As a primary plating medium, Brilliant Green Agar medium should be used to isolate Salmonella species. Kristensen and colleagues first described it as a selective isolation medium to Salmonella species. Kristensen et al. first described it as a selective isolation medium for Salmonella species. Kauffmann modified the formula to make it a highly selective plating media for the isolation and identification from salmonellae in feces, other pathological material, food and dairy products. Brilliant Green Agar should always be used in conjunction with other selective plating media like Deoxycholate Citrate Agar and Hektoen Enteric Agar. Salmonella Typhi is treated with Bismuth Sulphite.
Principle of Brilliant Green Agar
The medium is a combination of tryptone, peptone and yeast extract. It provides amino acids as well as long chains of protein and amino acids. The osmotic equilibrium is maintained by sodium chloride. The fermentable carbohydrate sources are lactose or sucrose. Phenol red is an acid-base indicator that gives a yellow color to sucrose and lactose fermenting bacteria. Within 18-24 hours of incubation, non-lactose fermenting bacteria produce white colonies to pinkish red colonies. The medium also contains bright green, which discourages the growth of most Gram-negative and Grampositive bacteria. Most of the inhibited species are Salmonella Typhi, Shigella species and Escherichiacoli.
Red-pink-white, opaque colonies with reddish-purple agar are surrounded by vibrant red zones in the agar. This is most likely salmonella (but it’s not Salmonella typhi).
Proteus and Pseudomonas species
They may form small red colonies.
Lactose/sucrose-fermenting organisms (normally inhibited)
Yellow to greenish-yellow colored colonies surrounded by intense yellow-green zones in the agar – Escherichia coli or Klebsiella/Enterobacter group.
Composition of Brilliant Green Agar
Final pH (at 25°C): 6.9±0.2
Preparation and Method of Use of Brilliant Green Agar
- Take 58.09 grams and 1000 ml purified/distilled water.
- To dissolve the medium completely, heat to boiling
- For 15 minutes, sterilize by using an autoclave at 15 lbs pressure (121degC). Avoid overheating.
- Keep it at 45-50 degrees Celsius
- Mix everything together and pour onto sterilized Petri dishes.
Examination of feces, or similar material, for salmonellae:
- Inoculate the Brilliant Green Agar plate. Inoculate plates of Tetrathionate Broth or Selenite Broth.
- For 18-24 hours, incubate the Brilliant Green Agar Plate at 35°C.
- Check the plates for suspicious colonies and use differential tests to identify them.
- If there are no non-lactose fermentationers on the primary plates cultures, inoculate Brilliant green agar and other media with enrichment cultures. Then proceed as in point 3.
Examination of foods
- Four 25g aliquots are to be pre-enriched in 75ml Buffered Peptone Water. Then, incubate for between 4-6 hours at 35°C.
- In each of the samples, add 75ml double-strength Selenite CystineBroth. Let it incubate for 24 hours at 43°C.
- Subculture to plates of Brilliant green Agar or Bismuth Sulphite agar.
- Place the plates in the oven at 35°C. After 24 hours, examine the Brilliant Green Agar and Bismuth Sulphite agar.
- Look out for salmonella-like colonies and confirm their identity using biochemical and serological testing.
Result Interpretation on Brilliant Green Agar
|Salmonella typhimurium||Good growth; red-colored colonies|
|Salmonella Abony||Good growth; pinkish white|
|Salmonella Enteritidis||Good growth; pinkish white|
|Salmonella Typhi||Poor-good; reddish pink|
|Escherichia coli||Inhibited or no growth; yellowish-green|
|Enterococcus faecalis||No growth to light growth; yellowish colonies with or without yellow halos|
Uses of Brilliant Green Agar
- Brilliant Green Agar can be used to isolate Salmonella species from clinical specimens.
- It is also recommended and approved by the APHA FDA. This medium is used to test clinical specimens.
- Due to its exceptional selectivity, heavy inocula can be used and samples that are heavily contaminated can be analysed.
- It is used to test for microbial limits and in conjunction with novobiocin to test food and pharmaceutical products.
- This medium is highly selective and should be used in conjunction with another less inhibitory medium to improve the chance of recovery. Cultures enriched with Tetrathionate or Selenite Broth are often plated on Brilliant green Agar. This is in addition to Bismuth Sulphite Agar and SS Agar.
- This medium may not be suitable for Shigella and Salmonella typhi species. You should consider alternative media.
- Proteus and Citrobacter species might mimic enteric pathogens through the production of small red colonies.
- To complete the identification of isolated colonies, biochemical and/or seological tests should be conducted.