Chapter 11: Campylobacter spp.

• Potential Food Safety Hazard
• Control Measures
• FDA Guidelines
• Growth
• Heat Resistance
• Analytical Procedures
• Food Sampling and Preparation of Sample Homogenate
• Isolation of Campylobacter species from food and water
• Commercial Test Products
• References

Contents

Potential Food Safety Hazard

Campylobacter is considered by many to be the leading cause of enteric illness in the United States (Nachamkin et al., 1992; Tauxe et al., 1988). Campylobacter species can cause mild to severe diarrhea, with loose, watery stools often followed by bloody diarrhea (Butzler, 1984; Nachamkin et al., 1992). 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 (Butzler, 1984; Klein et al., 1986; Linton et al., 1996; Patton et al., 1989; Tauxe et al., 1988; Tee et al., 1987).

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 (Black et al., 1988; Blaser et al., 1986; Butzler, 1984; Nachamkin et al., 1992; Tee et al., 1987). Only the mesophilic C. fetus is normally invasive. Thermophilic species (optimum 42°C) such as C. jejuni are occasionally invasive. The infections are manifested as meningitis, pneumonia, miscarriage, and a severe form of Guillain-Barré syndrome (Blaser et al., 1986; Nachamkin et al., 1992). Thermotolerant strains of C. fetus that grow at 42°C have been isolated from patients (Klein et al., 1986).

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 (Nachamkin et al., 1992). 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, (Abeyta and Kaysner, 1987; Abeyta, 1998; Castillo and Escartin, 1994; Clark and Bueschkens, 1986; Doyle and Schoeni, 1986; Fricker and Park, 1989; Klein et al., 1986; Mathewson et al., 1983; Nachamkin et al., 1992; Park and Sanders, 1992; Stern and Bolton, 1994; Tauxe et al., 1988).

C. jejuni can survive 2-4 weeks under moist, reduced-oxygen conditions at 4°C, often outlasting the shelf life of the product (except in raw milk products). They can also survive 2-5 months at -20°C, but only a few days at room temperature (Blaser et al., 1980; Castillo and Escartin, 1994; Clark and Bueschkens, 1986; Doyle and Schoeni, 1986; Fricker and Park, 1989; Nachamkin et al., 1992). 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 (Blaser et al., 1980; Nachamkin et al., 1992). Oxygen quenching agents in media such as haemin and charcoal as well as a microaerobic atmosphere and preenrichment can significantly improve recovery (Bark et al., 1996; Humphrey, 1986; Hunt et al., 1985; Hutchinson and Bolton, 1984; Park and Sanders, 1989; Stern and Bolton, 1994; Tran and Yin, 1997).

Campylobacters are microaerophilic, very small, curved, thin, Gram-negative rods (1.5-5 µm), with corkscrew motility. They often join to form zigzag shapes (Nachamkin et al., 1992; Smibert, 1984). Campylobacter spp. are currently identified by tests described by Harvey (1980) and Barret et al.(1988). PCR genus and species identification methods have been published (Harmon et al., 1997; Linton et al., 1996; Winters and Slavik, 1995).

For additional information contact Jan Hunt or Carlos Abeyta, FDA, P.O. Box 3012, Bothell, WA 98041-3012. Phone numbers: (425) 402-3171 (Hunt) or 483-4870 (Abeyta); e-mail: jhunt2@ora.fda.gov and cabeyta@ora.fda.gov. Tony Tran can be contacted at CFSAN, 200 C St., SW, Washington, DC 20204; phone no. is (202) 205-5253 and the e-mail address is TTT@cfsan.fda.gov.

Contents

Control Measures

Hazards from C. jejuni can be controlled by thoroughly cooking seafood and by stressing the importance of proper (and frequent) hand and equipment washing and sanitary food-handling practices. Since the infective dose of C. jejuni is thought to be small, time/temperature abuse of food products could result in this illness (Ward et al., 1997).

Contents

FDA Guideline

FDA to assess situations on a case by case basis.

Contents

Growth

Contents

Heat Resistance

Contents

Analytical Procedures

Contents

Food Sampling and Preparation of Sample Homogenate (Andrews and June, 1998)

The adequacy and condition of the sample or specimen received for examination are of primary importance. If samples are improperly collected and mishandled or are not representative of the sampled lot, the laboratory results will be meaningless. Because interpretations about a large consignment of food are based on a relatively small sample of the lot, established sampling procedures must be applied uniformly. A representative sample is essential when pathogens or toxins are sparsely distributed within the food or when disposal of a food shipment depends on the demonstrated bacterial content in relation to a legal standard.

The number of units that comprise a representative sample from a designated lot of a food product must be statistically significant. The composition and nature of each lot affects the homogeneity and uniformity of the total sample mass. The proper statistical sampling procedure, according to whether the food is solid, semisolid, viscous, or liquid, must be determined by the collector at the time of sampling by using the Investigations Operation Manual (FDA, 1993). Sampling and sample plans are discussed in detail in ICMSF (1986).

Whenever possible, submit samples to the laboratory in the original unopened containers. If products are in bulk or in containers too large for submission to the laboratory, transfer representative portions to sterile containers under aseptic conditions. There can be no compromise in the use of sterile sampling equipment and the use of aseptic technique. Sterilize one-piece stainless steel spoons, forceps, spatulas, and scissors in an autoclave or dry-heat oven. Use of a propane torch or dipping the instrument in alcohol and igniting is dangerous and may be inadequate for sterilizing equipment.

Use containers that are clean, dry, leak-proof, wide-mouthed, sterile, and of a size suitable for samples of the product. Containers such as plastic jars or metal cans that are leak-proof may be hermetically sealed. Whenever possible, avoid glass containers, which may break and contaminate the food product. For dry materials, use sterile metal boxes, cans, bags, or packets with suitable closures. Sterile plastic bags (for dry, unfrozen materials only) or plastic bottles are useful containers for line samples. Take care not to overfill bags or permit puncture by wire closure. Identify each sample unit (defined later) with a properly marked strip of masking tape. Do not use a felt pen on plastic because the ink might penetrate the container. Whenever possible, obtain at least 100 g for each sample unit. Submit open and closed controls of sterile containers with the sample.

Deliver samples to the laboratory promptly with the original storage conditions maintained as nearly as possible. When collecting liquid samples, take an additional sample as a temperature control. Check the temperature of the control sample at the time of collection and on receipt at the laboratory. Make a record for all samples of the times and dates of collection and of arrival at the laboratory. Dry or canned foods that are not perishable and are collected at ambient temperatures need not be refrigerated. Transport frozen or refrigerated products in approved insulated containers of rigid construction so that they will arrive at the laboratory unchanged. Collect frozen samples in pre-chilled containers.

Place containers in a freezer long enough to chill them thoroughly. Keep frozen samples solidly frozen at all times. Cool refrigerated samples, except shellfish and shell stock, in ice at 0-4ºC and transport them in a sample chest with suitable refrigerant capable of maintaining the sample at 0-4ºC until arrival at the laboratory. Do not freeze refrigerated products. Unless otherwise specified, refrigerated samples should not be analyzed more than 36 h after collection. Special conditions apply to the collection and storage of shucked, unfrozen shellfish and shell stock (APHA, 1985). Pack samples of shucked shellfish immediately in crushed ice (no temperature specified) until analyzed; keep shell stock above freezing but below 10ºC. Examine refrigerated shellfish and shell stock within 6 h of collection but in no case more than 24 h after collection. Further details on sample handling and shipment may be found in the Investigations Operation Manual (FDA, 1993) and the Laboratory Procedures Manual (FDA, 1989). The Investigations Operation Manual (FDA, 1993) contains sampling plans for various microorganisms. Some of those commonly used are presented here.

1. Sampling plans
1. Aerobic plate counts, total coliforms, fecal coliforms, Escherichia coli (including enteropathogenic strains), Staphylococcus spp., Vibrio spp., Shigella spp., Campylobacter spp., Yersinia spp., Bacillus cereus, and Clostridium perfringens
1. Sample collection. From any lot of food, collect ten 8 ounce (227 g) subsamples (or retail packages) at random. Do not break or cut larger retail packages to obtain an 8 ounce (227 g) subsample. Collect the intact retail unit as the subsample even if it is larger than 8 ounce (227 g).
2. Sample analysis. Analyze samples as indicated in current compliance programs.
2. Equipment and materials
1. Mechanical blender. Several types are available. Use blender that has several operating speeds or rheostat. The term "high-speed blender" designates mixer with 4 canted, sharp-edge, stainless steel blades rotating at bottom of 4 lobe jar at 10,000-12,000 rpm or with equivalent shearing action. Suspended solids are reduced to fine pulp by action of blades and by lobular container, which swirls suspended solids into blades. Waring blender, or equivalent, meets these requirements.
2. Sterile glass or metal high-speed blender jar. 1000 ml, with cover, resistant to autoclaving for 60 min at 121ºC.
3. Balance, with weights. 2000 g capacity, sensitivity of 0.1 g.
4. Sterile beakers. 250 ml, low-form, covered with aluminum foil.
5. Sterile graduated pipets. 1.0 and 10.0 ml.
6. Butterfield's phosphate-buffered dilution water (R11). Sterilized in bottles to yield final volume of 90 ± 1 ml.
7. Sterile knives, forks, spatulas, forceps, scissors, tablespoons, and tongue depressors. For sample handling.
3. Receipt of samples
1. The official food sample is collected by the FDA inspector or investigator. As soon as the sample arrives at the laboratory, the analyst should note its general physical condition. If the sample cannot be analyzed immediately, it should be stored as described later. Whether the sample is to be analyzed for regulatory purposes, for investigation of a foodborne illness outbreak, or for a bacteriological survey, strict adherence to the recommendations described here is essential.
2. Condition of sampling container. Check sampling containers for gross physical defects. Carefully inspect plastic bags and bottles for tears, pinholes, and puncture marks. If sample units were collected in plastic bottles, check bottles for fractures and loose lids. If plastic bags were used for sampling, be certain that twist wires did not puncture surrounding bags. Any cross-contamination resulting from one or more of above defects would invalidate the sample, and the collecting district should be notified (see 3-e, below).
3. Labeling and records. Be certain that each sample is accompanied by a completed copy of the Collection Report (Form FD-464) and officially sealed with tape (FD-415a) bearing the sample number, collecting official's name, and date. Assign each sample unit an individual unit number and analyze as a discrete unit unless the sample is composited as described previously in this chapter.
4. Adherence to sampling plan. Most foods are collected under a specifically designed sampling plan in one of several ongoing compliance programs. Foods to be examined for Salmonella, however, are sampled according to a statistically based sampling plan designed exclusively for use with this pathogen. Depending on the food and the type of analysis to be performed, determine whether the food has been sampled according to the most appropriate sampling plan.
5. Storage. If possible, examine samples immediately upon receipt. If analysis must be postponed, however, store frozen samples at -20°C until examination. Refrigerate unfrozen perishable samples at 0-4°C not longer than 36 h. Store nonperishable, canned, or low-moisture foods at room temperature until analysis.
6. Notification of collecting district. If a sample fails to meet the above criteria and is therefore not analyzed, notify the collecting district so that a valid sample can be obtained and the possibility of a recurrence reduced.
4. Thawing

Use aseptic technique when handling product. Before handling or analysis of sample, clean immediate and surrounding work areas. In addition, swab immediate work area with commercial germicidal agent. Preferably, do not thaw frozen samples before analysis. If necessary to temper a frozen sample to obtain an analytical portion, thaw it in the original container or in the container in which it was received in the laboratory. Whenever possible, avoid transferring the sample to a second container for thawing. Normally, a sample can be thawed at 2-5ºC within 18 h. If rapid thawing is desired, thaw the sample at less than 45ºC for not more than 15 min. When thawing a sample at elevated temperatures, agitate the sample continuously in thermostatically controlled water bath.

5. Mixing

Various degrees of non-uniform distribution of microorganisms are to be expected in any food sample. To ensure more even distribution, shake liquid samples thoroughly and, if practical, mix dried samples with sterile spoons or other utensils before withdrawing the analytical unit from a sample of 100 g or greater. Use a 50 g analytical unit of liquid or dry food to determine aerobic plate count value and most probable number of coliforms. Other analytical unit sizes (e.g., 25 g for Salmonella) may be recommended, depending on specific analysis to be performed. Use analytical unit size and diluent volume recommended for appropriate Bacteriological Analytical Manual method being used. If contents of package are obviously not homogeneous (e.g., a frozen dinner), macerate entire contents of package and withdraw the analytical unit, or, preferably, analyze each different food portion separately, depending on purpose of test.

6. Weighing

Tare high-speed blender jar; then aseptically and accurately (± 0.1 g) weigh unthawed food (if frozen) into jar. If entire sample weighs less than the required amount, weigh portion equivalent to one-half of sample and adjust amount of diluent or broth accordingly. Total volume in blender must completely cover blades.

7. Blending and diluting of samples requiring enumeration of microorganisms
1. All foods other than nut meat halves and larger pieces, and nut meal. Add 450 ml Butterfield's phosphate-buffered dilution water to blender jar containing 50 g analytical unit and blend 2 min. This results in a dilution of 10^-1. Make dilutions of original homogenate promptly, using pipets that deliver required volume accurately. Do not deliver less than 10% of total volume of pipet. For example, do not use pipet with capacity greater than 10 ml to deliver 1 ml volumes; for delivering 0.1 ml volumes, do not use pipet with capacity greater than 1.0 ml. Prepare all decimal dilutions with 90 ml of sterile diluent plus 10 ml of previous dilution, unless otherwise specified. Shake all dilutions vigorously 25 times in 30 cm (1 foot) arc in 7 s. Not more than 15 min should elapse from the time sample is blended until all dilutions are in appropriate media.
2. Nut meat halves and larger pieces. Aseptically weigh 50 g analytical unit into sterile screw-cap jar. Add 50 ml diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10⁰ dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.
3. Nut meal. Aseptically weigh 10 g analytical unit into sterile screw-cap jar. Add 90 ml of diluent (7-a, above) and shake vigorously 50 times through 30 cm arc to obtain 10^-1 dilution. Let stand 3-5 min and shake 5 times through 30 cm arc to resuspend just before making serial dilutions and inoculations.

Contents

Isolation of Campylobacter species from food and water (Hunt et al., 1998)

1. 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 Hutchinson and Bolton, (1984), Park and Sanders (1989), and Park and Sanders (1992).
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 cm² cover slips and immersion oil
14. Gas tank assembly (5% O₂, 10% CO₂, 85% N₂) and vacuum source (Figure 11-1).
15. Gassed jar system
1. Anaerobe jars, two types are available:
1. 3.4 L, Difco 1950-30-2 or Oxoid HP11 with vacuum-pressure gauge and Schrader valves. They may be used with either the gas tank/vacuum assembly or with gas generating envelopes.
2. 2.5 L BBL or EM Diagnostics (Remel, Lenexa, KS 66215) and 9.5 L BBL anaerobe jars without gauges or valves. These are used with gas-generating envelopes (2.5 L type such as Oxoid N025A or the BBL and EM gas paks).
3. 2.5 and 5.5 L anaerobe rectangular jars (International Bioproducts, 800-729-7611 or Mitsubishi Gas Chemical America, 212-752-4620).
4. Anaerobe pouches or pouch rectangular jars, 0.4 L, for 2-plate incubation (International Bioproducts or Mitsubishi). Pouches for 1-plate incubation are available from EM Diagnostics.
2. Campy gas-generating envelopes or pouches: for 3.4 L jars, Oxoid BR56 or CN035A; for 2.5 L and 9.5 L jars, Oxoid CN025A, Difco 1956-24-4, BBL 71040 or 71034 or EM Diagnostics 53013678; for rectangular jars, Mitsubishi 10-04; for 2-plate pouches, Mitsubishi 20-04 and 1-plate pouches, EM Diagnostics, 53-13699. The Oxoid CNO25A and the Mitsubishi and EM envelopes are used without water.

CAUTION: Only gas pacs used without water are compatible with the items in a.3) and a.4). The RECTANGULAR JARS CAN EXPLODE IF USED WITH THE OXOID BR56, BBL OR DIFCO ENVELOPES.

3. An Anerobe gas pak (1 only) can be used with a 9.5 L BBL anaerobe jar. A single pak in a large container reduces the oxygen level to ~ 5% and produces other gases promoting Campylobacter growth. Either gas pak type (does/does not require water) can be used.
16. Air incubators, 25 ± 2, 30 ± 2, 37 ± 2, and 42 ± 1ºC.
17. Water bath, preferably shaker type, range 30-42ºC or coliform bath set to 37 and 42ºC. Shaker water bath should have flask clamps, 250 or 500 ml if gassed flasks are used. Shaker water bath may be used either with bubbler system or gassed flask system. Static water bath can be used only with the bubbler system.
18. Shaker air incubator or air incubator with shaker platform (alternative to shaker water bath)
19. Shaking gassed flask or bag system (Figure 11-2)
1. Bags, see Figure 11-2. Metalized poly pouches, Associated Bag Company, Milwaukee, WI, 800-926-6100. Use 6 x 8 inch (15.2 x 20.3 cm) for 100 ml enrichments and 8 x 10 inch (20.3 x 25.4 cm) bags for 250 ml enrichments. Larger bags also are available. Bags are not sterile but can be radiation sterilized.
2. Vacuum flasks, 250 or 500 ml, with rubber stopper and foam-plugged vacuum tubing on the side arm, sterilized (see Figure 11-2). Further information on assembly and use is contained in BAM, 7th ed., 1992, chapter 7.
20. Bubbler system (Figure 11-3). Two gas delivery valve systems are available.
1. Evaporator/concentrator manifold with Y-connector, available in 6 or 12 position sets (AFC International, Inc., Downers Grove, IL; 800-952-3293). See sections D-3 for use and F-1 for assembly instructions.
1. Plastic luer-lock stopcocks. Stopcocks lock into the outlet valves.
2. Plastic aquarium tubing, 3/16" (0.5 cm) inner diameter
2. Nupro S-series fine metering valves with 1/8" Swage-lok compression fittings and 1/8" (0.3 cm) teflon or poly-flo tubing. Two to 4 inch (5.1-10.2 cm) long pieces of 3/16" (0.5 cm) inner-diameter aquarium tubing (equal to number of valves). See sections. D-3 and F-2 for instructions on operating and assembling this bubbler unit. These components are available from local valve suppliers or contact Indianapolis Valve and Fitting Co, Indianapolis, IN; 317-248-2468, for information on the nearest supplier.
3. Enrichment broth container (use either):
1. 400 ml or larger stomacher or stomacher filter bags, twist ties and stainless steel baskets
2. 250 or 500 ml Erlenmeyer flasks, foam plugged with foil wrap and sterilized. Two inch (5.1 cm) square Parafilm pieces and weighted rings or a platform with clamps. For instructions for assembly and use, see Figure 11-2 and BAM, 7th ed., 1992, chapter. 7.
4. Plastic 1 ml sterile pipets
21. Water analysis apparatus
1. Zetapor filters, 45 µm (Cuno, Meriden, CT, 800-243-6894; no substitutions), 47-293 mm (depending on filtering unit size), autoclaved separately from filtering unit
2. For 47 mm filter apparatus:
1. Teflon-faced borosilicate glass 47 mm holder(s) and filter clamp forceps, sterilized
2. Manifold, 6-12 place, for multiple subsamples
3. Vacuum flask, 1-4 L, and if a manifold is used, a rubber stopper with a 6-8 inch (15.2-20.3 cm) plastic tube inserted and a hose connecting the plastic tube to the manifold. Another hose connects the flask side arm to a vacuum source.
3. For 90, 142, or 293 mm filter apparatus
1. 90, 142, or 293 mm filtering unit with 3 foot (91.4 cm) hose attached at top, sterilized
2. Vacuum flask, 4-6 L, set up as in b-3 above, except the hose attached to the vacuum flask's stopper tube connects to the filtering unit outlet port
2. Media, biochemicals and reagents (section G, except where otherwise indicated)
1. Media
1. Campylobacter Enrichment Broth (Bolton formula, Oxoid AM7526 or Malthus Diagnostics LAB-135, Malthus Diagnostics, North Ridgeville, OH; 216-327-2585) with lysed horse blood and antibiotic supplement (Oxoid NDX131 or Malthus Diagnostics X131). Alternatively, antibiotic supplement may be prepared from individual components (G-1). (M28a)
2. Campylobacter isolation agars (use either)
1. Abeyta-Hunt-Bark (A-H-B) agar (G-2)(M29a)
2. Modified Campy blood-free agar (mCCDA)(G-2)(M30a)
3. Abeyta-Hunt-Bark agar (G-2), without antibiotics
4. Heart infusion agar (HIA) slants (M59)
5. 0.1% Peptone diluent (R56)
6. Semi-solid medium, modified, for biochemical identification (G-5)
1. Neutral red (NR) solution, glycine, NaCl, cysteine HCl, KNO₃
7. Triple sugar iron (TSI) agar slants (M149)
8. O-F glucose (M116), modified; prepare half the tubes with glucose and half without.
9. MacConkey agar (M91)
10. Culture shipping media
11. Cary-Blair medium (M31) or A-H slants with 5% filtered fetal bovine serum or lysed horse blood, w/o antibiotics (G-2)
12. Culture storage media
1. Semi-solid medium, modified, for short-term culture storage (G-4)(M30c)
2. Culture freezing media (G-3) for long-term storage (M30b)
2. Biochemicals and reagents
1. Hippurate (R33) and ninhydrin (R47) reagents
2. Nalidixic acid and cephalothin antibiotic disks (Difco)
3. Hydrogen peroxide, 3%
4. Fetal bovine serum (FBS), filtered (0.22 µm)
5. Oxidase reagent, liquid type preferred (R54)
6. Gram stain reagents (R32); counterstain with 0.5% carbol fuchsin (Difco)
7. Nitrate detection reagents A and B (R48)
8. Lead acetate strips (Difco)
9. Meritek Campylobacter identification kit (Meridian Diagnostics, Cincinnati, OH, 800-543-1980)
10. Sterile water, 1-2 L; 70% ethanol or 1000 ppm hypochlorite solution (G-6)
3. Sample preparation
1. Background information

Campylobacter spp. can survive, but not multiply, in food at refrigeration temperatures for 1-3 weeks, especially if foods (except raw milk) are in airtight containers. Their numbers decrease 2 logs upon freezing at -20ºC, but the surviving organisms can be recovered ³ 5 months. Samples should be analyzed for Campylobacter as soon as a sample package is opened; introduction of fresh oxygen adds significant stress to already weakened organisms.

Production of oxygen-neutralizing enzymes is decreased in microaerophiles, especially when cells are under stress. Therefore, oxygen-quenching compounds are added to the basic media. Protect enrichment broth from light after the media are prepared, as the haemin is light sensitive. Powdered bases and prepared media absorb oxygen during storage. Use freshly prepared media whenever possible. It can be used for up to 2 months. Keep caps tightly closed on all media.

Both the initial sample preparation and a 1:10 dilution are often needed for enrichment when high numbers of background flora (with broad species diversity) are present. With the sample dilution, antibiotics perform more effectively and Campylobacter cells can utilize the low-oxygen atmosphere more efficiently. If heavy background contamination is suspected, add 1:10 dilution enrichment. The following instructions include mandatory dilution enrichments for shellfish and eggs.

2. Preparation of Samples

Add 2 rehydrated vials of Bolton antibiotic additives and 50 ml lysed horse blood to 1000 ml Bolton broth base. Alternatively, antibiotic additives can be prepared from individual components (G-1).

1. All sample types except those listed in following (b-h)

Place filter bag in wire petri dish holder (type used in anaerobe jars). Hold bag lining in place with metal binder clip to prevent collapse during filling. Weigh 25 g sample (50 g if fruit or vegetables) into bag, and add 100 ml enrichment broth. Remove bag from holder, keeping clip attached and wrap twist-tie around top. Place bag(s) in basket or whirl-pak rack. Shake gently for 5 min or place on a table-top shaker set at 25 rpm.

After the rinsing step, hold 5 min Remove filter lining and allow it to drain a few s. If filter bag is not available, rinse sample in a sterile bag, and pour contents through a sterile, cheesecloth-lined funnel into the incubation bag or flask. When using metalized poly pouches for the gassed bag incubation, place filter liner from a stomacher bag into the pouch before weighing in the sample. NOTE: When analyzing acidic foods, such as chicken salad, adjust broth pH to 7.4 with 2N NaOH after the rinsing step.

2. Lobster tail or crab claws. Weigh 50-100 g into a filter-lined bag and rinse as in a, above.
3. Whole meat carcass or sample that cannot be easily reduced to 25 g (e.g., whole rabbit, lobster or larger piece of game meat)

Place sample into 3500 ml stomacher or other sterile bag. Add 200 ml 0.1% peptone water. Twist bag to seal, and swirl contents for 2-3 min. Tilt bag, and hold back food pieces to let rinse liquid flow to one corner. Sanitize a bottom bag corner with 1000 ppm hypochlorite solution or 70% ethanol; then rinse with sterile water. Aseptically cut corner of bag, and pour rinse through sterile cheesecloth-lined funnel into a 250 ml centrifuge bottle. Centrifuge at 16,000 x g for 15 min. Discard supernatant, and resuspend pellet in 10 ml 0.1% peptone water. Transfer 3 ml pellet mixture to 100 ml broth.

4. Liquid egg yolk or whole egg mixture

Divide sample into composites of two subsamples per composite, 25 g per sub. Weigh 25 g of each composite into 125 ml broth. After gently mixing, transfer 25 ml to another 100 ml broth. Analyze both the 1:6 and 1:48 dilutions.

5. Shellfish, shucked

In general, a minimum of 12 shellfish shall be taken in order to obtain a representative sample (APHA 1970, Recommended Procedures for the Examination of Sea Water and Shellfish). Depending on the size of the species, this will yield an approximately 100 to 200 g composite of shell liquor and meat. Collect the appropriate quantities of shell liquor and meats in a sterile blender or other suitable sterile container. Blend at low speed or stomach for 60 s. Remove 25 g shellfish homogenate for sample analysis to a Stomacher bag or 500 ml flask.

Add 225 ml enrichment broth. Transfer 25 ml of the mixture to a second 225 ml enrichment broth. Analyze both the 1:10 and 1:100 enrichments.

If enrichments are bubbled during incubation, leave them in bags or 500 ml flasks. If incubating in gassed bag or flask shaker system, use 6 x 10 inch (15.2 x 25.4 cm) metalized poly pouches or 500 ml vacuum flasks. If incubating in anaerobe jars, reduce volume/flask or bag to 125 ml by dividing each enrichment into two parts. The gas does not penetrate into a larger volume sufficiently to provide proper growth of campylobacters.

6. Water

Request investigators collect 2-4 liters for analysis. Upon collection, add 5 ml of 1 M sodium thiosulfate per liter of sample to chlorinated water. Filter smaller volume samples through 45 µm Zetapor filters, 47 mm diameter. These filters have a positive charge. The negatively charged Gram-negative organisms are more effectively retained in the filter. Filter larger volumes, especially those that are turbid, through 90 mm or larger diameter filters.

Place filter unit into autoclavable pan. If filter clogs, wear sterile gloves and open filter holder unit to aseptically remove filter with sterile forceps. Place filter into enrichment broth (see below). Place another sterile filter in unit, reassemble, and continue filtering. Use as many filters as needed per subsample. When analyzing sea or other salt water, flush excess salt off filter by running 100-1,000 ml (depending on filter size) sterile phosphate buffer through the filter as the last of the sample is going through the filter. Do this with every filter used for salt water analyses. Do not let filter become dry. Immediately transfer finished filter to broth. Campylobacters are very sensitive to drying and high salt concentrations.

Place filter(s) in broth in the enrichment container. When using large filters, fragment with a sterile pipet. Be sure the broth covers the filter(s).

Enrichments incubated in Campy gas in anaerobe jars should be 125 ml or less. Larger volumes should be divided into smaller amounts, aseptically dividing the filters.

7. Swabs

Pipet 10 ml enrichment broth into sterile 50 ml Erlenmeyer flasks with foil tops. Place one swab into each flask, aseptically breaking off the sticks below the top of the flask. Replace covers loosely. Place flasks in anaerobe jar. To fit two layers of flasks in jar, place a cardboard circle over bottom layer, leaving space around the cardboard's edge for gas circulation.

8. Milk, frozen dairy products
1. Raw milk. Instruct the investigator to test raw milk at the collection site by using a sterile pipette to place test portion onto pH test paper (pH 6-8 range). If the pH is below 7.6, add sterile 1-2 N NaOH and gently to adjust it to 7.5 ± 0.2. Immediately upon receipt in the laboratory, test the pH of the dairy sample with pH test paper and adjust to pH 7.5 ± 0.2 with sterile 1-2 N NaOH if necessary. Centrifuge a 50 g portion at 20,000 x g for 40 min. Discard supernatant and dissolve pellet (not fat layer) in 10 ml enrichment broth. Transfer pellet to 90 ml enrichment broth.
2. Other milk types and ice cream. Adjust pH as in raw milk. Centrifuge a 50 g portion at 20,000 x g for 40 min. Discard supernatant and dissolve pellet (not fat layer) in 10 ml enrichment broth. Transfer pellet to 90 ml enrichment broth.

Ice cream and other frozen dairy products: melt and aseptically remove any candy or other solids before weighing out.

3. Cheese. Weigh 50 g into a filter bag and add 50 ml 0.1% peptone. Stomach 15-30 s. Remove lining, letting it drain 5 s, and discard. Centrifuge and remove pellet to broth as in raw milk (h,1).
4. "Milk sock" or strainer (gauze piece used to filter out solids during milking). Place 50 g piece in 100 ml broth.
4. Preenrichment and enrichment (modified Park and Humphrey methods)
1. Pre-enrichments
1. 4 h pre-enrichment. If the age of the sample is known to be within 10 d of production or time of contamination, or if the sample is a dairy product, pre-enrich at 37ºC for 4 h. The pre-enrichment should be incubated under microaerobic 0conditions.
2. 5 h pre-enrichment. Use the 5 h method if any product has been refrigerated for >10 d. All water or shellfish samples are pre-enriched by the 5 h method.

Incubate at 30ºC for 3 h, then at 37ºC for 2 h. NOTE: Incubate microaerobically at 30°C unless using a non-shaking bath-bubbler system (D-3). Bubbling static enrichments at 30ºC fosters growth of anaerobes (D-3). Perform the 37ºC incubation under microaerobic conditions. This method yields greater recovery for severely stressed organisms.

3. General information concerning both methods. Set the shaker speed for bubbling enrichments to 50-60 rpm and to 175-200 rpm for gassed bags or flasks.
2. Enrichment (microaerobic, D-3)

After pre-enrichment, raise the temperature in the water bath or incubator to 42ºC. If analyzing for C. fetus, keep the temperature at 37ºC, even if a thermotolerant strain (growth at 42°C) was associated with the sample. Incubate shaking enrichments 23-24 h, except shellfish samples, which are incubated an extra 4 h. Dairy samples are incubated 48 h total. Incubate non-shaking enrichments 28-29 h, except shellfish, which are incubated 48 h. Incubate samples for C. fetus at 48 h (shaking) or 52 h (non-shaking).

3. Incubation and atmosphere modification methods for enrichments

Analysts may choose from three methods for generating microaerobic conditions in enrichment broth. These are: bubbling the gas mixture through broth, shaking enrichments to incorporate the gas, or incubating in evacuated and gassed anaerobe jars.

The first method uses the bubbler system that also can incorporate shaking the enrichments during incubation. The second uses heat-sealed, gassed, metalized poly pouches or evacuated and gassed flasks.

The third method is the evacuated and gassed anaerobe jar (or a jar that uses a Campy gas envelope). Choose this when other systems are not available. Exception: incubation of enrichments in 50 ml Erlenmeyer flasks (i.e., swabs), which can be accomplished only in the jar system.

The systems are described as follows:

1. Bubbler system

Double-bag enrichments to prevent bags from leaking (bags can tear during shaking). Place stomacher bags into stainless steel baskets (4-6/basket). Fill excess space in basket with water-filled dilution bottles. Place 1 ml plastic pipet tip end into each bag and fasten tightly with a twist-tie. Insert the plugged end of each pipet into the tubing connected to the bubbler valves.

Open the main gas tank valve and set the pressure to 4-6 pounds (27.6-41.4 kPa) with the regulator adjusting screw. This will give a flow rate of 2-3 bubbles/s into each bag (Figures 11-1 and 11-3). Ensure that the pipet tip in each bag is inserted 2/3 into the broth. Tie bubbler tubing for each enrichment loosely together above the baskets to keep the bags standing upright. Bring the water level of the bath up slightly higher than the level of the broths in the bags. Replenish water as needed during the incubation period.

Refer to the BAM, 7th ed., 1992, Chapter 7 for instructions on using Erlenmeyer flasks with the bubbler.

2. Shaking flask or bag system (use bags with an air shaker incubator)

1. Shaker bag system. Loosen the ring clamp holding the Schrader chuck and clip valve on the hose at the gas tank, and remove the valve. Insert the two-way connector attached to a length of 3/16 inch (0.5 cm) inner-diameter aquarium tubing. Open the main gas tank valve and set the regulator to 2 pounds (13.8 kPa) with the regulator adjusting screw. Place a sterile 1 ml plastic pipet in other end of the aquarium tubing, and keep pipet tip sterile by placing the tip end in a sterile bag (Figs. 1 and 2).

Heat seal each filled bag (metalized poly pouch) with a bag sealer. Cut a very small corner from the top of the sealed end. Squeeze air from bag by pressing area above liquid until the area is flat. Insert pipet into open corner of bag and open on-off valve on the gas hose. Fill area above the broth with gas. Repeat squeezing and gassing each bag 2 more times, ending with a gassing step. Quickly heat seal the corner of the bag shut. Place gassed bags into baskets lined with plastic bags. Set the basket(s) onto a shaker incubator platform. Set shaker speed to 175-200 rpm.

2. Shaker flask system. Refer to BAM, 7th ed., 1992, Chapter 7.
3. Gassed jar system. Place stomacher bags with the tops loosely closed with a twist tie in a gas jar. Amount of broth in each bag should not be over 125 ml. When using the 5.5 L rectangular jar, prepare a deep tray from foil and tape to contain the bottoms of the bags inside the jar.

1. Gas pak envelopes. Use 3 BBL Campy pak, Pack Plus or EM Anaerocult C gas-generating envelopes per 9.5 L BBL jar and 1 per small jar. With the 3.4 L Difco and Oxoid jars, use Difco or Oxoid gas pak envelopes, which are designed for use with a 3.4 L jar. Gas paks requiring water need to be used with a catalyst. With a 2.5 L rectangular jar use 1 gas pak (type not used with water); 3 for a 5.5 L jar. Or use 1 anaerobe gas pak in a 9.5 L BBL jar.
2. Gas tank and vacuum source. After tightening jar lid, attach chuck and clip valve of vacuum hose to valve indicated for vacuum on jar lid. Turn on vacuum and evacuate jar to 15-20 inches (381-508 mm) of Hg. Detach hose and tighten lid slightly if needed. Turn on tank and bring pressure to 6-8 pounds (41.4-55.2 kPa) with regulator adjusting screw (Figure 11-1). Attach chuck and clip valve on hose at tank to other valve on lid. Open tank on-off valve and fill jar to 4-5 pounds (27.6-34.5 kPa). Disconnect and close on-off valve. Repeat evacuation and gassing twice more, ending with gassing step. When opening jar, first release pressure by pressing down on lid valve stem with inoculating loop handle or similar object.
3. Guidelines for storing and maintaining jars. If a jar lid with gauges is knocked against a hard surface, a gauge can become misaligned. Mark new "0" place on gauge and adjust vacuum and gas readings accordingly.

Store jars with screw clamps placed in jars so that one end is lying over lip of jar bottom. Prop lid against clamp to allow free flow of air and prevent mold build-up from damp jar. Or clean jars between uses with 70% alcohol and dry before storing.

If a jar will not hold vacuum or gas pressure, check for the following: cracks in the jar bottom, cracked or missing rubber rings or seals in the lids or a faulty valve stem. Replacement valve stems and a Schrader extractor tool are available from the jars' distributors or bicycle shops. To replace stems, place prongs of extractor over valve stem and turn counter-clockwise until stem is removed. Drop new valve stem (pin-head side up) into valve and turn clockwise until meeting resistance.

4. Positive controls

Store Campylobacter cultures in freezing medium (G-3) at -70ºC. If cultures are used often, they can be kept at room temperature in semi-solid storage media (G-3). Control cultures can be ordered from ATCC. Labs should stock C. jejuni (ATCC 33560), C. coli (ATCC 33559), C. lari (ATCC 358221), and C. fetus (ATCC 27374).

Inoculate broth or agar positive controls from a frozen culture by rubbing a moistened sterile swab against the culture and breaking off the swab end into broth or swabbing agar plate. Incubate microaerobically.

To freeze a culture, grow it first on Abeyta-Hunt (A-H) horse blood agar without antibiotics. Inoculate plates generously and incubate under microaerobic conditions, 42ºC, 24 h. Incubate C. fetus cultures at 37ºC, 48 h. Mix enough freezing media to allow 1 ml/plate. Pipet 1.0 ml onto each plate, while wearing gloves. Use a sterile hockey stick to wash the growth to one end of each plate. Transfer washings to a sterile test tube. Pipet 0.5 ml of culture washings to cryotubes or sterile polypropylene test tubes. Store in -70ºC freezer. Freeze cultures in alcohol-dry ice bath to reduce freezer shock. Alcohol will remove most markers' identification, so mark tubes with tape labels on lids or use marker that will not be affected by alcohol.

When storing cultures in semi-solid medium, inoculate the medium at the surface and incubate loosely-capped tubes under microaerobic conditions, 24 h. See previous paragraph for proper incubation temperatures. After incubation, tighten caps and place away from direct light. Cultures can be stored up to 1-2 months with subsequent transfer.

NOTE: To ship cultures, grow the culture on A-H blood agar plates w/o antibiotics and swab off growth. Place swab in a sterile polypropylene screw-cap shipping tube filled with Cary-Blair media. Aseptically break off the excess swab stem and tighten the tube cap. Alternatively, grow culture on A-H agar slants (in shipping test tube) with 5% blood or fetal bovine serum, w/o antibiotics.

5. Isolation, identification and confirmation
1. Isolation procedure

After 24 and 48 h, streak enrichments onto either Abeyta-Hunt-Bark or modified CCDA agars. Make 1:100 dilution (0.1-9.9 ml 0.1% peptone) of each enrichment and streak undiluted and diluted portions. For shellfish, eggs, and other enrichments prepared as dilutions, streak from broths only. Transfer two loopfuls of enrichment broth to each plate and then streak for isolation. Protect plates from light.

Place plates in anaerobe jars (½ full if possible). Do not delay bringing jar to microaerobic conditions. Use either evacuation and gassing method, Campy gas paks with jars or 1 anaerobe pak with a 9.5 liter

BBL large jar (see D.3.c.-gassed jar system). Incubate at 42ºC, 24-48 h. Check for growth at 24 h. If analyzing for C. fetus, incubate at 37ºC for 48-72 h.

The inoculated agars may be incubated at a range of 37-42ºC, but thermophilic campylobacters show more rapid growth at higher temperature. NOTE: When preparing agar in plates, dry plates overnight on bench. If plates must be used the same day, place them in 42ºC incubator for several h. Do not dry in a hood with lids open. Even very brief drying of surface will inhibit Campylobacter growth.

2. Identification

Campylobacter colonies on agar are round to irregular with smooth edges. They can show thick translucent white growth to spreading, film-like transparent growth. Pick one typical colony per plate and prepare wet mount slide. To prepare, emulsify pick in drop of saline or buffer on slide. Cover each with 22 x 22 mm cover slip and examine without oil under dark-field optics at 63X or with oil under phase-contrast at 1000X. Store plates to be picked at 4ºC under microaerobic conditions if analysis is not begun quickly.

If neither type of scope is available, prepare wet mounts as follows: Emulsify a colony pick in 0.1 ml of contrast stain (50-50 mix of Gram's crystal violet to saline or buffer). After 3-5 min, prepare a wet mount and view under a 1000X oil-immersion light microscope. Compare with a positive control culture. Campylobacter cells are curved, 1.5-5 µm long, usually in chains resembling zigzag shapes (any length). Cells picked from agar often demonstrate only "wiggly" motility, whereas those from broth swim rapidly in corkscrew motion. About 10% of strains are nonmotile. Older or stressed cells have decreased motility and may show coccoid forms. Wear gloves or wash hands and disinfect microscope stage and lens after completing wet mounts. An infective dose can be acquired from cell suspensions that leak from slides.

If organisms appear typical, restreak to Abeyta-Hunt-Bark agar without antibiotics, two colonies/sub. Confirm only one plate/sub. Choose the plate with the least background growth. Refrigerate isolation agar plates microaerobically in case repicking is necessary. Incubate restreaked picks at 42ºC, 24-48 h, microaerobically (37ºC for C. fetus). Continue to restreak as necessary to obtain a pure culture. One or two plates can be incubated using the pouch-bag or pouch-jar systems.

3. Confirmation

Perform catalase and oxidase tests from growth on restreaked blood plate. Place loopful of growth from slant in a drop of 3% H₂O₂. Bubbles indicate positive catalase test. Rub loopful of growth on filter dampened with oxidase reagent. If reagent turns purple, it is oxidase-positive. Colonies grown on charcoal agar plates can give a false-negative reaction. All Campylobacter spp. are oxidase-positive. Perform Meritec Campylobacter kit test. This kit reacts with C. jejuni/coli/lari (not C. fetus) but will not differentiate among them. C. lari gives the slowest or a negative reaction. If colony pick is presumptively positive (even if Meritek kit is negative), proceed to biochemical tests for confirmation and species identification.

4. Biochemical tests (Table 11-3)

Table 11-3. Biochemical tests.

Characteristic	C. jejuni	C. jejuni subsp. doylei	C. coli	C. lari	C. fetus subsp. fetus	C. hyointestinalis	"C. upsaliensis"b
Growth at 25ºC (77ºF)	-a	"	-	-	+	D	-0
Growth at 35-37ºC (95-98.6ºF)	+	+	+	+	+	+	+
Growth at 42ºC (107.6ºF)	+	"	+	+	D	+	+
Nitrate reduction	+	-	+	+	+	+	+
3.5% NaCl	-	-	-	-	-	-	-
H2S, lead acetate strip	+	+	+	+	+	+	+
H2S, TSI	-	-	D	-	-	+c	-
Catalase	+	+	+	+	+	+	-
Oxidase	+	+	+	+	+	+	+
MacConkey's agar	+	+	+	+	+	+	-
Motility (wet mount)	+(81%)	+	+	+	+	+	+
Growth in 1% glycine	+	+	+	+	+	+	+
Glucose utilization	-	-	-	-	-	-	-
Hippurate hydrolysis	+	+	-	-	-	-	-
Resistance to nalidixic acid	Sd	S	S	R	R	R	S
Resistance to cephalothin	R	R	R	R	Se	S	S

^aSymbols: +, 90% or more of strains are positive; -, 90% or more of strains are negative; D, 11-89% of strains are positive; R, resistant; S, susceptible.
^bProposed species name.
^cSmall amount of H₂S on fresh (<3 d) TSI slants.
^dNalidixic acid-resistant C. jejuni have been reported.
^eCephalothin-resistant C. fetus subsp. fetus strains have been reported.

NOTE: C. hyointestinalis requires H₂ for vigorous growth and grows very poorly in O₂, CO₂, N₂ gas mixture. Use Campy Pak gas generating envelopes without catalyst for all incubations if analyzing samples for this species. "C. upsaliensis" does not grow in the FDA medium because of its sensitivity to antibiotics. Please call for more information.

Chart information adapted from T.J. Barret, C.M. Patton, and G.K. Morris (1988). Lab. Med. 19:96-102.

All tests should include the following controls: C. jejuni (for hippurate and other tests) and C. lari (for antibiotic resistance and hippurate). If testing for C. fetus, also include C. fetus as a positive control.

Gram stain. Use 0.5% carbol fuchsin as counterstain. Campylobacter spp. are Gram negative.

Hippurate hydrolysis. Emulsify generous 2 mm loopful of growth from the restreaked pick on the nonselective or antibiotic inhibition plate to 0.4 ml 1% hippurate solution in 13 x 100 mm tube. Incubate 2 h in 37ºC water bath. Add 0.2 ml ninhydrin reagent (R47), agitate, and reincubate 10 min. Violet (not medium or pale purple) color is positive reaction. Only C. jejuni is hippurate-positive. Refrigerate hippurate solution up to 1 month and ninhydrin solution up to 3 months.

TSI reaction. Generously inoculate slant and stab butt of TSI (M149) slant from blood plate. Incubate under microaerobic atmosphere at 35-37ºC for 5 d. Eighty % of C. coli and a few C. lari produce H S at stab; C. jejuni does not produce H S. All22 Campylobacter spp. produce alkaline/alkaline reactions. Prepare slants no more than 7 d before use.

Glucose utilization test. Stab 2 tubes of O-F media (M116), 3 times in each tube from blood plate. One tube contains glucose and one contains base alone. Incubate 4 d under microaerobic atmosphere at 35-37ºC. Campylobacter spp. do not utilize glucose or other sugars and show no change in either tube.

Meritec-Campy test kit. Follow manufacturer's instructions to test colony from isolation agar plate (presumptive identification only), antibiotic sensitivity plate, or HIA slant. The kit is not a substitute for biochemical identification and is not a serotyping kit. If the kit does not produce a positive test, the culture might be another species of Campylobacter if other tests indicate Campylobacter.

Tests using diluted inoculum. Emulsify growth from colony into 5 ml 0.1% peptone and adjust turbidity to McFarland No. 1. Use this suspension to inoculate the following tests.

1. Antibiotic inhibition. Swab an Abeyta-Hunt-Bark agar plate without antibiotics with the suspension and drop nalidixic acid and cephalothin disks onto opposite sides of plate. Incubate microaerobically, 24-48 h, 37ºC. Any size zone indicates sensitivity.
2. Growth temperature tolerance. Using loopful of diluted culture, streak a line across each of 3 plates of Abeyta-Hunt-Bark agar. Inoculate up to 4 cultures or lines per plate. Incubate one plate at 25ºC, one at 35-37ºC, and one at 42ºC under microaerophilic atmosphere for 3 d. More growth than the initial inoculum is a positive test.
3. Growth on MacConkey agar (M91). This alternative test is not necessary to identify C. jejuni, C. coli, or C. lari, but is useful to identify other species. Streak loopful from diluted culture across MacConkey agar plate, 4 cultures per plate. Incubate under microaerophilic atmosphere, at 37ºC for 3 d. Record positive or negative growth. Agar plates should be not more than 3 d old.
4. Growth in modified semisolid media (G-5). Inoculate surfaces of the following biochemicals with 0.1 ml diluted culture. Incubate microaerobically all semisolid media at 35-37ºC for 3 d, except nitrate media, which are incubated 5 d. Growth will be in a narrow band pattern just under the surface.

1% glycine. Record ± growth.

3.5% NaCl. Record ± growth.

H₂S from cysteine. Inoculate cysteine medium and hang a lead acetate strip from top, keeping cap loose. Do not let strip touch medium. Blackening of strip, even slightly, is positive reaction.

Nitrate reduction. After 5 d, add nitrate reagents A and B (R48). Red color is positive reaction.

Send identified cultures to Jan Hunt or Carlos Abeyta, at the address in the chapter introduction.

6. Bubbler apparatus assembly (Two systems available)
1. Concentrator/evaporator apparatus

Insert and twist the luer stopcocks into the outlet valves. Connect the intake valves to the Y-connector with two 3/16"-diameter aquarium tubing pieces; then connect the Y-connector to the gas tank (Figure 11-5) with a longer length of tubing. Determine the length of aquarium tubing pieces needed to reach from the unit's outlet valves to the enrichment bags or flasks. Cut one piece/valve and attach to each outlet port. Fasten the unit to a board or rack. Bubbler flow is determined by screws on the intake valves. Close stopcocks on unused outlet valves. More concentrator/ evaporator units can be added by splitting the gas line to the tank with connectors. It can also be mounted in a 42°C air incubator with a shaking platform placed inside and the gas line connected through the wall of the incubator. A second line should go to a bubbler in a 37°C incubator.

2. Fine metering S-series Nupro valve/Swage-lok apparatus

Determine how the valves should be arranged and connect with appropriate lengths of teflon tubing. Leave one outlet port/valve. Determine the length of the teflon tubing pieces needed to reach from the valves (when mounted) to the enrichment containers. Cut one piece/valve and attach to each outlet port. Place 2" long pieces of 3/16" aquarium tubing over the end of each outlet valve tubing piece. This enables insertion of 1 ml pipets. Mount the assembled bubbler unit to a board placed at the back of the water bath or on a rack suspended over the bath. It can also be mounted in a 42°C air incubator with a shaking platform placed inside and the gas line connected through the wall of the incubator. A second line should go to a bubbler in a 37°C incubator.

7. Media
1. Campylobacter enrichment broth (Bolton formula), Oxoid AM-7526 (manufactured by Med-Ox Chemicals Ltd.) or Malthus Diagnostics Lab-135. (M28a)
1. Enrichment Broth Base

Meat Peptone	10 g
Lactalbumin Hydrolysate	5 g
Yeast Extract	5 g
NaCl	5 g
Haemin	0.01 g
Sodium Pyruvate	0.5 g
a -Ketoglutamic Acid	1 g
Sodium Metabisulphite	0.5 g
Sodium Carbonate	0.6 g
Distilled Water	1000 ml

Final pH, 7.4 ± 0.2.

Thoroughly mix 27.61 g in 1 L water and let soak 10 min swirl again and autoclave 15 min at 121ºC (in screw-capped bottles if possible). Before use, add 50 ml lysed horse blood and 2 rehydrated [5 ml per vial 50:50 sterile filtered H₂0-Ethanol solution] vials of Campylobacter enrichment broth (Bolton formula) supplement (Oxoid NDX131 or Malthus Diagnostics X-131). If supplement is not available add 4 ml each of antibiotic concentrates (formulas below, solutions made separately).

Store powdered media in a tightly fastened container in a cool, dry area to reduce oxygen infusion and peroxide formation, which can inhibit recovery of microaerophiles. Use prepared broth within 1 month of preparation (preferably less than 2 weeks).

2. Lysed Horse Blood

Use fresh blood and freeze to lyse upon receipt. To freeze, resuspend blood cells gently and pour ~40 ml portions into sterile 50 ml disposable centrifuge tubes. Freeze at -20ºC. Thaw and refreeze once more to complete lysis. Store blood up to 6 months. Unused portions can be refrozen several times.

3. Campylobacter Enrichment Broth Supplements (Prepare each solution separately)
1. Sodium cefoperazone. Weigh 0.5 g into 100 ml distilled water in volumetric flask and dissolve. Only prepare amount required and filter-sterilize, 0.22 µm, using a syringe filter if # 25 ml. Store solution 5 d at 4ºC, 14 d at -20ºC, and 5 months at -70ºC. Freeze in sterile plastic tubes or bottles. Powder may be purchased from Sigma or obtained free from Pfizer by writing Roering Division, Pfizer, 235 E. 42nd St., New York, NY 10017. Request 2-4 g for in vitro use. Final concentration in the media is 20 mg/liter.
2. I. Trimethoprim lactate (Sigma Cat. No. T0667). Dissolve 0.66 g in 100 ml distilled water, and filter. May be stored 1 year at 4ºC. Add 4 ml/liter to yield a final concentration of 20 mg/liter Trimethoprim. or

II. Trimethoprim (Sigma Cat. No. T7883)[a lower cost alternative]. To prepare Trimethoprim (TMP)-HCl: add 0.5 g TMP to 30 ml 0.05N HCl at 50°C (stir until dissolved using hot plate with magnetic stirrer). Adjust volume to 100 ml with distilled water. Add 4 ml/liter to yield a final concentration of 20 mg/liter Trimethoprim.

3. Vancomycin (Sigma). Dissolve 0.5 g in 100 ml distilled water and filter. Store up to 2 months at 4ºC. Because of short shelf life, prepare smaller amounts. Add 4 ml/liter for a final concentration of 20 mg/liter.
4. Cycloheximide. Dissolve 1.25 g in 20-30 ml ethanol in a 100 ml volumetric flask and bring to line with water. Filter-sterilize. Store at 4ºC up to 1 year. Add 4 ml for final a concentration of 50 mg/liter.
2. Isolation agars
1. Abeyta-Hunt-Bark Agar (M29a)

Heart infusion agar (Difco)	40 g
Yeast extract	2 g
Distilled water	1 L

Autoclave 15 min at 121ºC. Final pH, 7.4 " 0.2. Cool and add sodium cefoperazone (6.4 ml if using broth preparation or 4 ml of the agar preparation [below]), 4 ml rifampicin, 4 ml amphotericin B, and 4 ml FBP.

After pouring plates, dry plates overnight on bench. If plates must be used the same day, place them in 42ºC incubator for several h. Do not dry in a hood with lids open. Even very brief surface drying will inhibit Campylobacter growth.

1. Sodium cefoperazone. Prepare as described for broth for final concentration of 32 mg/liter, adding 6.4 ml to agar. OR dissolve 0.8 g in 100 ml water in a 100 ml volumetric, filter and add 4 ml to agar.
2. Rifampicin. Dissolve 0.25 g slowly into 60-80 ml alcohol in a 100 volumetric, swirling repeatedly. When powder is dissolved completely, bring to the line with distilled water. Store up to 1 year at -20ºC. Final concentration is 10 mg/liter. Add 4 ml per liter.
3. Amphotericin B. Dissolve 0.05 g in water in a 100 ml volumetric flask and bring to the line. Filter sterilize and store at -20 C for 1 year. Final concentration is 2o mg/liter. Add 4 ml per liter.
4. FBP. Dissolve 6.25 g Sodium pyruvate in 10-20 ml distilled water. Pour into a 100 ml volumetric. Add 6.25 g Ferrous sulfate and 6.25 g Sodium metabisulfite. Bring to the line with distilled water and filter sterilize. Use 4 ml/liter agar. FBP is light sensitive and absorbs oxygen rapidly. Only prepare the amount needed. 10-25 ml amounts can be filtered with a 0.22 u syringe filter. Freeze unused portions in 5 ml amounts at -70ºC as soon as possible after preparation. It can be stored at -70ºC for 3 months or -20ºC for 1 month.
5. Modified Campylobacter Blood-Free Selective Agar Base (CCDA) (M30a)

CCDA agar base (OXOID)	45.5 g
Yeast extract	2 g
Distilled water	1 L

Autoclave 15 min at 121ºC. Final pH, 7.4 " 0.2. Cool and add of sodium cefoperazone (6.4 ml of strength used for broth or 4 ml of the solution for A-H agar), 4 ml rifampicin, and 4 ml amphotericin B. See A-H-B directions for precautions when drying plates.

3. Freezing medium. (M30b) Bolton broth base (9.5 ml), 1 ml fetal bovine serum (filtered, 0.22 µm) and 1 ml glycerol (10%). Mix well before use.
4. Semi-Solid Medium, modified, for Culture Storage (M30c)

Campylobacter enrichment broth (Bolton)(Oxoid AM-7526)	27.6 g
Agar	1.8 g
Sodium citrate	0.1 g
Distilled water	1 L

Mix ingredients, pH to 7.4 " 0.2, boil and dispense 10 ml per 16 X 125 screw-cap tube. Autoclave 121ºC, 15 min. Keep tubes tightly capped during storage. Do not add horse blood or antibiotics.

5. Semisolid Medium, modified, for Biochemical Identification

Campylobacter Enrichment Broth (Bolton)(Oxoid AM-7526) without blood or antibiotics	27.6 g
Agar	1.8 g
Distilled water	1 L
Neutral red solution (see below)	10 ml

Biochemicals (see below)

Mix ingredients, except the neutral red solution, boil and separate batch into 4 portions of 250 ml. Add 2.5 ml neutral red to each of 3 portions and omit neutral red from the 4th portion. Add one biochemical (sections b-e below) to one of each 250 ml portion. Final pH of each portion, 7.4 "0.2. Dispense 10 ml per 16 x 125 mm screw-cap tube. Autoclave 121ºC, 15 min.

1. Neutral red solution, 0.2%. Dissolve 0.2 g neutral red in 10 ml EtOH in a 100 ml volumetric and bring to line with distilled water.
2. Potassium nitrate, 1%. Add 10 g/L or 2.5 g/250 ml semi-solid mixture without neutral red.
3. Glycine, 1%. Add 10 g/liter or 2.5 g/250 ml semi-solid mixture with neutral red.
4. NaCl, 3.5%. Add 30 g/liter or 7.5 g/250 ml semi-solid mixture with neutral red.
5. Cysteine-HCl, 0.02%. Add 0.2 g/liter or 0.05 g/250 ml semi-solid mixture with neutral red.

Contents

Commercial Test Products

Table 11-4. Commercial test products for Campylobacter spp.

Test	Analytical Technique	Approx. Total Test Time1	Supplier
AccuPROBE®  Campylobacter Culture Identification Test	Nucleic acid hybridization	16-24 h	Gen-Probe  Contact: Vivian Jonas 10210 Genetic Center Dr.  San Diego, CA  92121  Phone: 619/410-8828 E-mail: vivianj@gen-probe.com Web: www.gen-probe.com
API Campy	Biochemical reactions	24 h	bioMérieux Inc. Contact:  bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO 63042  Phone: 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com
EIAFoss Campylobacter	Combination ELISA and Immuno Magnetic Separation	48 h	Foss North America, Inc. 7682 Executive Dr. Eden Prairie, MN 55344 Phone: 612/974-9892 E-mail: sales@fossnorthamerica.com Web: www.fossnorthamerica.com
GENE-TRAK Campylobacter Assay	Nucleic acid hybridization	50 h	GENE-TRAK Systems  Contact: Linda Dragone  94 South St.  Hopkinton, MA  01748  Phone: 508/435-7400
Probelia PCR System  [Used to identify C. jejuni and C. coli]	Polymerase chain reaction	24 h	BioControl Systems, Inc.  Contact: Robin Forgey  12822 SE 32nd St.  Bellevue, WA  98005  Phone: 425/603-1123  E-mail: info@rapidmethods.com  Web: www.rapidmethods.com
Vidas CAM	Enzyme linked fluorescent assay	48 h	bioMérieux Inc. Contact:  bioMérieux Industry 595 Anglum Rd.  Hazelwood, MO 63042  Phone: 314/731-8500  E-mail: usa@na.biomerieux.com Web: www.biomerieux.com

¹Includes enrichment

Contents

References

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