Brilliant Green Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

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3 years ago

Brilliant Green Agar: Introduction, Principle, Composition, Preparation, Procedure, Colony Morphology, Uses and Keynotes

Introduction of Brilliant Green Agar

Brilliant Green Agar is a selective medium for the isolation of Salmonellae other than Salmonella Typhi from feces, foods, and dairy products.

Principle of Brilliant Green Agar

The incorporation of peptone, tryptone, and yeast extract makes the medium highly nutritious. It supplies amino acids and long chains of peptides. Sodium chloride is responsible for the osmotic equilibrium. Lactose and sucrose both are fermentable carbohydrate sources. Phenol red is an acid-base indicator. It provides yellow color to lactose and or sucrose fermenting bacteria. The brilliant green of the medium inhibits the growth of the majority of Gram-negative and Gram-positive bacteria and it favors salmonellae. Microbes like Salmonella Typhi, Shigella species, Escherichia coli, Pseudomonas species, Proteus species, and Staphylococcus aureus are mostly inhabited. Non-lactose fermenting bacteria show white to pinkish-red colonies within 18-24 hours of incubation. Salmonella Typhi and Shigella species may not grow on this medium. However, Proteus, Pseudomonas, and Citrobacter species may mimic enteric pathogens by developing small red colonies.

Composition of Brilliant Green Agar

Ingredients Gms/Litre
Peptone: 5.0
Tryptone: 5.0
Yeast extract: 3.0
Lactose: 10.0
Sucrose: 10.0
Sodium chloride: 5.0
Phenol red: 0.08
Brilliant green: 0.0125
Agar: 20.0
pH after sterilization (at 25°C) 6.9±0.2

Preparation of Brilliant Green Agar

Suspend 5.809 grams in 100 ml purified/distilled or deionized water.
Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
After autoclaving, leave for cooling to 45-50°C.
Pour agar into each plate and leave plates on the sterile surface until the agar has solidified.
Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Brilliant Green Agar

Store at 2-8ºC and away from direct light.
Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
The product is light and temperature sensitive. Protect it from light, excessive heat, moisture, and freezing.

Test Requirements

Test specimens ( samples or growth of bacteria)
Inoculating loop
Bunsen burner
Incubator
Control strains (Salmonella Typhimurium
ATCC 14028 and Escherichia coli ATCC 25922 )

Procedure of Brilliant Green Agar

Allow the plates to warm at 37°C or to room temperature.
Leave the agar surface to dry before inoculating.
Inoculate and streak the specimen as soon as possible after collection.
If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
Streak for isolation with a sterile loop.
Incubate plates aerobically at 35-37ºC for 24-48 hours.
Examine colony characteristics.

Result- Interpretation of Brilliant Green Agar

Salmonellae (except Salmonella Typhi): Good-luxuriant growth

Salmonella Typhimurium ATCC 14028: Good-luxuriant growth

Escherichia coli ATCC 25922: None to poor growth

Staphylococcus aureus ATCC 25923: No growth

Colony Morphology of Brilliant Green Agar

Salmonella Typhimurium ATCC 14028: Pinkish white
S. Abony NCTC 6017:Pinkish white
S. Enteritidis ATCC 13076: Pinkish white
Escherichia coli ATCC 25922 (If grown): Yellowish green ( as shown in figure B)

Uses of Brilliant Green Agar

It is used for selective isolation of salmonellae( except Salmonella Typhi).
It is also used in the microbial limits test and with novobiocin for testing food and pharmaceutical products.

Keynotes on Brilliant Green Agar

Even being Brilliant Green Agar highly selective, it is recommended that this culture medium should be used along with a less inhibitory medium to increase the chances of recovery. Often cultures enriched in Selenite or Tetrathionate Broth are plated on Brilliant Green Agar along with Bismuth Sulphite Agar, Salmonella-Shigella (SS) Agar, MacConkey medium.
Kristensen et al first described Brilliant Green Agar as a primary plating medium for the isolation of Salmonella species. It is also for the differentiation of paratyphoid B from other Gram-negative enteric bacteria.
Later Kauffmann further modified it for the isolation of Salmonella from feces specimens.
The color and clarity of prepared Brilliant Green Agar are Greenish brown clear to slightly opalescent gel forms in Petri plates.