Brilliant Green Agar with Novobiocin

Cat. no. G175 Brilliant Green Agar with Novobiocin, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. J127BX XLT-4 Agar/Brilliant Green Agar with Novobiocin, 15x100mm Biplate, 10ml/10ml 100 plates/box

INTENDED USE

Hardy Diagnostics Brilliant Green Agar with Novobiocin is recommended for the selective isolation of Salmonella spp., other than S. typhi , from non-clinical samples.

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

SUMMARY

Brilliant Green Agar, Modified was formulated by Edel and Kamplemacher of the Netherlands Institute for Public Health, Utrecht and originally proposed as a selective medium for the isolation of Salmonella from pig feces and minced meat. (8,9) The medium has been widely utilized in Europe and used in the Standard European Community and by the International Standards Organization. (7,10) More selective than Deoxycholate Citrate Agar and other brilliant green media, Brilliant Green Agar, Modified inhibits the growth of Pseudomonas aeruginosa and Proteus spp., which may resemble the growth of Salmonella spp., thus facilitating the isolation of Salmonella colonies.

Brilliant Green Agar, Modified is recommended for the selective isolation of Salmonella spp., other than S. typhi , from clinical and non-clinical specimens. The medium is appropriate for subculture from selective enrichment media. The addition of novobiocin to the medium reduces the incidence of nuisance flora commonly cultured in conjunction with Salmonella .

FORMULA

Ingredients per liter of deionized water:*

Lactose 10.0gm
Sucrose 10.0gm
Casein Peptone 5.0gm
Animal Tissue Peptone 5.0gm
Gelatin Peptone 5.0gm
Sodium Chloride 5.0gm
Yeast Extract 3.0gm
Disodium Phosphate 1.0gm
Phenol Red 0.09gm
Monopotassium Phosphate 0.52gm
Brilliant Green 4.7gm
Novobiocin 2.0ml
Agar 12.0gm

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

For Meat and Meat Products :

1. Weigh out 25.0gm of the sample into a sterile blender.

2. Add 225ml of Buffered Peptone Water (Cat. no. U142) to the blender and macerate the sample and buffer to homogenize the mixture to yield 15,000 to 20,000 revolutions.

3. Asceptically transfer the contents of the blender jar to a sterile 500ml flask and incubate at 35 to 37ºC. for 16 to 20 hours.

4. Transfer 10ml samples to 100ml of Tetrathionate Broth Base (Cat. no. U165) supplemented with Iodine-Iodide Solution (Cat. no. Z129 or Z139).

5. Incubate at 42 to 43ºC. for 48 hours.

For Sewage and Polluted Water : (for Salmonella spp. other than S. typhi )

1. Inoculate 25ml aliquotes of the sample into 25ml of double strength Buffered Peptone Water and incubate samples at 35 to 37 ºC. for 18 hours.

2. Transfer 1ml samples into 10ml of Tetrathionate Broth Base (Cat. no. K65) supplemented with Iodine-Iodide Solution (Cat. no. Z129 or Z139)

3. Incubate at 43ºC. for 48 hours.

Subculture :

1. Subculture from the Tetrathionate Broth at 18 to 24 hours and at 48 hours onto Brillian Green Agar, Modified.

2. Incubate plates at 35 to 37ºC. for 18 to 24 hours.

3. Examine plates for typical Salmonella colony morphology.

INTERPRETATION OF RESULTS

Typical Salmonella colonies will produce 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.

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.

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.

LIMITATIONS

Organisms other than Salmonella spp., such as Morganella morgani and some Enterobacteriaceae , may grow on this medium.

Fermentation reactions, seroagglutination and other confirmatory tests should be carried out on all colonies that are presumptive for Salmonella .

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, other culture media such as Buffered Peptone Water (Cat. no. U142), Tetrathionate Broth Base (Cat. no. K65 or U165), as well as supplements, such as Iodine-Iodide Solution (Cat. no. Z129 or Z139), etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC ® 14028


A 18-24hr 35°C Aerobic Growth; red to pink-white colonies with red zones
Escherichia coli
ATCC ® 25922
B 18-24hr 35°C Aerobic Partial inhibition; small yellow to yellow-green colonies
Staphylococcus aureus
ATCC ® 25923
B 18-24hr 35°C Aerobic Inhibited

USER QUALITY CONTROL

Physical Appearance

Brilliant Green Agar with Novobiocin should appear slightly opalescent, and brownish-orange in color.

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Anon. 1975. Meat and meat products - detection of salmonellae (reference method). International Organization for Standardization. Geneva, Switzerland.

4. Anon. 1981. Microbiology - General Guidance on Methods for the Detection of Salmonella . Ref. method ISO 6579-1981(E). International Organization for Standardization. Geneva, Switzerland.

5. Anon. 2001. Milk and Milk Productss - Detection of Salmonella spp. ISO 6785. International Organization for Standardization. Geneva, Switzerland.

6. Atlas, R.M. 2010. Handbook of Microbiological Media , 4th ed. CRC Press, Inc. Boca Raton, FL.

7. Corry, J.E.L, G.D.W. Curtis, and R.M. Baird. 2003. Handbook of Culture Media for Microbiology . Elsevier Science B.V. Amsterdam, The Netherlands.

8. Edel, W. and E.H. Kamplemacher. 1968. Comparative studies on Salmonella isolation in eight European laboratories. Bull. Wld. Hlth. Org .; 39:487-491.

9. Edel, W. and E.H. Kamplemacher. 1969. Salmonella infections in nine European lanoratories using a standard technique. Bull. Wld. Hlth. Org .; 41:297-306.

10. Parry, P.T., L. Haysom, N.L. Thomas, and R. Davis.1982. A Manual of Recommended Methods for the Microbiological Examination of Poultry and Poultry Products . British Poultry Meat Association. London, U.K.

11. Read, JR., R.B. and A.L. Reyes. 1968. Variation in plating efficiency of salmonellae on eight lots of Brilliant Green Agar. Appl. Microbiol ; 16(5):746-748.

12. Reed, G.H. 1993. Foodborne illness (Part 2): Salmonellosis. Dairy, Food, Environ. San . 13:706.

13. Vassilliadis, P, J. Trichopoulos, V.K. Papadakis and Ch. Serie. 1979. Brilliant Green Deoxycholate Agar as an improved selective medium for the isolation of Salmonella . Ann. Soc. belge. Med. trop .; 59:117-120.


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