Violet Red Bile Agar With Lactose (VRBL) ISO

Peptone provides nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group. Lactose is the fermentable carbohydrate providing carbon and energy. Bile salts and crystal violet inhibit Gram-positive bacteria. Neutral red is a pH indicator. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Bacteriological agar is the solidifying agent.

It is convenient to use the pour plate method.

Lactose fermenters form red colonies with red-purple halos. Occasionally the cocci of the intestinal tract can develop as small, punctiform red colonies. The purplish red colonies (sometimes surrounded by a reddish zone of precipitated bile) are considered as typical colonies of coliforms and do not require further confirmation.

Atypical colonies (e.g. of smaller size), and all colonies derived from milk products that contain sugars other than lactose, may result in colonies with an appearance that looks similar to the typical coliforms. These colonies should be confirmed in tubes of Brilliant Green Bile Broth (LC-1228).

Preparation

Suspend 41.5 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. DO NOT OVERHEAT. Cool to 44-47 °C and use immediately.

According to ISO 4832

For the enumeration of coliforms according to ISO 4832:

1. It is recommended to prepare two dishes for the liquid product and/or for each dilution chosen.
2. With a sterile pipette transfer 1 mL of liquid product or the appropriate dilutions to the center of each dish. Use another sterile pipette to inoculate each dilution into the dishes.
3. Pour about 15 mL of VRBL medium, at 44 °C to 47 °C, into each Petri dish. The time elapsing between the end of the preparation of the initial suspension (or of the 10⁻¹ dilution if the product is liquid) and the moment when the medium is poured into the dishes should not exceed 15 min.
4. Carefully mix the inoculum with the medium and allow the mixture to solidify with the Petri dishes standing on a cool horizontal surface.
5. Also prepare a control plate with the medium for checking its sterility.
6. After complete solidification, pour about 4 mL of VRBL medium, at 44 °C to 47 °C, onto the surface of the inoculated medium. Allow to solidify as described above.
7. Invert the prepared dishes and incubate them in the incubator set at 30 °C or 37 °C (as agreed) for 24 ± 2 h.
8. In order to confirm the presumptive coliform colonies, inoculate five colonies of each atypical type, if available, into tubes of Brilliant Green Lactose Bile Broth (LC-1228).
9. Incubate the tubes in the incubator set at 30 °C or 37 °C (as agreed) for 24 ± 2 hours.
10. Consider as coliforms the colonies that show gas formation in the Durham tube.

• ISO 4832. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coliforms - Colony-count technique.
• Collins, J. Milk and Food Tech 18:169, 1955.
• Hartman, J. Milk and Food Tech 23:43. 1960.
• Speck, M.L. (ed) 1976. Compendium of Methods for the Microbiological Examination of Foods (APHA).