Brilliant Green Bile Broth 2% ISO

Enzymatic digest of casein provides nitrogen, vitamins, minerals and amino acids essential for growth. Lactose is the fermentable carbohydrate providing carbon and energy. Ox bile and brilliant green inhibit Gram-positive bacteria and most Gram-negative bacteria except coliforms. They also prevent the growth of the anaerobic lactose-fermenters such as Clostridium perfringens, which could give false positive reactions.

This medium is recommended by the ISO 4831 and ISO 4832 standards for the confirmation of coliforms. The production of gas at 30 °C or 37 °C confirms the presence of coliforms.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 40 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. Dispense into tubes with Durham gas collecting tubes for gas detection and sterilize in autoclave at 121 °C for 15 minutes. AVOID OVERHEATING.

When the sample has 1 mL or less volume, dispense medium in volumes of 10 mL. To analyze samples of 10 mL, dissolve 80 grams of the medium in a liter of distilled water and distribute in the same manner.

Instructions for use

For the confirmation of coliforms according to ISO 4831 and 4832:

1. Inoculate the typical colonies from the incubated plates of VRBL (LC-1093) into tubes of Brilliant Green Bile Broth 2%.
2. Incubate the tubes at a temperature of 30 °C or 37 °C for 24-48 hours.
3. Consider as coliforms the colonies that show gas formation in the Durham tubes.

Note: in the case of milk and milk products, the temperature of incubation is 30 °C.

For the confirmation of Escherichia coli:

1. Incubate Brilliant Green Bile Broth 2% at 44 ± 1 °C for 48 hours.
2. Turbidity in the broth and gas production in the inverted tube are positive signs.
3. An indole production test at 44.5 °C is also carried out in Peptone Tryptone Water (LC-1403).

• ISO 4831:2006 Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of coliforms - Most probable number technique.
• ISO 4832:2006 Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of coliforms - Colony-count technique.
• Standard Methods for the Examination of Water and Sewage, 9th. Edition 195. 1946.
• Standard Methods for the Examination of Dairy Products, 9th. Edition 152. 1948.