BRILLIANT GREEN BILE BROTH (BGBB)

INTENDED USE

Brilliant Green Bile Broth is used for the detection and enumeration of coliform, heat-tolerant coliform
bacteria and Escherichia coli (Mackenzie test) in milk, milk-based products and other food products,
drinking water and waste water.

HISTORY

Research and development of a culture medium inhibiting microorganisms other than coliform
bacteria has long interested bacteriologists. In 1926, Dunham and Schoenlein studied the proportions
of bile and brilliant green which could give good results. Jordan showed that this medium was better
than Lactose Broth for the detection of coliform bacteria in water. For the control of milk
pasteurization, MacCrady and Langevin satisfactorily used Brilliant Green Bile Broth for the detection
of coliform bacteria. Mackenzie verified that the brilliant green concentration was sufficient to
effectively inhibit the culture of lactose-fermenting anaerobes, in particular Clostridium perfringens.

PRINCIPLES

- The simultaneous presence of ox bile and brilliant green inhibit almost all Gram-positive organisms
and Gram-negative bacteria other than coliforms.
- The brilliant green concentration was specifically determined in order to prevent the growth of
lactose-fermenting anaerobes at 44°C, which avoids false positives.
- Development of coliform bacteria is shown by turbidity and gas production in the Durham tubes, as
a result of lactose fermentation.

PREPARATION

Single strength medium

- Dissolve 40.0 g of dehydrated medium (BK002) in 1 liter of distilled or deionized water.
- Stir slowly until complete dissolution.
- Dispense in appropriately-sized tubes containing a Durham tube.
- Sterilize in an autoclave at 121°C for 15 minutes.
- After cooling, the Durham tubes should not contain trapped air.

NOTE :
M.U.G. (4-methylumbelliferyl-β-D-glucuronide) can be added to the medium in order to detect
Escherichia coli (refer to the monograph on MUG 50 mg Supplement, BS024).

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Double strength medium
- Dissolve 80.0 g of dehydrated medium (BK002) in 1 liter of distilled or deionized water.
- Stir slowly until complete dissolution.
- Dispense 10 mL appropriately-sized tubes (without a Durham tube) at 10 mL per tube.
- Sterilize in an autoclave at 121°C for 15 minutes.

INSTRUCTIONS FOR USE

Single strength medium

- Cool the medium to 25°C.
- Inoculate the tubes prepared as described or use ready-to-use media (BM011) with 1 mL of
inoculum and its serial tenfold dilutions.

Double strength medium

- Cool the medium to 25°C.
- Inoculate the tubes with 10 mL of inoculum.
- Cover with a plug of sterile agar (Bacteriological Agar type E), previously maintained at 44-47°C.

Incubation
- For coliform bacteria, incubate for 24 and 48 hours at 30 or 37°C, depending on the analysis
protocol followed.
- For heat-tolerant coliform bacteria, incubate for 24 and 48 hours at 44°C.
- Transfer one inoculating loop of double strength culture to a single strength tube and incubate for
an additional 24 to 48 hours before reading.

RESULTS

Lactose fermentation, which results in the production of gas in the Durham tubes (volume at least
equal to 1/10 of the tube volume) in less than 48 hours, indicates the presence of coliform bacteria.
The bacteria can be identified by subculturing from the gas-producing tube on suitable agar media :
ƒ BCP lactose agar (BK023)
ƒ EMB agar (BK056).
The presence of Escherichia coli can be confirmed by carrying out the Mackenzie test as follows :
ƒ Inoculate a loop from the gas-producing tubes into a tube of BGBB with a Durham tube
and also in a tube of Peptone Water (BK084).
ƒ Incubate at (44.0 ± 0.5)°C in a controlled water bath for 24 and 48 hours.
Under these conditions, Escherichia coli simultaneously presents the following characteristics :
ƒ growth on BGBB : positive and gas-producing.
ƒ indole production (Ehrlich-Kovacs reagent) : positive.

NOTE :
In the context of the protocol described in NF ISO 4832, it may be necessary to confirm atypical
colonies obtained on VRBL agar through their gas-producing characteristics as observed in Brilliant
Green Bile Broth (BGBB).

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TYPICAL COMPOSITION of single strength medium
(can be adjusted to obtain optimal performance)

For 1 liter of medium :

- Tryptone .........................................................................................10.0 g
- Bacteriological ox bile ....................................................................20.0 g
- Lactose ..........................................................................................10.0 g
- Brilliant green ..............................................................................13.3 mg

pH of the ready-to-use medium at 25°C : 7.2 ± 0.2.

QUALITY CONTROL

- Dehydrated medium : greenish powder, free-flowing and homogeneous.

- Prepared medium : green solution, limpid.
- Typical culture response after 48 hours of incubation at 30°C :

Microorganisms Growth Gas production

(1)
Escherichia coli ATCC® 25922 excellent, score 2 ≥ 5 mm (Durham tube)
(1)
Citrobacter freundii ATCC 43864 excellent, score 2 ≥ 5 mm (Durham tube)
Enterococcus faecalis ATCC 29212 partially inhibited, negative
score 0-1
Staphylococcus aureus ATCC 25923 inhibited, score 0
(2)
Escherichia coli RIVM WR1 good ≥ 5 mm (Durham tube)
(2)
Enterococcus faecalis CCM 2541 inhibited negative
(1)
inoculum ≤ 102 microorganisms)
(2)
according to NF T90-461

STORAGE / SHELF LIFE

Dehydrated medium : 2-30°C.

- The expiration date is indicated on the label.
Prepared medium (benchmark value*) :
- Media in tubes : 3 months at 2-8°C, shielded from light.
Ready-to-use media in tubes containing Durham tubes,
MUG 50 mg Supplement :
- Store between 2-8°C, shielded from light.
- The expiration dates are indicated on the labels.

PACKAGING Code

Ready-to-use media :
- 50 x 10 mL tubes BM01108
Dehydrated medium :
- 500 g bottle BK002HA
MUG 50 mg Supplement :
- 10 vial pack BS02408

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BIBLIOGRAPHY

DUNHAM, H.G. and SCHOENLEIN , H.W.. 1926. Stain Technology, 1 : 129.

JORDAN. 1927. Journal of American Water Works Association, 18 : 337.

McCRADY and LANGEVIN. 1932. Journal of Dairy Science, 15 : 321.

MACKENZIE, E.F.W., TAYLOR, W.E., and GILBERT, W.E.. 1948. Recent experiments in the rapid identification of
Bacterium coli type I. Journal of General Microbiology, 2 : 197-204.

Journal Officiel du 21 Septembre 1968. Méthodes officielles de prélèvement et d’analyse bactériologiques des glaces et
crèmes glacées (Arrêté du 30 Août 1968).

Rodier, J. 1984. L’analyse de l’eau. Dénombrement des coliformes, coliformes fécaux, et Escherichia coli présumés. Dunod
7ème Ed., 793-798.

NF T 90-413. Octobre 1985. Essais des eaux. Recherche et dénombrement des coliformes et des coliformes
thermotolérants. Méthode générale par ensemencement en milieu liquide (NPP).

XP CEN ISO/TS 11133-2 (V 08-104-2). Janvier 2004. Microbiologie des aliments. Guide pour la préparation et la production
des milieux de culture. Partie 2 : Guide général pour les essais de performance des milieux de culture.

NF T 90-461. Juillet 2001 et NF T 90-461/A1. Juin 2005. Qualité de l’eau. Microbiologie. Contrôle qualité des milieux de
culture.

NF ISO 4832 (V 08-015). Juillet 2006. Microbiologie des aliments. Méthode horizontale pour le dénombrement des
coliformes. Méthode par comptage des colonies.

NF ISO 4831 (V 08-016). Octobre 2006. Microbiologie des aliments. Méthode horizontale pour la recherche et le
dénombrement des coliformes. Technique du nombre le plus probable.

*Benchmark value refers to the expected shelf life when prepared under standard laboratory conditions following manufacturer’s instructions. It is provided as a
guide only and no warranty, implied or otherwise is associated with this information.

The information provided on the package or instructions take precedence over the formulations or instructions described in this document.
The information and specifications contained in this technical data sheet date from 2009-02-16.
They are susceptible to modification at any time, without warning.
Code document : BK002/A/2003-01 : 6.

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Biokar Diagnostics – Rue des Quarante Mines – ZAC de Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France
Tél : + 33 (0)3 44 14 33 33 – Fax : + 33 (0)3 44 14 33 34 – www.biokar-diagnostics.com