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VIOLET RED BILE GLUCOSE (VRBG) AGAR (ISO)

Code: CM1082

Violet Red Bile Glucose Agar (VRBGA) (ISO) is for the detection and enumeration of Enterobacteriaceae in food, animal feed and environmental samples. This medium meets the formulation and performance criteria laid down in ISO 21528^1.2 and ISO 11133:2014³.

*Adjusted as required to meet performance standards

Directions
Suspend 38.5g of VRBGA (ISO) dehydrated medium in 1 litre of distilled water. Bring to the boil while mixing. Continue to boil for up to 2 minutes or for the minimum time necessary to dissolve completely. DO NOT AUTOCLAVE. Cool to 44-47°C and use within 4 hours. Mix well before pouring.

Description
Various groups of bacteria or individual species are used to provide evidence of poor hygiene, inadequate processing or post-processing contamination of foods. Some bacteria, such as Escherichia coli, are present in the gastrointestinal tract of many animals, including humans, so their presence can be used to indicate potential faecal contamination.

One of the most common groups of bacteria employed as indicator organisms by the food industry are the coliforms, which can be regarded as a subgroup within the Enterobacteriaceae. However, in an attempt to improve assessment of food safety and quality by the food industry, there has been a gradual move towards testing for total Enterobacteriaceae rather than limiting testing to coliforms. In processed foods, particularly foods subjected to heat treatment, these organisms can provide a reliable indication of process failure, under-processing or post-processing contamination.

Mossel et al³ modified Violet Red Bile Lactose Agar by adding glucose to improve recovery of all Enterobacteriaceae. Later work by Mossel and Cowell^4,5 demonstrated that lactose could be omitted, resulting in the formulation known as VRBGA.

Enterobacteriaceae rapidly ferment glucose and so reduce the pH of the medium, which produces purple/pink coloured colonies due to the inclusion of the neutral red indicator and crystal violet in the medium. These colonies are usually surrounded by purple halos of precipitated bile salts. Crystal violet and bile salts inhibit the growth of Gram-positive flora.

Technique
According to ISO 11133:2014³, VRBGA (ISO) should deliver a productivity ratio of ≥0.5 for E. coli when compared to the same inoculum grown on a reference medium (Tryptone Soya Agar). VRBG Agar (ISO) is tested in line with the requirements of ISO 11133:2014³. Quality certificates, indicating organisms tested and productivity, are available for every batch.

Refer to the appropriate standard method for full instructions. The following is an overview of ISO 21528-2:2004².

1. Prepare the sample to be tested according to the instructions in the relevant part of ISO 6887⁷ or the specific standard as appropriate.
2. Take two Petri dishes and transfer to each 1.0ml of the test sample or test suspension. Repeat the process with decimal dilutions of the sample if required.
3. Within 15 minutes, add approximately 10ml of molten VRGBA (ISO), at 44-47°C. Carefully mix the medium with the inoculum by horizontal movements and allow the medium to cool.
4. Once the agar has solidified, pour an additional 15ml of medium onto the surface of the inoculated plate to prevent spreading growth.
5. Incubate for 24 ± 2 hours at 37°C.
6. Count the total number of characteristic colonies. Enterobacteriaceae form purple/pink coloured colonies larger than 0.5mm in diameter, usually surrounded by a halo.
7. Randomly select five purple/pink colonies for confirmation.
8. Streak onto Nutrient Agar, and incubate at 37°C for 24 ± 2 hours. Select a well-isolated colony from each plate for confirmation using oxidase and fermentation tests.

Storage conditions and Shelf life
VRBGA (ISO) should be stored in the tightly capped original container at 10-30°C. When stored as directed, the un-opened product will remain stable until the expiry date printed on the packaging.

Locally prepared VRBGA (ISO) plates can be stored for up to 2 weeks when made from CM1082 according to the manufacturer’s instructions and stored at 2-8°C, out of direct sunlight. A longer shelf life may be attainable, but should be validated under the relevant, local manufacturing and storage conditions.

Appearance
Dehydrated medium: Straw-pink, free-flowing powder
Prepared medium: Purple gel

Quality Control

* This organism is also available as a Culti-Loop®

According to ISO 11133:2014³, VRBGA (ISO) should deliver a productivity ratio of ≥ 0.5 for E. coli when compared to the same inoculum grown on a reference medium (Tryptone Soya Agar). VRBGA (ISO) is tested in line with the requirements of ISO 11133:2014³. Quality certificates, indicating organisms tested and productivity, are available for every batch.

Precautions
VRBGA (ISO) should be prepared, cooled to 44-47°C and used within 4 hours.

VRBGA (ISO) is for laboratory use only. Do not use the dehydrated medium beyond the stated expiry date or if the product shows any sign of deterioration.

Refs
1. ISO 21528-1:2004 Microbiology of food and animal feeding stuffs - Horizontal methods for the detection and enumeration of Enterobacteriaceae - Part 1: Detection and enumeration by MPN technique with pre-enrichment
2. ISO 21528-2:2004 Microbiology of food and animal feeding stuffs - Horizontal methods for the detection and enumeration of Enterobacteriaceae - Part 2: Colony-count method

3. ISO 11133:2014 Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media.
4. Mossel, Eelderink, Koopmans and van Rossem. 1978. Lab Practice 27:1049
5. Mossel, Eelderink, Koopmans and van Rossem. 1979. J. Food Protect. 42:470
6. Cowell, N.D. and Morisetti, M.D. 1969 J. Sci. Fd. Agric., 20:573
7. ISO 6887 series Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination

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