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Technical Data
Brilliant Green Bile Broth 2% M121
Intended Use:
Recommended for detection and confirmation of coliform bacteria in water, waste water, food, milk and dairy products.
Composition**
Ingredients g/L
Peptone 10.000
Lactose 10.000
Bile# 20.000
Brilliant green 0.0133
Final pH ( at 25°C) 7.2±0.2
**Formula adjusted, standardized to suit performance parameters
# - Equivalent to Oxgall
Directions
Suspend 40.01 grams in 1000 ml purified/distilled water. Heat if necessary to dissolve the medium completely. Distribute in
fermentation tubes containing inverted Durhams tubes and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
For preparation of double strength it is recommended to heat the dissolved broth (80.02 grams per litre) at 100°C for 30
minutes. Do not autoclave double strength broth.
Principle And Interpretation
Brilliant Green Bile Broth 2% is one of the most widely used medium for the detection of coliform bacteria in water,
wastewater, foods, and milk and dairy products. This medium is formulated as per APHA (1,2,3) for the presumptive
identification and confirmation of coliform bacteria (4,5). This medium is also recommended by the ISO Committee for
enumeration of coliforms by most probable number technique (6).
Peptone serves as a source of essential nutrients. Lactose is the fermentable carbohydrate. Bile inhibits gram-positive bacteria
whereas the gram-negative bacteria are inhibited by brilliant green. Production of gas from lactose fermentation is detected
by incorporating inverted Durham's tube, which indicates the positive evidence of faecal coliform since non faecal coliforms
growing in this medium do not produce gas. Further gas production in EC broth (M127) at 45°C used as a confirmation of
faecal coliform. Gram-positive spore formers may produce gas if the bile or brilliant green inhibition is weakened by reaction
with food material.
During examination of water samples, growth from presumptive positive tubes showing gas in Lactose Broth (M026) or
Lauryl Tryptose Broth (M080) is inoculated in Brilliant Green Bile Broth 2% (M121). Gas formation within 48 ± 2 hours
confirms the presumptive test (1).
Type of specimen
Food and dairy samples; Water samples
Specimen Collection and Handling:
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (3,7,8).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards
(9). After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions :
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection.
Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established
guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations :
1. Do not autoclave double-strength broth.
2. Gram-positive sporing organisms may produce gas if the bile/brilliant green inhibition is attenuated by foodmaterial.
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at
recommended temperature.
Quality Control
Appearance
Cream to pale green coloured homogeneous free flowing powder.
Colour and Clarity of prepared medium
Emerald green coloured, clear solution without any precipitate.
Reaction
Reaction of 4.0% w/v aqueous solution at 25°C. pH : 7.2±0.2
pH
7.00-7.40
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-48 hours.
Organism Inoculum Growth Gas
(CFU)
Bacillus cereus ATCC >=104 inhibited
10876
Escherichia coli ATCC 50-100 good-luxuriant positive reaction
25922 (00013*)
# Klebsiella aerogenes 50-100 good-luxuriant positive reaction
ATCC 13048 (00175*)
Enterococcus faecalis 50-100 none-poor negative reaction
ATCC 29212 (00087*)
Staphylococcus aureus >=104 inhibited
subsp. aureus ATCC
25923 (00034*)
Key : (*) Corresponding WDCM numbers (#) Formerly known as Enterobacter aerogenes
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 15-25°C. Use before expiry
date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to
prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may
lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of
ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow
established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must
be decontaminated and disposed of in accordance with current laboratory techniques (9,10).
Reference
1. Lipps WC, Braun-Howland EB, Baxter TE,eds. Standard methods for the Examination of Water and Wastewater, 24th
ed. Washington DC:APHA Press; 2023.
2. Richardson G., (Ed.), 1985, Standard Methods for the Examination of Dairy Products, 15th Ed, APHA, Washington, D.C.
3. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of
Foods, 5th Ed., American Public Health Association, Washington, D.C.
4. McCrady and Langerin, 1932, J. Dairy Science, 15:321.
5. McCrady, 1937, Am. J. Publ. Health, 27:1243.
6. International Organization for Standardization (ISO), 1991, Draft ISO/DIS 4831.
7. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed.,
Washington D.C.
8. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed.,
APHA Inc., Washington, D.C.
9. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
10. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual
of Clinical Microbiology, 11th Edition. Vol. 1
Revision : 05/2024
Please refer disclaimer Overleaf.
HiMedia Laboratories Technical Data
Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and
other related HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the best
of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to specifications and information related
to the products at any time. Products are not intended for human or animal or therapeutic use but for laboratory,diagnostic, research or further
manufacturing use only, unless otherwise specified. Statements contained herein should not be considered as a warranty of any kind, expressed or
implied, and no liability is accepted for infringement of any patents.
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