Brilliant Green Agar Base, Modified
Intended Use:
Recommended for selective isolation of Salmonellae other than Salmonella Typhi from faeces, food, dairy products.
Composition**
Ingredients Gms / Litre
Proteose peptone 10.000
Yeast extract 3.000
Lactose 10.000
Sucrose 10.000
Sodium chloride 5.000
Phenol red 0.080
Brilliant green 0.0125
Agar 20.000
Final pH ( at 25°C) 6.9±0.2
Directions
Suspend 29.0 grams in 500 ml purified / distilled water. Heat to boiling to dissolve the medium completely. Sterilize by
autoclaving at 15 lbs pressure (121°C) for 15 minutes. AVOID OVERHEATING. Cool to 45-50°C. For more selectivity,
aseptically add rehydrated contents of 1 vial of S Selective Supplement (FD068). Mix well before pouring into sterile Petri
plates.
Principle And Interpretation
Salmonella species cause many types of infections, from mild self-limiting gastroenteritis to life threatening typhoid fever.
The most common form of Salmonella disease is self-limiting gastroenteritis with fever lasting less than 2 days and diarrhoea
lasting less than 7 days. Brilliant Green Agar Base, Modified, as a primary plating medium for isolation of Salmonella
species was first described by Kristensen et. al. (1) and further modified by Kauffmann (2). Brilliant Green Agar is also
recommended by APHA (3,4) FDA (5) and described in EP, BP and IP (6,7,8).
This medium contains brilliant green, which inhibits growth of majority of Gram-negative and Gram-positive bacteria.
Salmonella Typhi, Shigella species Escherichia coli, Pseudomonas species, Staphylococcus aureus are mostly inhibited.
Clinical specimens can be directly plated on this medium. However, being highly selective, it is recommended that this
medium should be used along with a less inhibitory medium to increase the chances of recovery. Often cultures enriched in
Selenite or Tetrathionate Broth is plated on Brilliant Green Agar along with Bismuth Sulphite Agar, SS Agar, MacConkey
Agar.
The medium contains proteose peptone and yeast extract as sources of carbon, nitrogen, vitamins, amino acids and essential
nutrients. The two sugars namely lactose and sucrose serve as energy sources. Fermentation of lactose and/or sucrose in the
medium results in the formation of acidic pH which is detected by phenol red indicator. Sodium chloride maintains the
osmotic equilibrium. Brilliant green helps to inhibit the contaminating microflora. The medium can further supplemented
with sulphaacetamide (1g/l) and sodium mandelate (0.25g/l) to inhibit contaminating microorganisms when the sample
is suspected to contain large number of competing organisms along with Salmonella species. Non-lactose fermenting
bacteria develop white to pinkish red colonies within 18 - 24 hours of incubation.
Type of specimen
Clinical : Faeces; Foodstuffs & dairy samples; Water samples; Pharmaceutical samples.
Specimen Collection and Handling
For clinical samples follow appropriate techniques for handling specimens as per established guidelines (12,13).
For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (3,4).
For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local standards (5) .
Warning and Precautions
In Vitro diagnostic use. For professional use only. 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 clinical specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
1. Though this medium is selective for Salmonella other species of Enterobacteriaceae may grow.
2. Salmonella Typhi and Shigella species may not grow on this medium.
3. Moreover Proteus, Pseudomonas and Citrobacter species may mimic enteric pathogens by producing small red colonies.
4. Further confirmation has to be carried out on presumptive Salmonella isolates.
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
Light yellow to light pink homogeneous free flowing powder
Gelling
Firm, comparable with 2.0% agar gel.
Colour and Clarity of prepared medium
Greenish brown clear to slightly opalescent gel forms in Petriplates
Reaction
Reaction of 5.8% w/v aqueous solution at 25°C. pH : 6.9±0.2
pH
6.70-7.10
Cultural Response
Cultural response was carried out after an incubation at 30-35°C for 24-48 hours. Recovery rate is considered as 100% for
bacteria growth on Soyabean Casein Digest Agar.
Organism Inoculum Growth
(CFU)
Recovery Colour of
Colony
Escherichia coli ATCC
25922 (00013*)
50 -100 none-poor 0 -10 %
Escherichia coli ATCC 8739 50 -100 none-poor 0 -10 %
none-poor 0 -10 %
=10 inhibited 4 0%
Staphylococcus
aureus subsp. aureus
ATCC 6538 (00032*)
=10 inhibited 4 0%
Salmonella Typhi
ATCC 6539
50 -100
Salmonella Typhimurium
ATCC 14028 (00031*)
50-100
reddish pink
pinkish white
yellowish green
yellowish green
Salmonella Enteritidis
ATCC 13076 (00030*)
50 -100
fair-good 30 -40 %
good-luxuriant =50 %
luxuriant =50 %
Salmonella Abony
NCTC 6017 (00029*)
50-100 good-luxuriant =50 %
pinkish white
pinkish white
yellowish green
(00012*)
Escherichia coli NCTC 9002 50 -100
Staphylococcus aureus
subsp. aureus ATCC
25923 (00034*)
Key : *Corresponding WDCM numbers.
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°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 clinical
sample must be decontaminated and disposed of in accordance with current laboratory techniques (12,13).
