Violet Red Bile Glucose Agar (VRBG Agar)

Intended Use

Violet Red Bile Glucose Agar is used for detecting and enumerating Enterobacteriaceae in food and dairy products. Meets United States Pharmacopeia (USP), European Pharmacopoeia
(EP) and Japanese Pharmacopoeia (JP)1-3 performance specifications, where applicable.

Summary and Explanation

The Enterobacteriaceae group includes lactose-fermenting coliform bacteria, lactose-nonfermenting strains of E. coli, and lactose-nonfermenting species, such as Salmonella and Shigella.
When examining some foods, it is desirable to detect Enterobacteriaceae rather than the coliform bacteria.4 Enterobacteriaceae are glucose-fermenting bacteria. Mossel et al.5 modified lactose-containing Violet Red Bile Agar by adding glucose to improve the recovery of Enterobacteriaceae. Later work by Mossel et al.6,7 demonstrated that lactose could be omitted,
resulting in the formulation known as Violet Red Bile Glucose Agar (VRBGA). Violet Red Bile Glucose Agar is recommended for the detection and enumeration of Enterobacteriaceae in food and dairy products.8,9 Violet Red Bile Glucose Agar is also listed in the USP as the recommended solid medium for use in the isolation of bile-tolerant gram-negative bacteria from nonsterile pharmaceutical products.1 The Violet Red Bile Glucose Agar formulation is available as a dehydrated culture medium, as prepared plated media, and in Difco™ Hycheck™ Enterobacteriaceae hygiene contact slides. Hycheck™ Enterobacteriaceae slides are double-sided paddles containing both Violet Red Bile Glucose Agar and Tryptic Soy Agar surfaces for immersing into fluids or for sampling surfaces.

Principles of the Procedure

Violet Red Bile Glucose Agar contains pancreatic digest of gelatin as a source of carbon, nitrogen, vitamins and minerals. Yeast extract supplies B-complex vitamins which stimulate bacterial
growth. Glucose is a carbohydrate. Bile salts and crystal violet inhibit gram-positive bacteria. Glucose fermenters produce red colonies with red-purple halos (bile precipitation) in the presence of neutral red, a pH indicator. Sodium chloride maintains the osmotic balance. Agar is the solidifying agent.

Formula

Violet Red Bile Glucose Agar
Approximate Formula* Per Liter
Yeast Extract................................................................ 3.0 g
Pancreatic Digest of Gelatin............................................ 7.0 g
Bile Salts No. 3............................................................. 1.5 g
Glucose...................................................................... 10.0 g
Sodium Chloride........................................................... 5.0 g
Neutral Red................................................................. 0.03 g
Crystal Violet.............................................................. 2.0 mg
Agar........................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 41.5 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for no more than
2 minutes to completely dissolve the powder. DO NOT AUTOCLAVE.
3. Test samples of the finished product for performance using stable, typical control cultures.

Sample Collection and Handling

For food samples, follow appropriate standard methods for
details on sample collection and preparation according to sample type and geographic location.8,9 

For pharmaceutical samples, refer to the USP for details on sample collection and preparation for testing of nonsterile products.1Procedure

For food samples, refer to appropriate standard references for details on test methods using Violet Red Bile Glucose Agar.8,9 For pharmaceutical samples, refer to USP General Chapter <62> for details on the examination of nonsterile products and tests for isolating Enterobacteriaceae using Violet Red Bile Glucose Agar.1 This medium can be used in spread or pour plate procedures, with or without an overlay. In addition, this medium can be used as an overlayer for spread plates to both prevent swarming colonies and to provide semi-anaerobic conditions that suppress the growth of nonfermentative gram-negative organisms. Stab inoculation procedures can also be used with this medium.

Expected Results

Enterobacteriaceae ferment glucose, produce acid products and form red to dark purple colonies surrounded by red-purple halos.

Limitation of the Procedure

When used in the pour plate procedure, the medium should be freshly prepared, tempered to 47°C, and used within 3 hours.

User Quality Control Escherichia coli

NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Violet Red Bile Glucose Agar
Dehydrated Appearance: Pink-beige to pink, free-flowing, homogeneous
                                   (may contain small dark particles).
Solution:                       4.15% solution, soluble in purified water upon
                                   boiling. Solution is reddish purple, very slightly
                                   to slightly opalescent.
Prepared Appearance:    Reddish purple, very slightly to slightly opalescent.
Reaction of 4.15%
Solution at 25°C:          pH 7.4 ± 0.2
Violet Red Bile Glucose Agar (prepared)
Appearance:                Reddish to purple and opalescent.
Reaction at 25°C:         pH 7.4 ± 0.2

Cultural Response
Violet Red Bile Glucose Agar
Prepare the medium per label directions. Using the pour plate method,
inoculate and incubate at 35 ± 2°C for 18-24 hours. Using the streak
plate method, inoculate and incubate (*) cultures at 30-35°C and (**)
culture at 35-37°C for 18-24 hours.

Violet Red Bile Glucose Agar (prepared)
Inoculate plates and incubate E. coli strains and P. aeruginosa at 30-35°C for 18-24 hours. Incubate S. Typhimurium and S. aureus at 35-37°C for 18-24 hours.

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