The coliform bacteria group includes aerobic and facultatively anaerobic , gram-negative non-spore-forming bacilli that produce lactose, forming gas and acid at 35°C in less than 48 hours. The members of the Enterobacteriaceae constitute the majority of the group. However, other organisms that ferment lactose could include. Methods for detecting, enumerating and presumedly identify coliforms are employed in the testing of food as well as dairy products.1-3 One method to perform the presumptive test for coliforms is using the Violet Red Bile Agar. If colonies that are typical of coliforms appear after further testing, they are examined to confirm that they are coliforms.
Violet Red Bile Agar is used to count bacteria that cause coliforms in dairy products.
Composition of Violet Red Bile Agar (VRBA)
|Bile salts mixture||1.500|
Final pH (at 25°C): 7.4±0.2
Principle of Violet Red Bile Agar (VRBA)
It is based on the use of inhibitory compounds that are selective the bile and crystal violet, as well as indicators like lactose, and neutral red. The bile and crystal violet salts block the growth of the Gram positive bacteria that live in the bacteria. The conversion of lactose to acid is evident with the help of the pH indicators neutral red that changes color from red to that of a red by the precipitation of bile acid. This means that the growth of various undesirable organisms is stopped, and an attempt to identify bacteria that are sought is possible. Organisms that quickly attack lactose produce colonies of purple, with haloes of purple. Non-fermenters, or late lactose fermenters produce pale colonies with green 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 digests of animal tissues and yeast extract are sources of nitrogen, carbon vitamins, and other vital growth factors. Lactose is a fermentable carbohydrate and its utilization leads to the creation of acids. Neutral red indicators detect the acidity that is created. The bile and crystal violet aid in preventing the accompanying unrelated and gram-positive floral flora. Salts of sodium maintain the balance of the osmotic equilibrium.
Preparation and Method of Use of VRBA
- Submerge 41.53 grams in 1000ml purified distillate water.
- Stir the heat until it reaches a boil. to completely dissolve the medium.
- DO NOT AUTOCLAVE.
- Cool to 45-50°C, then pour into sterilized Petri plates that contain the inoculum.
- If you wish The medium can be sterilized using autoclaving at 15 pounds tension (121degC) in 15 minutes.
Presumptive testing for coliforms in solid medium
- Transfer 1mL of amount of test sample to an Petri dish.
- Add 10mL of Violet Red Bile Aggar (at 48°C) and stir to mix.
- Allow the medium to set prior to incubating at 35°C for 18 to 24 hours. You can use 32degC for dairy products.
- Check for colonies of purple-red, 0.5 mm in diameter (or greater) and surrounded by an area of bile acids.
- Continue to confirm the test results of typical colonies of coliforms.
Result Interpretation on Violet Red Bile Agar (VRBA)
- Lactose fermenters: Purple-red colonies, with or without a zone of precipitate around the colonies
- Lactose non fermenters: Colorless to transparent colonies Gram-positive cocci: Colorless, pinpoint colonies
|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 can be used to identification, detection and quantification of coli-aerogenes-producing bacteria in water as well as milk and other dairy products and clinical samples.
Limitations of Violet Red Bile Agar (VRBA)
- Violet Red Bile might not completely inhibit Gram-positive organisms. Conduct biochemical and Gram stain tests as required to determine the presence of isolates.
- The medium is able to produce gram-negative bacilli that are not those belonging to the Enterobacteriaceae. Conduct biochemical tests to determine isolates belonging to species or genus.
- Heating the medium for more than 2 minutes could reduce the capacity of the medium to support growth.
- Plates of the Violet Red Bile Agar are not to be kept incubated for more than 24 hours as microorganisms that have been partially inhibited could expand after prolonged incubation.
- To ensure maximum performance, you must prepare and utilize the medium within 24 hours.