TETRATHIONATE BROTH WITH BRILLIANT GREEN

Cat. no. K164 Tetrathionate Broth with Brilliant Green, 16x125mm Tube, 10ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Tetrathionate Broth with Brilliant Green, along with the addition of Iodine-Iodide Solution (Cat. no. Z129), is recommended for use as a selective media for the isolation of Salmonella spp. from pharmaceutical products, food, meat, and water.

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

SUMMARY

Muller first described the use of Tetrathionate Broth for the cultivation of Salmonella spp. (1) He found the medium to be inhibitory to lactose-fermenting Enterobacteriaceae while allowing unrestricted growth of Salmonellae. Schaeffer later demonstrated the medium to posses enrichment properties for Salmonellae. (2) In addition, formula modifications were made by Kauffman who reported enhanced recovery of Salmonella spp. while suppressing the growth of undesirable microorganisms. (3,4) However, Proteus and some other species of accompanying flora are not inhibited by this medium alone and are able to reduce tetrathionate. Consequently, using more than one selective agent in the media promotes the isolation of Salmonella from samples containing multiple serotypes. (5)

Tetrathionate Broth with Brilliant Green contains bile salts which are inhibitory to gram-positive and gram-negative microorganisms other than Salmonella spp. Iodine-Iodide Solution (Cat. no. Z129) is added to the prepared medium just prior to inoculation. The use of Iodine-Iodide Solution promotes the production of tetrathionate which inhibits the growth of coliforms and other enteric bacteria. (6,7) Calcium carbonate buffers the production of sulfuric acid generated during the reduction of tetrathionate. Sodium thiosulfate provides a source of sulfur. Finally, the addition of brilliant green to the medium aids in the suppression of predominantly gram-positive bacteria.

FORMULA

Ingredients per liter of deionized water:*

Tetrathionate Broth with Brilliant Green:
Sodium Thiosulfate 30.0gm
Calcium Carbonate 10.0gm
Pancreatic Digest of Casein 2.5gm
Peptic Digest of Animal Tissue 2.5gm
Bile Salts 1.0gm
Brilliant Green 0.1gm

Final pH 8.4 +/- 0.2 at 25ºC.

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

Iodine-Iodide Solution (Cat. no. Z129) not included:**
Iodine 15.0gm
Potassium Iodide 12.5gm
Deionized Water 50.0ml

** Sold separately.

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, 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. (5,7-10) 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. Shake the sample vigorously to resuspend any precipitate.

3. Immediately before inoculation, add 0.2ml of Iodine-Iodide Solution (Cat. no. Z129) to each tube.

4. Place 1gm of solid sample or 1ml of liquid sample into a Tetrathionate Broth with Brilliant Green tube. Swab specimens may be inserted directly into the broth.

5. Emulsify the specimen thoroughly.

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

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

8. Incubate aerobically at 35ºC.

9. Examine plates for pathogens in 18 to 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. (5-10)

LIMITATIONS

Due to the nutritional requirements and inhibitory characteristics of the microorganisms obtained, microbes other than salmonellae, such as Morganella morganii and some Enterobacteriaceae, may grow in this medium.

Fermentation, seroagglutination and other confirmatory tests should be performed on pure cultures of all presumptive Salmonella colonies recovered.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, Iodine-Iodide Solution (Cat. no. Z129), incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

The following organisms are routinely used for testing at Hardy Diagnostics:

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Salmonella enterica
ATCC ® 14028
I 18-24hr 35°C Aerobic Growth upon subculture to MacConkey
Escherichia coli
ATCC ® 25922
I 18-24hr 35°C Aerobic Partial to complete inhibition upon subculture to MacConkey
Enterococcus faecalis
ATCC ® 29212
I 18-24hr 35°C Aerobic Inhibited upon subculture to MacConkey

USER QUALITY CONTROL

REFERENCES

1. Compt. Rend. Soc. Biol. ; 89:434, 1923.

2. Zentr. Bakt. I. Abt. Orig. ; 133:458, 1935.

3. Zentr. Bakt. I. Abt. Orig. ; 119:148, 1930.

4. Zeit, Hyg. ; 117:26, 1935.

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

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

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

8. Anderson, N.L., et al. Cumitech 3B; Quality Control and Quality Assurance Practices in Clinical Microbiology , Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

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

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

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

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

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


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

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