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Code: CM0031

This medium is used to detect or confirm the presence of members of the coli-aerogenes group; the brilliant green content suppresses anaerobic lactose fermenters, such as Clostridium perfringens, and the medium is recommended for the 44°C confirmatory test for Escherichia coli.

* Adjusted as required to meet performance standards

Directions
Dissolve 40g in 1 litre of distilled water. Mix well, distribute into containers fitted with Durham’s tubes and sterilise by autoclaving at 121°C for 15 minutes.

Do not autoclave double stregnth medium. The alternative procedure is to heat the dissolved broth at 100°C for 30 minutes¹.

Description
This medium was formulated by Durham and Schoenlein² to selectively recover organisms of the coli-aerogenes group. The bile and brilliant green components inhibit the Gram-positive organisms, whilst the coli-aerogenes group are recognised by the rapid formation of gas during lactose fermentation³. It is important that the inhibitory agents in the medium are balanced with the nutrient and mineral components, so that Clostridium and Bacillus spores do not give false positive reactions in the medium i.e. gas formation. Brilliant Green Bile Broth is used in water, dairy and food analysis^4,5,6,7,8.

The addition of 4-methylumbelliferyl-β-D-glucuronide (MUG) (BR0071) to this medium will enhance the detection of Escherichia coli. See MUG Reagent (BR0071) under Biological Reagents for further details.

Technique
To indicate the presence of Escherichia coli, Brilliant Green Bile Broth is incubated at 44 ± 1°C for 48 hours. Turbidity in the broth and gas production in the inverted tube are positive signs. An indole production test at 44°C is also carried out in Tryptone Water (CM0087) or Peptone Water (CM0009) to confirm the identity of Escherichia coli.

In water plant control tests, where <1ml to 10ml volumes of water are used, it is important not to overdilute the medium. Thus 1ml or less volumes of water can be added to 10ml of Brilliant Green Bile Broth. For 10ml volumes of water, double strength Brilliant Green Bile Broth should be used in equal volumes. When incubated at 35°C for 48 hours, gas formation presumptively indicates coli-aerogenes organisms.

Food macerates are decimally diluted and added to the broth in the proportion 1:10. Double strength broth can be used for large volume samples.
Incubation is carried out; at 44°C for 48 hours to detect Escherichia coli; at 32°C for 25-48 hours to detect coli-aerogenes organisms^1,10; or at 4°C for 10 days to detect psychrotrophic coliform organisms.

The medium becomes turbid and yellowish-green in colour when bacteria are growing and, when accompanied by copious gas formation, there is presumptive evidence of coli-aerogenes organisms. Confirmatory tests should be carried out.

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 tubes of broth at 2-8°C.

Appearance
Dehydrated medium: Pale green, free-flowing powder
Prepared medium: Green solution

Quality Control

* This organism is available as a Culti-Loop®

Precautions
Do not autoclave double-strength broth.
Gram-positive sporing organisms may produce gas if the bile/brilliant green inhibition is attenuated by food material.

References
1. Baird R. M., Corry J. E. L. and Curtis G. D. W. Editors. (1987) `Pharmacopoeia of Culture media for Food Microbiology’ Internat. J. Food Microbiol. 5. 206-207.
2. Durham H. G. and Schoenlein H. W. (1926) Stain Techn. 1. 129-134.
3. Mackenzie E. F. W., Windle Taylor E. and Gilbert W. E. (1948) J. Gen. Microbiol. 2. 197-204.
4. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products. 14th Edn. APHA Inc. Washington D.C.
5. American Public Health Association (1992) Compendium of Methods for the Microbiological Examination of Foods. APHA Inc. Washington D.C.
6. American Public Health Association (1998) Standard Methods for the Examination of Water and Wastewater. 20th Edn. APHA Inc. Washington D.C.
7. Association of Official Analytical Chemists (1978) Bacteriological Analytical manual. 5th Edn. AOAC Washington D.C.
8. Labots H. and Galesloot Th. E. (1960) Rapp. Ned. Inst. Zuivelonderz. 25-31.
9. Joint Circular 20/82, Departments of the Environment (1982) incorporating EC Directive relating to the Quality of Water intended for human consumption. (80/778/EEC) HMSO London England.
10. Lightbody L. G. (1963) Aust. J. Dairy Techn. 18. 202-203.