Bolton Selective Supplement

Description

Campylobacter genus are Gram-negative, microaerophilic bacteria that can be present in milk, untreated water or undercooked food.

Injured organisms are not generally detected and therefore a recovery step must be included in examination procedures. This is of importance, particularly in the food industry as various processes such as heat, desiccation, preservation processes, pH changes, etc., cause sublethal injuries to Campylobacter. The broth is rich in nutrients and produces high resuscitation rates for sublethally injured bacteria and intense growth.

Meat peptone provides nitrogen, vitamins, minerals and amino acids essential for growth. Lactalbumin hydrolysate provides nitrogen, amino acids, vitamins, and carbon and is specially indicated for culture media for Campylobacter growth. Yeast extract is a source of vitamins, particularly the B-group. Sodium chloride supplies essential electrolytes for transport. Alpha-ketoglutaric acid is incorporated to satisfy the specific nutritional requirements of Campylobacter species. Sodium pyruvate is a source of energy for bacterial metabolism and aids in resuscitation of stressed organisms. Sodium carbonate is a pH regulator. Hemin provides X factor, which stimulates the growth of many microorganisms. The addition of Bolton Broth Selective Supplement inhibits the accompanying Gram-positive bacteria due to trimethoprim, Gram-negative bacteria due to vancomycin, cefoperazone and trimethoprim as well as yeasts and moulds due to amphotericin B.

Technique

Aseptically reconstitute 1 vial with 2.5 mL of sterile distilled water/ethanol 1:1. Mix gently until complete dissolution. Aseptically add one vial to 475 mL of Bolton Selective Enrichment Broth Base (LC-1441), autoclaved and cooled to below 47 °C, + 25 mL of sterile lysed horse blood. Mix well and distribute into sterile containers.

Instructions for use

For the detection and enumeration of Campylobacter spp. for samples with low numbers of campylobacters and low level of background microflora and/or stressed campylobacters according to ISO 10272:

• Add the test portion to the liquid enrichment medium Bolton Broth. In general, for preparing the initial suspension, combine a quantity of 10 g or 10 mL of the test portion with 90 mL of the Bolton Broth.
• Incubate in a microaerobic atmosphere at 37 °C for 4 to 6 h, then at 41.5 °C for 44 ± 4 h.
• From the enrichment culture obtained, inoculate two selective media, CCDA agar (LC-1129) and any other selective medium using different selective principles.
• Incubate the selective solid media at 41.5 °C in a microaerobic atmosphere for 44 h to detect the presence of suspect Campylobacter colonies.
• Examine the suspect Campylobacter colonies for morphology and motility using a microscope, subculture on a non-selective blood agar, and then confirm by detection of oxidase activity and an aerobic growth test at 25 °C.

Reconstitute the original freeze-dried vial by adding 2.5 mL of sterile distilled water/ethanol (50:50).

Note: each vial is sufficient to supplement 500 mL of Bolton Selective Enrichment Broth.

Physical/chemical control

Microbiological control

Reconstitute 1 vial as indicated in Composition; shake and dissolve completely.

Add 1 vial to 500 mL of medium base. DO NOT HEAT once supplemented.

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/Amd 1:2018.

Sterility control

Add 5 mL of the sample to 100 mL of TSB and to 100 mL of Thioglycollate.

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.

• ISO 10272-1:2017 Microbiology of the food chain. Horizontal method for detection and enumeration of Campylobacter spp. Part 1: Detection method.
• Post, D. E. (1995). Food-Borne Pathogens Monograph Number 3 Campylobacter.
• Bolton, F.J. (1995) Personal communication.
• Hunt, J.M. (1998) Campylobacter. In: F.D.A. Bacteriological Analytical Manual, 8th Edition (Rev. A) 7.01-7.27. AOAC, Arlington Va.