CRITERION™ BISMUTH SULFITE AGAR

Cat. no. C5210 CRITERION™ Bismuth Sulfite Agar 104gm
Cat. no. C5211 CRITERION™ Bismuth Sulfite Agar 500gm
Cat. no. C5212 CRITERION™ Bismuth Sulfite Agar 2kg
Cat. no. C5213 CRITERION™ Bismuth Sulfite Agar 10kg
Cat. no. C5214 CRITERION™ Bismuth Sulfite Agar 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ Bismuth Sulfite Agar is a highly selective and differential medium. It is recommended for the isolation of Salmonella species, especially S. typhi, from food and clinical specimens.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

Salmonellosis continues to be an important public health issue in the United States and worldwide. Despite efforts to control the occurrence of Salmonella in domesticated animals, Salmonella are primary pathogens of many animals (e.g., poultry, cows, pigs, reptiles, etc.) and are the principal source of non-typhoidal salmonellosis in humans.(12) Human infections with Salmonella are most commonly caused by ingestion of fecally contaminated food, water, or milk. Improper handling of poultry products is many times the cause of Salmonella-related gastroenteritis, with about one half of salmonellosis epidemics caused by contaminated poultry and poultry products. Non-typhoidal Salmonella usually causes an intestinal infection accompanied by diarrhea, fever, and abdominal cramps. Typically, it is mild and self-limiting, often lasting one week or longer. All ages are affected, with the highest incidence being in infants.(2,5,12)

Typhoid fever, caused by S. typhi, is a serious bloodstream infection common in developing countries. It is rare in the United States and most reported cases are related to foreign travel. Typhoid fever typically presents sustained, debilitating high fever and headache, without diarrhea. There is a long, and highly variable incubation period (1 to 6 weeks). It is transmitted by person to person contact or by fecally contaminated food and water.(2) Humans are the only known reservoir for S. typhi.(5)

In food testing, Bismuth Sulfite Agar is specified as one of the highly selective medias to for isolation of Salmonella species from dairy products. Raw milk has been associated with human outbreaks involving Salmonella. Salmonella infection in dairy cattle are common and outbreaks of bovine salmonellosis have been reported. Dairy cattle can acquire Salmonella infection from a variety of sources, including contaminated feed or water. Salmonella is incapable of surviving pasteurization. Therefore, its presence in pasteurized milk is usually caused by improper processing or post pasteurization contamination.(12)

Bismuth Sulfite Agar is selective due to the presence of inhibitors, and is differential on the basis of hydrogen sulfide production.(13) Beef extract and peptones provide nitrogen, vitamins and minerals. Dextrose is an energy source. Bismuth sulfite and brilliant green are selective agents, inhibiting most commensal gram-positive and gram-negative organisms other than Salmonella species and some Shigella species. Ferrous sulfate is an indicator for hydrogen sulfide production, which occurs when the H2 S produced by Salmonella reacts with the iron salt. This reaction causes a black or green metallic colony and brown or black precipitate.(2)

FORMULA

Gram weight per liter: 52.0gm/L
Pancreatic Digest of Casein 5.0gm
Peptic Digest of Animal Tissue 5.0gm
Beef Extract 5.0gm
Dextrose 5.0gm
Disodium Phosphate 4.0gm
Bismuth Sulfite Indicator 8.0gm
Ferrous Sulfate 0.3gm
Brilliant Green 0.025gm
Agar 20.0gm

Final pH 7.5 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original light greenish beige.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 52.0gm of the dehydrated culture media in 1 liter of distilled or deionized water. Stir to mix thoroughly.

2. Heat to boiling to dissolve completely, approximately 1 minute. Do not overheat.

3. Do not autoclave.

4. Cool to 45-50ºC.

5. Mix thoroughly before pouring into petri plates. Use poured plates the same day.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references.

LIMITATIONS

Incubation of Bismuth Sulfite Agar plates at higher temperatures (i.e., 43ºC.) can result in small, atypical Salmonella colonies. In some instances, higher temperatures will also reduce method sensitivity as well as show significantly lower recoveries.(6)

Typical S. typhi colonies usually develop within 24 hours, however, all plates should be incubated for 48 hours to allow growth of all typhoid strains.(13)

Bismuth Sulfite Agar plates should not be stored refrigerated (2-8ºC.) for longer than 2 days. After 3 days of storage there is a reduction of media selectivity, decreasing the number of Salmonella recovered. It is recommended that Bismuth Sulfite Agar plates be used on the day prepared.(13)

Most Shigella species are usually inhibited on Bismuth Sulfite Agar; however, S. flexneri and S. sonnei may exhibit growth.(13)

S. typhi and S. arizonae are the only enteric organisms to exhibit typical brown zones in the medium; although, S. arizonae is usually inhibited. Other members of the Enterobacteriaceae do not produce brown zones.(13)

It is important to streak for well isolated colonies; in heavy growth areas S. typhi appears light green and could be interpreted as negative growth for S. typhi.(13)

Colonies on Bismuth Sulfite Agar may be contaminated with other viable organisms; therefore, isolated colonies should be subcultured onto a less selective medium (i.e., MacConkey Agar).(13)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL

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

ATCC ® 14028
A
24-48hr
35°C
Aerobic
Growth; colonies are black or greenish-gray, may have sheen, no zones or halo effect
Escherichia coli
ATCC ® 25922
B
24-48hr
35°C
Aerobic
Partial to complete inhibition; brown-green colonies
Enterococcus faecalis
ATCC ® 29212
B
24-48hr
35°C
Aerobic
Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

CRITERION™ Bismuth Sulfite Agar powder should appear homogeneous, free-flowing, and light greenish-beige in color. The prepared media should appear opaque, with a flocculent precipitate, and gray-green 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. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

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

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

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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

7. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.

8. Atlas, R.M. 1997. Handbook of microbiological media, 2nd ed. CRC Press, Inc., Boca Raton, Florida.

9. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

10. Wilson, James W. and E.M.M'V. Blair. 1926. A combination of Bismuth and Sodium Sulphite affording an enrichment and selective medium for the Typhoid-Paratyphoid groups of bacteria. The Journal of Pathology and Bacteriology; 29:310-311.

11. Hajna, A.A. and S.R. Damon. 1956. New enrichment and plating media for the isolation of Salmonella and Shigella organisms. Applied Microbiology. 4:341-345.

12. Marshall, R.T., Ph.D. 1993. Standard Methods for the Examination of Dairy Products, 16th ed. American Public Health Association, 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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050916gr