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BRILLIANT GREEN AGAR

Code: CM0263

A selective medium for the isolation of salmonellae, other than Salmonella typhi.

* Adjusted as required to meet performance standards

Directions
Suspend 50g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.

Description
Brilliant Green Agar was first described as a selective isolation medium for Salmonella species by Kristensen et al.¹ Kauffmann² modified their formula to give a highly selective plating medium for the isolation and identification of salmonellae from faeces and other pathological material, and from food and dairy products. This medium was not designed for the isolation of Salmonella typhi or Shigella species and where these may be encountered, Brilliant Green Agar should be used in parallel with other selective plating media such as Desoxycholate Citrate Agar (Hynes) CM0227, Hektoen Enteric Agar CM0419, X.L.D. Agar CM0469. Bismuth Sulphite Agar (Modified) CM0201 is specifically recommended for Salmonella typhi.

The use of enrichment/selective broths prior to subculture on Brilliant Green Agar will improve the probability of isolating salmonellae. Tetrathionate Broth Base CM0029, Tetrathionate Broth USA CM0671, Selenite Broth Base CM0395 and Muller-Kauffmann Tetrathionate Broth Base CM0343 may be used in conjunction with Brilliant Green Agar.

Brilliant Green Agar corresponds to the medium recommended by the APHA^3,4 .

The addition of sulphonamides to Brilliant Green Agar helps improve the isolation of salmonellae⁵. To one litre of Brilliant Green Agar add 1.0g of sulphapyridine or 0.8g sulphadiazine and sterilise in the normal way.

Technique

Examination of faeces, or similar material, for salmonellae:

1. Heavily inoculate a Brilliant Green Agar plate. At the same time, inoculate other plating media and tubes of Selenite Broth and Tetrathionate Broth.
2. Incubate the Brilliant Green Agar plate for 18-24 hours at 35°C.
3. Examine the plates and identify suspect colonies using differential tests for serological methods.
4. If no non-lactose fermenters are observed on the primary plate cultures, inoculate Brilliant Green Agar and other media with the enrichment cultures - then proceed as in point 3.

Examination of foods

1. Pre-enrich four 25g aliquots of food in 75ml of Buffered Peptone Water CM0509 and incubate at 35°C for 4-6 hours.
2. Add to each sample 75ml of double-strength Selenite Cystine Broth CM0699 and incubate at 43°C for 24 hours.
3. Subculture to plates of Brilliant Green Agar and Bismuth Sulphite Agar (Modified) CM0201.
4. Incubate the plates at 35°C and examine the Brilliant Green Agar after 24 hours and the Bismuth Sulphite Agar after 48 hours.
5. Look for colonies with salmonella characteristics and confirm their identity with biochemical and serological tests.

Colonial characteristics

Non-lactose/sucrose-fermenting organisms
Red-pink-white opaque coloured colonies surrounded by brilliant red zones in the agar - most probably salmonella (but not Salmonella typhi).

Proteus and Pseudomonas species
These may grow as small red colonies.

Lactose/sucrose-fermenting organisms (normally inhibited)
Yellow to greenish-yellow coloured colonies surrounded by intense yellow-green zones in the agar - Escherichia coli or Klebsiella/Enterobacter group.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates of medium at 2-8°C.

Appearance
Dehydrated medium: Straw-green coloured, free-flowing powder
Prepared medium: Green-brown coloured gel

Quality control

* This organism is available as a Culti-Loop®

Precautions
Lactose-fermenting salmonella (Salmonella arizona) may be present in foods⁷.
Salmonella typhi and Shigella species may not grow on this medium. Use the cited alternative media.
Proteus, Citrobacter and Pseudomonas species may mimic enteric pathogens by producing small red colonies.

References
1. Kristensen M., Lester V. and Jurgens A. (1925) Brit. J. Exp. Pathol. 6. 291-297.
2. Kauffman F. (1935) Seit. F. Hyg. 177. 26-34.
3. American Public Health Association (1976) Compendium of Methods for the Microbiological Examination of Foods. APHA Inc. Washington D.C.
4. Downes and Ito (Ed.) Standard Methods for the Examination of Water and Wastewater,20th Ed APHA Washington D.C.
5. Osborn W. W. and Stokes J. L. (1955) Appl. Microbiol. 3. 295-301.
6. The United States Pharmacopaeia USP 28 2005.
7. Harvey R. W. S., Price T. H. and Hall L. M. (1973) J. Hyg. Camb. 71. 481-486.