BRILLIANT GREEN (BG) SULFA AGAR

Cat. no. G87 BG Sulfa Agar, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics BG Sulfa Agar is a selective medium recommended for the isolation of Salmonella spp., other than S. typhi and S. paratyphi , from food following an enrichment procedure.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

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) Osborne and Stokes showed that a small amount of sulfapyridine added to the medium restored its selective properties against Escherichia coli and Proteus spp., thereby improving Salmonella isolation.

Hardy Diagnostics BG Sulfa Agar incorporates phenol red as the pH indicator and brilliant green as an inhibitory agent against gram-positive microorganisms and gram-negative bacilli. Sulfapyridine 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 appears 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.

FORMULA

Ingredients per liter of deionized water:*

Lactose 10.0gm
Sucrose 10.0gm
Sodium Chloride 5.0gm
Pancreatic Digest of Casein 5.0gm
Peptic Digest of Animal Tissue 5.0gm
Yeast Extract 3.0gm
Phenol Red 0.08gm
Sulfapyridine 1.0gm
Brilliant Green 12.5mg
Agar 20.0gm

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.

PRECAUTIONS

PROCEDURE

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.

General Method:

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) using the four quadrant technique 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 Sulfa Agar 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 BG Sulfa Agar 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 show as yellow to yellow-green colonies with a yellow-green or green zone.

Escherichia coli may be partially inhibited and present as yellow to yellow-green colonies with a green halo.

Shigella spp. may exhibit partial to complete inhibition with colorless colonies.

Other non-lactose fermenting microorganisms may mimic enteric pathogens and present as red to pink-white colonies surrounded by red zones. Further biochemical testing is needed to fully identify these strains. (14)

LIMITATIONS

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, 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.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC ® 14028**
A 18-48hr 35°C Aerobic Growth; red to pink-white colonies with red zones
Escherichia coli
ATCC ® 25922
B 18-48hr 35°C Aerobic Partial inhibition; yellow to yellow-green colonies
Enterococcus faecalis
ATCC ® 29212**
B 18-48hr 35°C Aerobic Inhibited; no color change
Proteus mirabiles
ATCC ® 12453**
B 18-48hr 35°C Aerobic Inhibited; no color change

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

BG Sulfa Agar should appear slightly opalescent, and brownish-orange in color.

REFERENCES

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, 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; American Society for Microbiology, Washington, D.C.

14. Taylor, W.I. 1965. Am. J. Clin. Pathol. ; 44:471.


ATCC is a registered trademark of the American Type Culture Collection.

040116sw