TETRATHIONATE BRILLIANT GREEN BILE BROTH

Cat. no. K273 Tetrathionate Brilliant Green Bile Broth, 16x125mm Tube with Cap, 10ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Tetrathionate Brilliant Green Bile Broth is recommended for the selective enrichment of Salmonella species from pharmaceutical products, food stuffs, meat samples, and water.

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

SUMMARY

The Deutsches Arzneibuch (DAB), the European Pharmacopoeia and the Indian Pharmacopoeia recommend Tetrathionate Brilliant Green Bile Broth for the selective enrichment of Salmonella spp. from foods, water and other samples. (1-3) This medium is useful for inhibiting the growth of competing microflora while promoting the growth of target species. In addition, oxbile, which selects for enteric gram-negative pathogens, is incorporated into the medium. Thus, the use of an additional selective supplement such as iodine-iodide is not required with this product. Tetrathionate Brilliant Green Bile Broth may not inhibit the growth of some Proteus species. Subculturing to an appropriate selective solid media is necessary to observe typical colony characteristics.

Hardy Diagnostics Tetrathionate Brilliant Green Bile Broth contains brilliant green and oxbile that inhibit the growth of gram-positive, as well as some gram-negative, species. Potassium tetrathionate inhibits the normal flora of fecal specimens. Calcium carbonate buffers sulfuric acid that is produced upon reduction of tetrathionate. (4) Finally, sodium chloride maintains the osmotic balance and meat peptone serves as the nitrogen, carbon and general amino acid source.

FORMULA

Ingredients per liter of deionized water:*

Calcium carbonate 20.0gm
Potassium Tetrathionate 20.0gm
Meat Peptone 8.6gm
Oxbile 8.0gm
Sodium Chloride 6.4gm
Brilliant Green 0.07gm

Final pH 7.0 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration, 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. (5-8,9) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Specimens should be delivered to the laboratory within 2-3 hours. Special attention is required for stool samples. They should be collected early in the course of the disease and need to be cultured within two hours after collection. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Method of use:

1. The sample may be pre-enriched in Lactose Broth (Cat. no. K223).

2. Place 1gm of feces or 1ml of liquid stool into a Tetrathionate Brilliant Green Bile tube. Swab specimens may be inserted directly into the broth.

3. Emulsify the specimen thoroughly.

4. Incubate aerobically for 18 to 24 hours at 35ºC.

5. Place one to two drops of the incubated broth onto selective plated media for Salmonella spp. and streak for isolated colonies.

6. Incubate aerobically at 35ºC.

7. Examine plates for pathogens in 18-48 hours.

INTERPRETATION OF RESULTS

Culture analysis and colony characteristics are made from the media onto which the selectively enriched specimen is subcultured (see steps 5-7 above). Consult listed references for the interpretation of colonies and other biochemical reactions to identify growth of the organism. (5-9,13)

LIMITATIONS

The recovery of many Salmonella spp. is greatly jeopardized if stool specimens remain unpreserved for more than three hours before processing. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation.

Due to the selective nature of this product, Proteus spp. may not be inhibited. Observe colonies upon subculture to plated selective media for typical Salmonella cultural characteristics and biochemical reactions.

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
I 18-24hr 35°C Aerobic Growth upon subculture to MAC
Escherichia coli
ATCC ® 25922
I 18-24hr 35°C Aerobic Partial to complete inhibition upon subculture to MAC
Enterococcus faecalis
ATCC ® 29212
I 18-24hr 35°C Aerobic Inhibited upon subculture to MAC

USER QUALITY CONTROL

REFERENCES

1. European Pharmacopeia II, Kapitel VIII, 10.

2. Deutsches Arzneibuch (DAB), 10. Auflage, Kapitel VIII, 10.

3. Indian Pharmacopeia, Vol. II, 1996. Published by the Controller of Publications, New Dehli, Government of India, Ministry of Health and Family Welfare.

4. Nealson, K.H. 1978. Component of tetrathionate-containing and tetrathionate-producing culture media (TGB Broth) for buffering sulfuric acid produced on reduction of tetrathionate. Media component of luminous bacteria. Methods Enzymol. ; 57:154.

5. 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.

6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

7. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

8. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

9. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

10. American Public Health Association. 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. APHA, Washington, D.C.

11. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

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

13. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.


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