Brilliant Green Agar Modified ISO

Brilliant Green Agar Modified inhibits the growth of Pseudomonas aeruginosa and partially inhibits the growth of Proteus spp., which may be similar in appearance to Salmonella.

Salmonella can be present in small numbers and is often accompanied by considerably larger numbers of other Enterobacteriaceae or bacteria of other families. A pre-enrichment stage is used to permit the detection of low numbers of Salmonella or injured Salmonella.

Beef extract, casein peptone and meat peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group, essential for bacterial growth. Lactose and sucrose are the fermentable carbohydrates providing carbon and energy. Phenol red is the pH indicator. Brilliant green inhibits Gram-positive and most Gram-negative bacteria, except Salmonella. If the medium overheats, brilliant green may lose its properties. Bacteriological agar is the solidifying agent.

ISO 6579 recommends Brilliant Green Agar as a second selective medium.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 54.7 grams of the dehydrated medium in one liter of distilled water and leave for 15 minutes. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. AVOID OVERHEATING. DO NOT AUTOCLAVE. Dispense into appropriate containers.

Instructions for use

For detection of Salmonella spp. in food, animal feed, animal faeces, and environmental samples:

1. Pre-enrichment in non-selective liquid medium: inoculate the Buffered Peptone Water (LC-1402) with the sample or dilutions, and incubate at 34-38 °C for 18 h.
2. Enrichment in/on selective media: inoculate, with the culture obtained in the pre-enrichment stage, the Rappaport Soy Broth (Vassiliadis) (LC-1174) or the Modified Semisolid Rappaport Vassiliadis medium (MSRV) (LC-1376), and the Tetrathionate Broth (Muller-Kauffmann) (LC-1173). The Rappaport Soy Broth and the Modified Semisolid Rappaport medium are incubated at 41.5 °C for 24 h, and the Tetrathionate Broth at 37 °C for 24 h.
3. Plating out on selective solid media: from the selective enriched cultures, inoculate two selective isolation agars: XLD agar (LC-1274) and any other selective medium complementary to XLD agar (Salmonella Chromogenic Agar (LC-1122), Brilliant Green Agar, Bismuth Sulfite Agar (LC-1011), DCLS Agar (LC-1045), Desoxycholate Citrate Agar (LC-1067), Hektoen Enteric Agar (LC-1030), Salmonella Shigella Agar (LC-1064) and XLT4 Agar (LC-1159)). Incubate the XLD plates inverted at 37 °C for 24 ± 3 h. Incubate the second selective medium in accordance with the manufacturer's instructions.
4. Confirmation: subculture colonies of presumptive Salmonella and confirm their identity by biochemical and serological tests.

For detection of Salmonella spp. in water samples:

1. Pre-enrichment in non-selective medium: inoculate the Buffered Peptone Water (LC-1402) with the sample or dilutions, and incubate at 36 ± 2 °C for 18 ± 2 h.
2. Enrichment in selective media: inoculate, with the culture obtained in the pre-enrichment stage, the Rappaport Soy Broth (Vassiliadis) (LC-1174) and the Tetrathionate Broth (Muller-Kauffmann) (LC-1173). The Rappaport Soy Broth is incubated at 41.5 ± 1 °C and the Tetrathionate Broth at 37 ± 1 °C, both of them for 24 ± 3 hours.
3. Plating out on selective solid media: from the selective enriched cultures, inoculate two selective isolation agars: XLD agar (LC-1274) and any other selective medium complementary to XLD agar (for instance, Brilliant Green Agar or Bismuth Sulfite Agar (LC-1011)). Incubate the XLD plates inverted at 36 ± 2 °C for 24 ± 3 hours. Incubate the second selective medium in accordance with the manufacturer's instructions.
4. Confirmation: subculture colonies of presumptive Salmonella and confirm their identity by biochemical and serological tests.

• UNE-EN-ISO 6579 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp.
• ISO 19250 Water quality - Detection of Salmonella spp.