VIOLET RED BILE GLUCOSE AGAR

Code: CM0485

a glucose-containing selective medium for the detection and enumeration of Enterobacteriaceae in food products

Typical Formula*	gm/litre
Yeast extract	3.0
Peptone	7.0
Sodium chloride	5.0
Bile Salts No.3	1.5
Glucose	10.0
Neutral red	0.03
Crystal violet	0.002
Agar	12.0
pH 7.4 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 38.5g in 1 litre of distilled water. Bring to the boil. Continue to boil for 2 minutes or for the minimum time necessary to dissolve completely and ensure that there are no remaining flecks of unmelted agar. No further sterilisation is necessary or desirable. Mix well and dispense into tubes or dishes.

Description
Results from tests that may be applied to water to detect coli-aerogenes organisms as possible indicators of faecal contamination possess far less significance when applied to raw foods. In the examination of foodstuffs, detection of a more defined group of organisms, the Enterobacteriaceae, that ferment glucose to produce acid and/or gas has been recommended^1,2. In addition to coliforms this group includes salmonellae and shigellae, which do not ferment lactose, and enterotoxigenic Escherichia coli. It also contains organisms, such as Klebsiella and Citrobacter, which are more resistant to heat than coliforms and are therefore better indicators of failure of processes that use minimal heat.

The difficulties of measuring the total Enterobacteriaceae content of foodstuffs have been studied by Mossel et al.³, who showed that the addition of glucose to an existing medium for the detection of coliforms improves the performance. They added 10g per litre of glucose to crystal violet neutral red bile lactose agar (Violet Red Bile Agar CM0107), and named the modified formulation MacConkey Glucose Agar.

Further work by Mossel et al.^4,5 showed that the lactose could be omitted resulting in the formulation of Violet Red Bile Glucose Agar. The continued inclusion of lactose would not provide test results leading to more accurate identification. Exclusion of lactose renders the medium more economical to make as less weight is required per litre.

Media that contain bile salts have an intrinsic toxicity for Enterobacteriaceae, even for cells that have not been under stress^{6,7,8,9,10,11}.

Considerable differences have been observed among six commercial preparations of Violet Red Bile Agar^4,5 with regard to productivity for Enterobacteriaceae¹², and the intensity of their metabolism. In conjunction with Oxoid the components of the medium were examined and Mossel drafted a specification as follows:

1. Approved media have to be clear and yield colonies of satisfactory size. They have to give reproducible counts of typical colonies of Enterobacteriaceae.
2. When challenged for intrinsic toxicity by an anaerobic metabolic test ¹³ using a strain of Yersinia enterocolitica (Serotype 03) as a sensitive indicator, media must promote adequate growth, acid formation and, where required, adequate gas formation.
3. Media have to satisfy the confirmation rate of typical colonies, i.e. the number of colonies confirmed as Enterobacteriaceae divided by the number of colonies tested.

Violet Red Bile Glucose Agar has been developed to satisfy all of these criteria.

Technique
Prepare a series of dilutions of the samples so that at least one will be included that will yield 100-200 colonies from a 1ml aliquot. Transfer 1ml aliquots of each dilution to 9cm Petri dishes using 2 plates for each dilution. Add 15ml of medium, cool to 47°C. Gently swirl the plates 3 times clockwise and 3 times anti-clockwise. After the medium has solidified overlay with 10ml of the same medium and leave to solidify. Invert the dishes and incubate at >42°C for 18 hours, 32°C for 24-48 hours or 4°C for 10 days depending on the groups of Enterobacteriaceae to be recovered ¹⁵.

The agar overlay ensures anaerobic conditions which suppress the growth of non-fermentative Gram negative bacteria. It also encourages the fermentation of glucose which favours the formation of clearly visible purple colonies, surrounded by a purple-pnk halo.

Characteristic appearance of colonies
Round, purple-pink, 1-2mm diameter surrounded by purple haloes.
Although colony size is generally 1-2mm, size can be affected by a number of influences and all purple-pink colonies should be counted. Confirmation of the identity of these colonies must be made by further tests.

Storage conditions and Shelf Life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C and use as freshly as possible.

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

Quality control

Positive control:	Expected results
Escherichia coli ATCC® 25922 *	Good growth; purple/pink colonies with halo
Negative controls:
Staphylococcus aureus ATCC® 6538*	No growth
Enterococcus faecalis ATCC® 29212	No growth