BOLTON BROTH

Cat. no. U83 Bolton Broth, 500ml Wide Mouth Polycarbonate Bottle, 225ml 10 bottles/box

INTENDED USE

Hardy Diagnostics Bolton Broth is recommended for the selective enrichment of Campylobacter spp. from food and dairy products. This medium aids in the resuscitation of sublethally damaged cells of Campylobacter , while inhibiting the growth of undesirable gram-positive and gram-negative microorganisms, yeasts, and molds.

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

SUMMARY

Campylobacter spp. are widely distributed in the environment and are a major cause of bacterial gastroenteritis. Many species commonly reside in the intestinal tract of a wide range of domestic and wild animals: cattle, sheep, pigs and birds. Consequently, foods derived from these animals can become contaminated with Campylobacter spp. when prepared under suboptimal conditions. (5,6) Poultry is the most common source of bacterial gastroenteritis; however, cross-contamination to other foods prepared in common areas with infected meat products are also responsible for infection. (6)

Several species of campylobacters are thermotolerant and microaerophilic; these microorganisms can continue to grow optimally at temperatures up to 42ºC. and prefer to live in oxygen-reduced environments. The most common strains of thermotolerant infectious campylobacters are Campylobacter jejuni and Campylobacter coli . (5,6)

Cells of Campylobacter spp. can be difficult to culture as these bacteria may be infectious in very low numbers but are often found in the presence of a high number of competing microorganisms. (6) More importantly, cells may become sublethally damaged during the processing of contaminated food products and, thus, are difficult to isolate. Traditional methods of culture and subculture have utilized selective enrichment medias, combined with the use of antibiotics to suppress the growth of competitors, and biochemical assays. (6)

Hardy Diagnostics Bolton Broth as an enrichment media was developed to optimize the growth of Campylobacter spp. from contaminated food and dairy products. This medium contains enzymatic digests of animal tissues, lactalbumin hydrolysates and yeast extract which provide essential nutrients like vitamins, amino acids and other nitrogenous compounds. Sodium metabisulphite and sodium pyruvate reduce toxic compounds and increase the recovery rate and aero-tolerance of the culture. Alpha-ketoglutaric acid provides a stimulus to metabolism, whereas sodium carbonate neutralizes acids that may form in the culture. The osmotic balance is further maintained by the addition of sodium chloride. The antibiotics vancomycin, cefoperazone and trimethoprim inhibit the growth of gram-positive and gram-negative microorganisms. Cycloheximide largely reduces the growth of yeast and molds. Finally, the incubation temperature of 41.5ºC., after the initial incubation step, increases the selectivity of this medium making it easier to detect the presence of infectious Campylobacter spp.

FORMULA

Ingredients per liter of deionized water:*

Enzymatic Digest of Animal Tissues 10.0gm
Lactalbumin Hydrolysate 5.0gm
Yeast Extract 5.0gm
Sodium Chloride 5.0gm
a -Ketoglutaric Acid 1.0gm
Sodium Carbonate 0.6gm
Sodium Pyruvate 0.5gm
Sodium Metabisulfite 0.5gm
Hemin 0.01gm
Horse Blood, Lysed 50.0ml

Bolton Broth Supplement Solution
Cycloheximide 50.0mg
Cefoperazone 20.0mg
Vancomycin 20.0mg
Trimethoprim 20.0mg

Final pH 7.4 +/- 0.3 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, discoloration, hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

1. Mix 11.0gm of food sample into 100ml Bolton Broth or 25.0gm of food sample into 225ml of Bolton Broth and mix to combine thoroughly.

2. Incubate sample for 4 hours at 35ºC. Continue incubation for an additional 14 to 44 hours at 42ºC.

3. Subculture to appropriate isolation medium for further analysis. Consult listed references for more information. (2-4,8,9)

INTERPRETATION OF RESULTS

Due to the selectivity of the medium and the elevated incubation temperature, growth upon subculture indicates a positive reaction; partial to complete inhibition upon subculture indicates a negative reaction.

LIMITATIONS

For identification, the organism must be in pure culture. Morphological, biochemical and /or serological tests should be performed for final identification. Consult appropriate references for detailed information and recommended procedures. (2-4,8,9)

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
Campylobacter jejuni
ATCC ® 33291**
A 24-48hr 35°C Micro Growth; upon subculture
Escherichia coli
ATCC ® 25922**
B 24-48hr 35°C Aerobic Inhibited; partial to complete inhibition upon subculture

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

USER QUALITY CONTROL

Physical Appearance

Bolton Broth should appear clear, and deep brick-red in color.

REFERENCES

1. Anderson, N.L., et al. 2005. 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. Krieg, et al. 1984. Bergey's Manual of Systematic Bacteriology , Vol. I. Williams & Wilkins Co., Baltimore, MD.

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

5. Heisick, J. 1985. Comparison of Enrichment Broths for Isoaltion of Campylobacter jejuni. Appl. Environ. Microbiol. ; 50:1313-1314.

6. Baylis, C.L., S. MacPhee, K.W. Martin, T.J. Humphre, and R.P. Betts. 2000. Comparison of three enrichment media for the isolation of Campylobacter spp. from foods. J. Appl. Microbiol. ; 89:884-891.

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

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

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

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

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

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