PI 9212, Rev New, October 2008

BRILLIANT GREEN AGAR (ISO) (9212)

Intended Use

Brilliant Green Agar (ISO) is used for the selection and differentiation of Salmonellae (other than S. typhi) from foods or feedstuffs.

Product Summary and Explanation Salmonellosis continues to be an important public health problem worldwide. Infection can result from consumption of raw, undercooked, or improperly processed foods contaminated with Salmonella. Kristensen, Lester, and Jurgens first described the use of Brilliant Green Agar as a primary plating medium for the isolation of Salmonella.

1 The report described the medium as useful for the differentiation of “paratyphoid

B” from other intestinal Gram-negative bacilli.1 Kauffmann modified their formula and used Brilliant Green Agar

in addition to Tetrathionate Broth for the isolation of Salmonella from stool specimens.2 Brilliant Green Agar

(ISO) was developed from a formula developed by Rijks Instituut voor de Volksgezondheid (National Institute for Public Health), Utrecht.

3,4 This medium has shown increased inhibition of Escherichia coli and Proteus spp.,

Pseudomonas spp is inhibited, with increased recovery of Salmonella species.5

Brilliant Green Agar (ISO) conforms to ISO Standards.

6,7,8

Principles of the Procedure Enzymatic Digest of Casein and Enzymatic Digest of Animal Tissue provide sources of nitrogen, amino acids, and carbon. Yeast Extract supplies vitamins required for organism growth. Sodium Phosphate is the buffering agent. Lactose and Sucrose are the carbohydrates in the medium. Brilliant Green inhibits Gram-positive bacteria and most Gram-negative bacilli other than Salmonella spp. Phenol Red is the pH indicator and turns the medium yellow with the formation of acid when lactose and/or sucrose is fermented. Agar is the solidifying agent.

Formula / Liter Enzymatic Digest of Animal Tissue........................................ 5.0 g Peptone ................................................................................ 10.0 g Yeast Extract .......................................................................... 3.0 g Lactose ................................................................................ 10.0 g Sucrose ................................................................................ 10.0 g Sodium Phosphate, dibasic ................................................... 1.0 g Sodium Phosphate, monobasic ............................................. 0.6 g Phenol Red .......................................................................... 0.09 g Brilliant Green .................................................................. 0.0047 g Agar ..................................................................................... 12.0 g Final pH: 6.9 ± 0.2 at 25°C

Formula may be adjusted and/or supplemented as required to meet performance specifications.

Precautions 1. For Laboratory Use. 2. IRRITANT. Irritating to eyes, respiratory system, and skin.

Directions 1. Suspend 52 g of the medium in one liter of purified water. 2. Heat with frequent agitation and boil for one minute to completely dissolve the medium. 3. DO NOT AUTOCLAVE.

Quality Control Specifications

Dehydrated Appearance: Powder is homogeneous, free flowing, and beige with a green tint.

PI 9212, Rev New, October 2008

Prepared Appearance: Prepared medium is brown-red, trace to slightly hazy, and slightly opalescent.

Expected Cultural Response: Cultural response on Brilliant Green Agar (ISO) at 35 ± 2°C and examined for growth after 18 - 24 hours incubation.

Microorganism Approx.

Inoculum

(CFU)

Expected Results

Growth Reaction

Salmonella typhimurium ATCC® 14028 10 - 100 Fair to good Red colonies

Proteus mirabilis ATCC® 29906 1000 Partial to complete inhibition

Pink colonies, no swarming if present

Escherichia coli ATCC® 25922 1000 Partial to complete inhibition

Yellow-green colonies if present

The organisms listed are the minimum that should be used for quality control testing.

Test Procedure (as outlined by Edel and Kampelmacher)4

1. Add one part of the sample to 20 parts of Muller Kauffmann Tetrathionate Broth Base (9221). 2. After agitation, incubate Muller Kauffmann Tetrathionate Broth Base into a 45°C waterbath for 15 minutes

only. 3. Transfer the inoculated flask to a 43°C incubator. 4. Subculture the Muller Kauffmann Tetrathionate Broth Base to Brilliant Green Agar (ISO) after 18 – 48

hours using the streak method. 5. Incubate Brilliant Green Agar (ISO) at 35°C for 18 – 24 hours.

Results Typical Salmonella spp. colonies are red. Serological confirmation is required.

Storage Store sealed bottle containing the dehydrated medium at 2 - 30°C. Once opened and recapped, place container in a low humidity environment at the same storage temperature. Protect from moisture and light.

Expiration Refer to expiration date stamped on the container. The dehydrated medium should be discarded if not free flowing, or if appearance has changed from the original color. Expiry applies to medium in its intact container.

Limitations of the Procedure 1. Lactose-fermenting Salmonella species may be present in foods. 2. Salmonella typhi and Shigella species may not growth on this medium. 3. Proteus, Citrobacter, and Pseudomonas species may mimic enteric pathogens by producing small red

colonies.

Packaging

Brilliant Green Agar (ISO) Code No. 9212A 500 g

9212B 2 kg

9212C 10 kg

References 1. Kristensen, M., V. Lester, and A. Jurgens. 1925. On the use of trypsinized casein, brom thymol blue, brom cresol purple, phenol

red and brilliant green for bacteriological nutrient media. Br. J. Exp. Pathol. 6:291.

2. Kauffmann, F. 1935. Weitere Erfahrungen mit den kombinierten Anreicherungsverfahren Fur Salmonellabacillen. Z. Hyg.

Infektionskr. 117:26.

3. Edel, W., and E. H. Kampelmacher. 1968. Bull. Wld Hlth. Org. 39:487-491.

4. Edel, W., and E. H. Kampelmacher. 1969. Bull. Wld. Hlth. Org. 41:297-306.

5. Read, R. B., and A. L. Reyes. 1968. Appl. Microbiol. 16:746-748.

PI 9212, Rev New, October 2008

6. Anon. 1975. International Organization for Standardization. Meat and meat products – detection of Salmonella. Ref. Method, ISO 3565-1975(E).

7. Anon. 1981. International Organization for Standardization. Microbiology – General guidance on methods for the detection of

Salmonella. Ref. method, ISO 6579-1981 (E).

8. Anon. 2001. International Organization for Standardization. Milk and milk products – detection of Salmonella species, ISO 6785.

Technical Information Contact Acumedia Manufacturers, Inc. for Technical Service or questions involving dehydrated culture media preparation or performance at (517)372-9200 or fax us at (517)372-2006.