Bolton Broth Base ISO

Description

The meat peptone and lactalbumin hydrolysate supply the carbon and nitrogen for growth. Sodium chloride provides osmotic balance and the sodium carbonate neutralizes the acidity generated by the microbial growth. Yeast extract and ketoglutaric acid act as growth factors. The inclusion of sodium metabisulfite, sodium pyruvate and haemin neutralises toxic compounds that may form in the culture medium due to the action of oxygen and avoids the need for a microaerobic atmosphere. Lysed horse blood is necessary to neutralize trimethoprim antagonists present in the medium.

The selectivity of the enrichment step is optimized with the Selective Supplement: vancomycin is active against Gram-positive microorganisms. Cefoperazone is predominantly active against Gram-negative bacteria. Trimethoprim acts against a wide variety of Gram-positive and Gram-negative cells and cycloheximide or amphotericin B are efficient fungicides.

Technique

Introduce a quantity (mass or volume) into nine times its volume of Bolton Selective Enrichment Broth so as to obtain a test sample/medium ratio of 1:10 (w/v or v/v) and homogenize.

Bolton Selective Enrichment Broth does not require incubation in a microaerobic environment, but must be used in screw-topped containers which are filled leaving a headspace of less than 20 mm, and have tightly closing caps.

Incubate the initial suspension at 37 °C for 4-6 hours, then at 41.5 °C for 44 ± 4 hours.

For the isolation and identification techniques, refer to ISO or BAM (Bacteriological Analytical Manual) methods.

Note: to complete the culture medium, add 5% of lysed horse blood and Campylobacter Bolton Selective Supplement (LC-6070).

Note: to complete the base medium, add 5% lysed horse blood and the Campylobacter selective supplement according to Bolton (LC-6070).

LC-4675, 125 mL bottles

Physical/chemical control

Microbiological control

Prepare tubes - inoculate with 100 ± 20 CFU for growth promotion or 10⁴-10⁶ CFU for selectivity.

Microaerophilia. 37 ± 1 °C for 5 ± 1 h; then 41.5 ± 1 °C for 44 ± 4 h.

Subculture after incubation onto appropriate media.

Microbiological control according to ISO 11133:2014.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

• BAYLIS, C.L., (editor) (2007) Manual of Microbiological Methods for the Food and Drinks Industry. 5th ed. Method 3.3.1:2007. CCFRA. Chipping Campden. UK.
• BOLTON, F.J. (2000) Methods for isolation of campylobacters from humans, animals, food and water. In "The increasing incidence of human campylobacteriosis" Report and Proceedings of a WHO Consultation of Experts. Copenhagen Denmark 21-25 November 2000, WHO/CDS/CSRAPH 2001. p. 87-93.
• BOLTON, F.J., D. COATES, P.M. HINCHCLIFFE & L. ROBERTSON (1983) Comparison of selective media for isolation of Campylobacter jejuni/coli. J. Clin. Pathol. 36:78-83.
• BOLTON, F.J., D. COATES & D.N. HUTCHINSON (1984) The ability of Campylobacter media supplements to neutralize photochemically induced toxicity and hydrogen peroxide. J. Appl. Bacteriol. 56:151-157.
• CORRY, J.E.L., H. IBRAHIM ATABAY, S.J. FORSYTHE & L.P. MANSFIELD (2003) Culture Media for the isolation of campylobacters, helicobacters and arcobacters. In "Handbook of Culture Media for Food Microbiologists". J.E.L. Corry et al. (Eds.) Elsevier Science B.V. Amsterdam.
• DOYLE, M.P. & D.J. ROMAN (1982) Recovery of Campylobacter jejuni and C. coli from inoculated foods by selective enrichment. Appl. Environm. Microbiol. 43:1343-1353.
• FDA (Food and Drug Administration) (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International. Gaithersburg. Maryland. USA.
• HUNT, J.M., C. ABEYTA & T. TRAN (1998) Campylobacter. In: FDA BAM 8th ed. (Rev. A) 7.01-7.027 AOAC International. Gaithersburg. MD. USA.
• ISO 10272-1 Standard (2017) Microbiology of the food chain - Horizontal Method for detection and enumeration of Campylobacter spp. - Part 1: Detection method.
• ISO 10272-2 Standard (2017) Microbiology of the food chain - Horizontal Method for detection and enumeration of Campylobacter spp. - Part 2: Colony count technique.
• ISO 11133:2014/Amd 1:2018. Microbiology of food, animal feed and water. Preparation, production, storage and performance testing of culture media.
• STERN, N.J., J.E. LINE & H.C. CHEN (2001) Campylobacter in "Compendium of methods for the Microbiological Examination of Foods" 4th ed. F.P. Downes & K. Ito (Eds.) APHA, Washington. DC. USA.