Brilliant Green agar with Sulfadiazine
|Cat. no. J131||XLT-4 Agar / Brilliant Green Agar with Sulfadiazine, 15x100mm Biplate, 10ml/10ml||10 plates/bag|
Hardy Diagnostics Brilliant Green Agar with Sulfadiazine is recommended for the isolation of Salmonella spp., other than S. typhi and S. paratyphi , from food samples, especially eggs, following an enrichment procedure.
This product is not intended to be used for the diagnosis of human disease.
In 1925, Kristensen et al. described the first use of Brilliant Green Agar as a primary plating medium for the isolation of Salmonella spp. (8) Kauffmann later modified the formula for use in conjunction with tetrathionate broth. (6) In 1955, Osborne and Stokes described the sulfa-modification to further enhance the selective properties of the medium; they found that whole egg and egg yolk products neutralize traditional brilliant green medium and considerably reduced its effectiveness in selecting for Salmonella . (11)
Hardy Diagnostics Brilliant Green Agar with Sulfadiazine incorporates phenol red as the pH indicator and brilliant green as an inhibitory agent against gram-positive microorganisms and gram-negative bacilli other than Salmonella . Sulfadiazine is a sulfonamide antibiotic effective against gram-positive and gram-negative microorganisms. Casein peptone, digest of animal tissue and yeast extract provide nitrogen, vitamins, and essential minerals for growth. Organisms that ferment lactose and/or sucrose exhibit yellow to yellow-green colonies surrounded by a yellow-green zone. Salmonella spp. appear as red to pink-white colonies surrounded by a red zone in the medium. The red coloration indicates the lack of lactose or sucrose utilization.
Ingredients per liter of deionized water:*
|Pancreatic Digest of Casein||5.0gm|
|Peptic Digest of Animal Tissue||5.0gm|
Final pH 6.9 +/- 0.2 at 25ºC.
* Adjusted and/or supplemented as required to meet performance criteria.
STORAGE AND SHELF LIFE
Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.
Specimen Collection: Consult listed references for information on specimen collection. (1-3,5,10,12) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium in order to maintain viability of the organisms.
1. Allow the medium to warm to room temperature prior to inoculation.
2. Inoculate the medium from a previously inoculated selective enrichment broth (e.g. Tetrathionate Broth with Brilliant Green, Cat. no. K164) to produce isolated colonies. A heavy inoculum should be used since the medium is quite inhibitory. (3) Alternatively, if a swab is being used, roll the swab over a small area near the edge of the plate and proceed with the streak method as outlined to obtain isolated colonies. In addition, it is recommended that a non-selective medium (e.g. MacConkey Agar, Cat. no. G35) be streaked in parallel to increase the recovery of gram-negative microorganisms and to characterize other organisms present in the sample.
3. Incubate plates aerobically at 35ºC. for up to 48 hours and examine for typical colony morphology.
Procedure for Testing Food Samples: (2,4)
1. Allow the medium to warm to room temperature as above. Weigh out 25gm of the food sample to be tested into a sterile blender jar. Add 225ml of Nutrient Broth (Cat. no. U149) and blend to mix well. Incubate the sample for 18-24 hours at 35ºC.
2. Transfer 1ml of the incubated nutrient broth to a tube of Tetrathionate Broth with Brilliant Green (Cat. no. K164) or Selenite Cysteine Broth (Cat. no. K69). Incubate both broths for 18-24 hours at 35ºC.
3. Transfer a loopful (10ul) of each broth to separate plates of BG Agar with Sulfadiazine and Bismuth Sulfite Agar (Cat. no. C5211) and incubate plates aerobically at 35ºC.
4. Examine plates at 18-24 hours for typical Salmonella colonial morphology. If growth is not observed, reincubate the plates for an additional 24 hours.
INTERPRETATION OF RESULTS
Typical Salmonella colonies on Brilliant Green Agar with Sulfadiazine appear as red to pink-white with red zones. The red coloration of the medium indicates that lactose or sucrose was not utilized.
Lactose or sucrose fermenting microorganisms not completely inhibited by the medium will appear as yellow to yellow-green colonies with a yellow-green or green zone.
Escherichia coli may be partially inhibited and, if present, will produce yellow to yellow-green colonies with a green halo.
Shigella spp. may be partially to completely inhibited, but if present they will appear as colorless colonies.
Other non-lactose fermenting microorganisms may mimic enteric pathogens and produce red to pink-white colonies surrounded by red zones. Further biochemical testing is needed to fully identify these strains. (14)
Salmonella typhi , S. paratyphi and Shigella spp. do not grow adequately on this medium, limiting its effectiveness as a screening tool for stool cultures.
The medium is highly selective. Inoculating a less selective medium, such as MacConkey, Hektoen Enteric, or other suitable media in conjunction with an enrichment broth, is recommended for best results in isolating all enteric pathogens.
Colonies of other non-lactose fermenting or slow lactose fermenting species, such as Proteus or Pseudomonas , may mimic the growth of enteric pathogens. (14)
The normal coloration of this medium is brownish-orange, yet it may become more red to bright red upon incubation. The medium will return to its normal color when allowed to equilibrate to room temperature.
MATERIALS REQUIRED BUT NOT PROVIDED
Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.
|Test Organisms||Inoculation Method*||Incubation||Results|
ATCC ® 14028
|A||18-24hr||35°C||Aerobic||Growth; red to pink-white colonies with red zones|
ATCC ® 25922
|B||18-24hr||35°C||Aerobic||Partial inhibition; yellow to yellow-green colonies|
ATCC ® 12453
|B||18-24hr||35°C||Aerobic||Partial inhibition; small red colonies, no swarming|
USER QUALITY CONTROL
Brilliant Green Agar with Sulfadiazine should appear slightly opalescent, and brownish-orange in color.
1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.
2. D'Aoust, J.Y. and A.M. Sewell. 1986. Slow Rehydration for Detection of Salmonella spp. in Feeds and Feed Ingredients. Appl. and Environ. Micro. ; Vol. 51, No. 6, p. 1220-1223.
3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.
4. Health Protection Branch. 1978. Methods for the Isolation and Identification of Salmonella from Foods. Acceptable (HPB) method MFA-20. Health and Welfare Canada, Ottawa.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
6. Kauffmann, F. 1935. Z. Hyg. Infektionskr. ; 117:26.
7. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.
8. Kristensen, M., et al. 1925. Br. J. Exp. Pathol. ; 6:291.
9. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.
10. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.
11. Osborne, W.W. and J.L. Stokes. 1955. A Modified Selenite Brilliant-Green Medium for the Isolation of Salmonella from Egg Products. Appl. Microbiol. ; 3:295.
12. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.
13. Sack, R.B., et al. 1980. Cumitech 12 , Laboratory diagnosis of bacterial diarrhea .; ASM Press, Washington, D.C.
14. Taylor, W.I. 1965. Am. J. Clin. Pathol. ; 44:471.
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