
# Klebsiella aerogenes
ATCC 13048 (00175*)
50-100 luxuriant =50% pink to pinkish
red
50-100 luxuriant =50% pinkish red
with bile
precipitate
Salmonella Enteritidis
ATCC 13076 (00030*)
50-100 luxuriant =50% Colourless to
orangish yellow
Staphylococcus aureus
subsp.aureus ATCC
25923 (00034*)
=104 inhibited 0%
Escherichia coli ATCC
25922 (00013*)
Key : (*) Corresponding WDCM numbers. (#) Formerly known as Enterobacter aerogenes
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the
label. On opening, product should be properly stored dry, after tightly capping the bottle in order 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.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with
clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (11,12).
