Brilliant Green Agar- Composition, Principle, Preparation, Results, Uses

Brilliant Green Agar is a selective and differential medium for the isolation of Salmonella species other than S. Typhi and S. Paratyphi from clinical specimens, foods, and dairy products. It contains the following ingredients and their quantities per liter of distilled water:

• Peptone: 5.0 g
• Tryptone: 5.0 g
• Yeast extract: 3.0 g
• Lactose: 10.0 g
• Sucrose: 10.0 g
• Sodium chloride: 5.0 g
• Phenol red: 0.08 g
• Brilliant green: 0.0125 g
• Agar: 20.0 g

The final pH of the medium is 6.9 ± 0.2 at 25°C.

Brilliant Green Agar is a selective and differential medium that inhibits the growth of most Gram-positive and Gram-negative bacteria, except for some Salmonella species. It also distinguishes between lactose/sucrose-fermenting and non-lactose/sucrose-fermenting bacteria based on the color change of the pH indicator phenol red.

Non-lactose/sucrose-fermenting organisms are those that cannot utilize lactose or sucrose as a source of energy. They do not produce acid from these carbohydrates and therefore do not change the color of the phenol red indicator in the agar. These organisms typically belong to the genus Salmonella, which is the main target of this medium.

Lactose and sucrose are the fermentable carbohydrate sources in Brilliant Green Agar. Phenol red serves as an acid-base indicator that changes color depending on the pH of the medium. When lactose and/or sucrose are fermented by bacteria, they produce acidic by-products that lower the pH of the medium. This causes the phenol red indicator to turn yellow.

To prepare Brilliant Green Agar, you need to follow these steps:

1. Suspend 58.09 grams of the dehydrated medium in 1000 ml of purified or distilled water.
2. Heat the mixture to boiling with stirring to dissolve the medium completely.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Avoid overheating as it may affect the performance of the medium.
4. Cool the medium to 45-50°C and mix well.
5. Pour the medium into sterile Petri plates and let them solidify.
6. Store the plates in a refrigerator at 2-8°C until use.

To use Brilliant Green Agar for the isolation of Salmonella species, you need to follow these steps:

1. For examination of feces or similar material, heavily inoculate a Brilliant Green Agar plate with the specimen using a sterile loop or swab. At the same time, inoculate other plating media and tubes of Selenite Broth and Tetrathionate Broth for enrichment.
2. Incubate the Brilliant Green Agar plate for 18-24 hours at 35°C.
3. Examine the plate for suspect colonies that are red-pink-white in color with a red zone around them. Confirm their identity using biochemical and serological tests.
4. If no non-lactose fermenters are observed on the primary plate cultures, inoculate Brilliant Green Agar and other media with the enrichment cultures and repeat steps 2 and 3.
5. For examination of foods, pre-enrich four 25g aliquots of food in 75ml of Buffered Peptone Water and incubate at 35°C for 4-6 hours.
6. Add to each sample 75ml of double-strength Selenite Cystine Broth and incubate at 43°C for 24 hours.
7. Subculture to plates of Brilliant Green Agar and Bismuth Sulphite Agar.
8. Incubate the plates at 35°C and examine the Brilliant Green Agar after 24 hours and the Bismuth Sulphite Agar after 48 hours.
9. Look for colonies with Salmonella characteristics and confirm their identity with biochemical and serological tests.

On Brilliant Green Agar, the colonies of different bacteria can be distinguished by their color and appearance. The following table summarizes the common results:

Brilliant Green Agar is a useful medium for the isolation and identification of Salmonella species other than S. Typhi and S. Paratyphi from clinical specimens, such as feces, urine, blood, and pus. It is also recommended by APHA FDA and is in accordance with United States Pharmacopoeia for microbiological procedures for absence of specified microorganisms-nutritional and dietary supplements. This medium is employed in testing clinical specimens, dairy products, food, and pharmaceutical products. It can be used in conjunction with other media, such as Selenite Broth, Tetrathionate Broth, Bismuth Sulphite Agar, SS Agar, and MacConkey Agar, to increase the chances of recovery and differentiation of Salmonella species.

• Brilliant Green Agar is a highly selective medium that inhibits the growth of most Gram-negative and Gram-positive bacteria, except some Salmonella species. Therefore, it should be used in conjunction with other less selective media to increase the recovery rate of potential pathogens.
• Brilliant Green Agar may not support the growth of some Salmonella strains, such as S. Typhi and S. Paratyphi, which are the causative agents of typhoid and paratyphoid fever, respectively. These strains are more sensitive to the inhibitory effect of brilliant green dye and may be overgrown by other bacteria.
• Brilliant Green Agar may also fail to differentiate some non-Salmonella bacteria that can produce red-pink-white colonies with red zones on the agar. These include Proteus, Citrobacter, and Pseudomonas species, which may mimic Salmonella by producing hydrogen sulfide or acid from lactose or sucrose.
• Brilliant Green Agar is not suitable for the cultivation of anaerobic bacteria, which require special conditions to grow. Therefore, it should not be used for the isolation of anaerobic pathogens, such as Clostridium species.
• Brilliant Green Agar may lose its selectivity and differential properties if overheated during sterilization or storage. Therefore, it should be prepared carefully and stored at a cool temperature away from light. It should also be checked for signs of deterioration, such as discoloration or contamination, before use.

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