BAM: CampylobacterReturn to BAM table of contents1January 2001Bacteriological Analytical ManualChapter 7CampylobacterAuthors: Jan M. Hunt, Carlos Abeyta and Tony TranRevision History: Updated and revised: 2000-DEC-29 Media Instructions Modified on 2001-MAR-08 Isolation of Campylobacter Species from Food and WaterCampylobacter is considered by many to be the leading cause of enteric illness in the United States (20,26). Campylobacter species can cause mild to severe diarrhea, with loose, watery stools often followed by bloody diarrhea (7,20). C. jejuni, C. coli, and C. lari account for more than 99% of the human isolates (C. jejuni 90%). Other species have been associated with human illness in recent years (7,17,18,23,26,27).Campylobacter species are highly infective. The infective dose of C. jejuni ranges from 500 to 10,000 cells, depending on the strain, damage to cells from environmental stresses, and the susceptibility of the host (4,6,7,20,27). Only the mesophilic C. fetus is normally invasive. Thermophilic species (optimum 42C) such as C. jejuni are occasionally invasive. The infections are manifested as meningitis, pneumonia, miscarriage, and a severe form of Guillain-Barr syndrome (6,20). Thermotolerant strains of C. fetus that grow at 42C have been isolated from patients (17).Campylobacters are carried in the intestinal tract of a wide variety of wild and domestic animals, especially birds. They can establish a temporary asymptomatic carrier state, as well as illness, in humans. This is especially prevalent in developing countries (20). Consumption of food and water contaminated with untreated animal or human waste accounts for 70% of Campylobacter-related illnesses each year. The foods include unpasteurized milk, meats, poultry, shellfish, fruits, and vegetables, (1,8-11,17,19,20,22, 25,26).C. jejuni can survive 2-4 weeks under moist, reduced-oxygen conditions at 4C, often outlasting the shelf life of the product (except in raw milk products). They can also survive 2-5 months at -20C, but only a few days at room temperature (5,8-11,20). Environmental stresses, such as exposure to air, drying, low pH, heating, freezing, and prolonged storage, damage cells and hinder recovery to a greater degree than for most bacteria. Older and stressed organisms gradually become coccoidal and increasingly difficult to culture (5,20). Oxygen quenching agents in media such as hemin and charcoal as well as a microaerobic atmosphere and preenrichment can significantly improve recovery (2,14-16,21,25,28).Campylobacters are microaerophilic, very small, curved, thin, Gram-negative rods (1.5-5 m), with corkscrew motility. They often join to form zigzag shapes (20,24). Campylobacter spp. are currently identified by tests described by Harvey (13) and Barret et al.(3). PCR genus and species identification methods have been published (12,18,30).For additional information contact Carlos Abeyta, FDA, PRLNW, Bothell, WA 98041-3012. Phone number: (425) 483-4890.SAMPLE PREPARATION FLOWCHART FOR VARIOUS FOODS AND WATER (SECTION C.2.a-h)*for samples produced or processed 10 days previously.for frozen samples or samples produced or processed 10 days previously.A. Equipment and materials 1. Balances, 6000 g capacity, accurate to 0.1 g; and 200 g capacity, accurate to 0.0001 g 2. Sterile stomacher bags, 400 and 3500 ml bags and 400 ml filter bags (other bag types and sizes described in refs. 16, 21, and 22) 3. Whirl-pak bag racks and stainless steel baskets 4. Bench top shaker 5. Centrifuge, refrigerated, capable of 20,000 x g 6. Polypropylene or stainless steel 250 ml centrifuge bottles and 50 ml centrifuge tubes, sterile 7. Large funnels with cheese cloth linings, sterile (for whole seafood and meat samples or if filter bags are unavailable) 8. White or orange grease pencils to mark blood-free agar plates 9. 50 ml sterile conical centrifuge tubes 10. Plastic 5-10 ml tubes with screw cap lids, sterile 11. Cryotubes, 1 ml, sterile 12. Phase-contrast microscope, with 100X oil immersion objective or dark-field microscope with 63X objective or light microscope with 1000X objective 13. Microscope slides, 1 cm sq cover slips and immersion oil 14. Gas tank assembly (5% O2, 10% CO2, 85% N2) and vacuum source (Fig. 1) 15. Microaerobic container system a. Anrobe jars and bags: 1. Jars with vacuum-pressure gauge and Schrader valves. They may be used with either the gas tank/vacuum assembly or with gas-generating envelopes. 3.4 L, (Difco 1950-30-2 or Oxoid HP11) may be used with either the gas tank/vacuum assembly or with gas-generating envelopes. 2. Jars without a gauge or valves (2.5 liter BBL or EM Diagnostics Remel, Lenexa, KS 66215 and 9.5 liter BBL) anaerobe jars. These are used with gas-generating envelopes (2.5 liter type such as Oxoid N025A or the BBL and EM gas paks). 3. Rectangular jars 2.5 and 5.5 liter anaerobe rectangular ja