SOP: Isolation of Thermophilic Campylobacter from Food

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method isolates the thermotolerant and microaerophilic bacteria of the genus Campylobacter from raw poultry products, raw meat unprocessed, dairy products (with the exception of cheeses), molluscan shellfish, and environmental samples (stainless steel) to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. This revised method replaces MFLP-46, dated March 2002.

Although the method is only approved for the food products listed above, it is assumed it could be used with other foods. For commodities outside the application, it is imperative that the commodities be properly validated following the criteria in the Compendium of Analytical Methods (refer to Development of Methods in Volume 1 of the Compendium of Analytical Methods) so that the method is fit for purpose.

This method determines the presence of viable thermophilic Campylobacter species in food products. The procedure consists of four stages. A portion of the product is selectively enriched under microaerophilic conditions, where the initial handling of the food at the enrichment stage varies according to the type of food under investigation. The enrichment broth is then plated onto the specified selective agar medium and incubated under specific conditions of time, temperature and atmosphere. Under these conditions, viable Campylobacter cells multiply and form visible colonies which can be identified. Presumptive positive colonies are purified and confirmed by using phase-contrast microscopy, biochemical tests, rapid identification kits or molecular based methods (e.g., PCR, DNA hybridization etc.).

Apparatus

• Stomacher, blender or equivalent
• Refrigerated centrifuge capable of 16,000 × g (for milk products)
• Gas mixture (10% CO₂, 5% O₂, 85% N₂), CampyPak 2 envelope (BBL), Gas Generating Kit for Campylobacter (Oxoid), Anaerocult C (Merck), BD GasPak EZ campy container system, or equivalent
• CO₂ indicator kit
• Incubator capable of maintaining temperatures of 37 °C and 42 °C
• Anaerobic jars (optional)
• Shaking incubator (37 °C and 42 °C) (optional)
• Tri-gas incubator, or CO₂ incubator (37 °C and 42 °C)
• Phase contrast microscope
• Glass slides and coverslips
• Cryotubes
• Ultra-low temperature freezer (−65 °C to −80 °C)
• Liquid nitrogen (optional)
• Orbital shaker (depending on the identification method selected)

Reagents

Media and reagents listed below are commercially available and are to be prepared and sterilized according to the manufacturer’s instructions. The formulations of the individual media are given in Appendix G of the Compendium and in the Atlas Handbook of Microbiological Media (see References).

• 0.1% peptone water or buffered peptone water (optional)
• Park and Sanders Enrichment Broth [Supplement A (10 mg/L vancomycin, 10 mg/L trimethoprim lactate), B (32 mg/L sodium cefoperazone and, optionally, 100 mg/L cycloheximide) and lysed horse blood (50 mL/L)] — the broth is prepared immediately prior to testing by adding the lysed horse blood and both supplements to the Brucella broth
• Campylobacter Agar with Charcoal and Desoxycholate plus supplement (CCDA) (Modified CCDA-Preston Oxoid)
• Preston Agar (also known as Campylobacter selective Agar Base (Preston))
• Brucella Broth
• Brucella Semi-solid Medium
• Mueller Hinton Agar (MH)
• Mueller Hinton Agar with 5% Blood (MHB)
• Wilkins Chalgren Anaerobe Blood Agar (transport medium; the transport procedure below prints “Wilkins Chalgren Anaerobic Blood Agar”)
• Positive controls: C. jejuni, C. coli and C. lari

Optional reagents (depending on the identification method selected)

• Identification test kits (API and/or VITEK 2)
• SIM media
• Cysteine medium
• Lead acetate strips
• Each of the following biochemicals prepared in a base of Brucella Semi-solid Media: 1% glucose, 1% nitrite, 1% glycine and 3.5% sodium chloride
• Nitrate reagents [N(1-naphthyl)-ethylene diamine-2 HCl and Sulfanilic Acid]
• Oxidase test strips or Tetramethylparaphenylenediamine-2 HCl reagent
• Nalidixic acid (30 µg) disks, Cephalothin (30 µg) disks (Difco) or E-test strips (Oxoid)
• Sodium hippurate solution (+ ninhydrin solution)
• Indoxyl acetate disks (available commercially: Remel or equivalent)
• Dimethyl sulfoxide
• Hydrogen peroxide

Media formulations

• 0.1% Peptone Water
• To prepare: dissolve 0.1 g of peptone in distilled water and make up to 100 mL
• Buffered Peptone Water (BPW)
• Basal Medium
• Peptone — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Disodium phosphate — 3.5 g
• Potassium dihydrogen phosphate — 1.5 g
• Distilled water — 1.0 L
• Final pH 7.2 ± 0.2
• Complete Medium: dissolve the ingredients in 1.0 L of distilled water; dispense into appropriate glassware; autoclave at 121°C for 15 min
• Campylobacter Agar with Charcoal and Deoxycholate (CCDA; also known as Modified CCDA Preston)
• Basal Medium (per litre)
• Meat extract — 10.0 g
• Peptone — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Bacteriological charcoal — 4.0 g
• Casein hydrolysate — 3.0 g
• Sodium deoxycholate — 1.0 g
• Ferrous sulphate (FeSO₄) — 0.25 g
• Sodium pyruvate — 0.25 g
• Agar — 12.0 g
• Distilled water — 1.0 L
• pH 7.5 ± 0.2
• Supplements
• Sodium cefoperazone — 0.032 g
• Amphotericin B — 0.010 (unit not stated in the source)
• Distilled water — 10.0 mL
• Complete Medium: dissolve all basal components in 1.0 L distilled water; boil to dissolve completely; autoclave at 121 °C for 15 min; cool to 50 °C; dissolve the supplement components in distilled water, mix thoroughly, filter sterilise through a 0.2 µm filter, and add aseptically to the medium; mix well and pour into sterile Petri plates
• Preston Agar (Campylobacter Selective Medium)
• Basal Medium
• Beef extract — 10.0 g
• Peptone — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Agar — 12.0 g
• Distilled water — 940 mL
• Final pH: 7.5 ± 0.2
• Antibiotics Supplement
• Polymyxin B — 5000.0 IU
• Rifampicin — 0.01 g
• Trimethoprim lactate — 0.01 g
• Cycloheximide — 0.1 g
• Acetone:distilled water (50:50) — 10 mL
• Supplement
• Lysed horse blood — 50.0 mL
• Complete Medium: suspend the basal medium ingredients in 940 mL of distilled water; mix thoroughly and heat to boiling to dissolve completely; autoclave at 121 °C for 15 min; cool to 45–50 °C; aseptically add 50.0 mL of lysed horse blood and 10.0 mL of the antibiotic supplement; mix thoroughly and dispense into sterile Petri dishes
• Antibiotic supplement: add the antibiotic components to 10.0 mL of 50:50 acetone:distilled water and filter sterilize
• Brucella Broth
• Basal Medium
• Tryptone — 10.0 g
• Peptic digest of animal tissues — 10.0 g
• Dextrose — 1.0 g
• Yeast extract — 2.0 g
• Sodium chloride (NaCl) — 5.0 g
• Sodium bisulfite (NaHSO₃) — 0.1 g
• Distilled water — 1.0 L
• Final pH 7.0 ± 0.2
• Complete Medium: suspend the ingredients in 1.0 L of distilled water; boil to dissolve completely; autoclave at 121°C for 15 min; dispense
• Brucella Semi-solid Medium
• Basal Medium
• Tryptone — 10.0 g
• Peptic digest of animal tissues — 10.0 g
• Dextrose — 1.0 g
• Yeast extract — 2.0 g
• Sodium chloride (NaCl) — 5.0 g
• Sodium bisulfite (NaHSO₃) — 0.1 g
• Neutral red — 0.02 g
• Agar — 1.6 g
• Distilled water — 1.0 L
• Final pH 7.0 ± 0.2
• Complete Medium: mix the ingredients in distilled water; heat with frequent agitation and boil for about 1 min; dispense 5.0–7.0 mL into screw-cap tubes (16 × 125 mm); autoclave at 121°C for 15 min
• Mueller-Hinton Agar (MH)
• Basal Medium
• Beef, infusion from — 300 g
• Casamino acids, technical — 17.5 g
• Soluble starch — 1.5 g
• Agar — 17.0 g
• Distilled water — 1.0 L
• pH 7.3 ± 0.2
• Complete Medium: suspend the ingredients in distilled water; heat to boiling to dissolve completely; autoclave at 121 °C for 15 min; cool and dispense
• Note: when used for halophilic Vibrio spp., add NaCl to a final concentration of 2–3%
• Mueller-Hinton Agar with 5% Blood (MHB)
• To prepare: prepare as for Mueller-Hinton Agar above; after autoclaving and cooling, aseptically add blood to a final concentration of 5% (v/v); mix and pour into plates
• Wilkins Chalgren Anaerobe Blood Agar
• Basal Medium
• Pancreatic digest of casein — 10.0 g
• Gelatin peptone — 10.0 g
• Sodium chloride (NaCl) — 5.0 g
• Yeast extract — 5.0 g
• Glucose — 1.0 g
• L-Arginine — 1.0 g
• Sodium pyruvate (C₃H₃O₃Na) — 1.0 g
• Hemin — 5.0 mg
• Menadione (vitamin K) — 0.5 mg
• Agar — 10.0 g
• Tween 80 — 1.0 mL
• Distilled water — 950 mL
• pH: 7.1 ± 0.2
• Supplement
• Lysed horse blood — 50.0 mL
• Complete Medium: suspend the basal medium ingredients in distilled water and bring the volume to 950 mL; mix thoroughly; gently heat to boiling to dissolve the agar; autoclave at 121 °C for 15 min; cool to 50–55 °C; aseptically add 50.0 mL of lysed horse blood; mix thoroughly and dispense into sterile Petri dishes
• Biochemical test media in a base of Brucella Semi-solid Medium
• To prepare: add glucose, nitrite, glycine and sodium chloride to Brucella Semi-solid Medium to final concentrations of 1%, 1%, 1% and 3.5% (w/v) respectively

Sample preparation

Collection of samples

• Raw milk samples: supplement with (a) 5% cysteine or (b) 0.01% sodium bisulfite and either 0.15% sodium thioglycolate or an atmosphere of 100% nitrogen. During transportation and storage, sample units of frozen products shall be kept frozen.
• Environmental samples: follow the procedures in MFLP-41 (Environmental Sampling for the Detection of Microorganisms) for obtaining and transporting samples. Environmental samples should be placed into appropriate transport media to ensure survival during transportation to testing facilities.

Handling of samples

• In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen, depending on the nature of the product; thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death
• Analyse samples as soon as possible after receipt in the laboratory, because Campylobacter is extremely sensitive to oxygen, particularly at room temperature; Campylobacter is also sensitive to freezing, thawing and drying conditions

Preparation of plates

• After preparing plates, leave them overnight at room temperature to ensure the plates are adequately dried; alternatively, plates can be dried in a laminar flow hood for a short period of time just prior to plating — excess moisture on agar plates will result in spreading colonies

Procedure

Analyse each sample unit individually. The test shall be carried out in accordance with the following instructions.

Enrichment

Include pure cultures as positive and negative controls and an uninoculated broth as a sterility (blank) control, respectively
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Optionally, primary enrichment may be performed with agitation: shake the contents of the flask or bag through all steps at 100–120 cycles/min using a shaking incubator; incubation duration and microaerobic conditions remain unchanged
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For the incubation times stated, the following flexibility guidelines apply: incubation times of 24 h are ± 2 h; incubation times of 48 h are ± 4 h
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Fresh raw meat and poultry samples: cut sample into pieces of 0.3–0.5 cm³ and add 25 g into a 250 mL flask or bag containing 100 mL of Park and Sanders Enrichment Broth (alternatively, the rinse method below can be used); incubate under microaerobic conditions at 37 °C for 3–4 h, then continue incubation at 42 °C under microaerobic atmosphere for a total of 24 and 48 h
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Rinse samples (raw whole chicken carcasses, meat pieces, boned meat pieces, or samples that cannot easily be reduced to 25 g): place the sample (up to 1.5 kg) into a large sterile plastic bag and add 200 mL of 0.1% cold (4 °C) peptone water (or buffered peptone water); shake the contents gently and manually massage for approx. 2 min; inoculate 30 mL of rinse into 30 mL of double strength Park and Sanders Enrichment Broth and follow the same incubation and plating
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Dairy products (with the exception of cheeses): pour 100 mL or g of milk into a centrifuge bottle; verify pH and adjust if necessary to 7.5 ± 0.2; centrifuge at 16,000 × g for 20 min at 4 °C; remove the fat layer with a sterile spatula and discard the fat and liquid; suspend the pellet in 100 mL Park and Sanders Enrichment Broth and follow the same incubation and plating
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Molluscan shellfish: weigh a 25 g sample into a blender jar or stomacher bag containing 100 mL of Park and Sanders Enrichment Broth, and blend or stomach at low speed for 30 sec; follow the same incubation and plating
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Frozen foods: thaw frozen samples at 4 °C, or under time and temperature conditions which prevent microbial growth or death; after thawing, cut sample into pieces of 0.3–0.5 cm³ and add 25 g into a 250 mL flask or bag containing 100 mL of Park and Sanders Broth with lysed horse blood and Supplement A; incubate under microaerobic conditions at 37 °C for 3–4 h; add Supplement B to the enrichment broth and continue incubation at 42 °C under microaerobic atmosphere for a total of 24 and 48 h; optionally, shake the contents of the flask or bag through all steps at 100–120 cycles/min using a shaking incubator; then plate as below
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Environmental surface samples: transfer each swab into a 250 mL flask or bag containing 100 mL of Park and Sanders Enrichment Broth and follow the same incubation and plating

Note: If the samples have been stressed, particularly by freezing, incubate the inoculated Park and Sanders media (Brucella broth + blood + Supplement A) for 3–4 h before adding Supplement B. When analyzing acidic foods, adjust broth pH to 7.4 with 2 N NaOH after the rinsing step.

Plating

After 24 ± 2 h and 48 ± 4 h incubation, streak two 10 µL loopfuls per plate of the culture onto CCDA agar and Preston agar
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For fresh poultry, a single agar type (CCDA or Preston) is sufficient; novel chromogenic and other isolation agars may be used, but in conjunction with the plating media above
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Incubate the agar plates at 37 ± 1 °C for 24 to 72 h under microaerobic conditions

Procedure for the microaerobic atmosphere

Thermophilic Campylobacter are strict microaerophiles, and this requirement must be taken into account in the enrichment and plating procedures. Use CO₂ indicators in the jar and/or in the incubators. Optimal atmospheric conditions can be achieved by one of the following procedures, depending upon availability of equipment.

• Replacement of air with a gas mixture of 5% O₂, 10% CO₂, and 85% N₂, or use of gas generator ion envelope kits according to the manufacturer’s instructions for the number of envelopes per volume of jar:
• Place flasks or stomacher bags (up to five according to the anaerobic jar size — do not overfill the jar) containing enrichment broth or agar plates into an anaerobic jar or an air-tight cabinet without catalyst; evacuate the air from the jar or cabinet up to about 610–635 mm or 24–25 inches Hg vacuum and refill the jar with the gas mixture; repeat the entire procedure at least once; jars or cabinets can subsequently be placed in a shaking incubator, in order to allow for maximal gas exchange (optional); if anaerobic jars with gas generator envelopes are being used, follow the manufacturer’s instructions for use
• Use of tri-gas or CO₂ incubators:
• Enrichment broths and plates can be incubated in a tri-gas incubator with a gas mixture consisting of 5% O₂, 10% CO₂, and 85% N₂; plates can be incubated in a CO₂ incubator with a CO₂ level of 10%

Identification

For maintaining cultures or for the basal medium for biochemical tests, use Brucella Semi-solid medium (Brucella Broth supplemented with 0.15 to 0.2% agar and 0.002% neutral red in tubes. Tubes of this semi-solid medium can be incubated aerobically. Do not tighten the screw caps of the tubes during aerobic incubation. Since Campylobacter grow near the surface of the medium (10 mm zone), inoculate the culture into this zone.

Note: Use a fresh culture for biochemical and identification tests. All three controls strains should be used when performing biochemical tests.

From each selective agar medium, select 3 suspect colonies (on Preston/CCDA: smooth, convex, translucent, colorless to greyish-cream-colored and pin-point to 2–4 mm in diameter, or often spreading colonies; on CCDA colonies may develop a characteristic metallic sheen)
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Streak the same suspect colonies onto CCDA and MHB plates; incubate plates at 37 °C microaerobically for 1–2 days and check for purity
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Examine a wet mount preparation of each colony using a phase contrast microscope: young cells of Campylobacter are small (0.2–0.8 μm wide by 1.5–5 μm long) and S-shaped, and possess a characteristic corkscrew-like motility; old cells grown for more than 72 h or cells exposed to air often change to coccoid forms (plate, cell morphology and motility patterns are critical in the identification of the organism)
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Hippurate hydrolysis test: inoculate a loopful of a 24 h culture from MHB into 0.5 mL of sterile sodium hippurate solution in a small capped tube; incubate tubes, including positive (C. jejuni), negative (C. coli) and uninoculated controls, aerobically at 37 °C for a minimum of 3 h; overlay 0.2 mL of ninhydrin solution (do not mix); development of a deep purple color within 5 min indicates a positive result — only tubes that do not develop any coloration should be considered negative; development of a slight bluish color should be further investigated using an alternate test
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Continue with the Gram stain, catalase test, oxidase test, and nalidixic acid / cephalothin resistance test (these may be done sequentially or concurrently; refer to Table 1 for typical biochemical reactions; proceed only with typical isolates)
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Gram stain: standard methods are used; for Campylobacter species, carbol-fuchsin is more effective than safranin as a counter stain — if safranin is used, the exposure time may be extended to 3 min or longer
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Catalase test: mix a portion of a colony from the recently incubated CCDA plate with a drop of 3% hydrogen peroxide (H₂O₂) on a clean glass slide, or drop H₂O₂ directly onto the culture, provided all other tests have been done first; development of bubbles is indicative of a positive reaction
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Oxidase test: smear a portion of a colony from the recently incubated CCDA plate onto an oxidase strip; the oxidase test is positive if the cell mass turns dark purple in 5–10 seconds
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Nalidixic acid / cephalothin resistance / sensitivity: swab an MHB plate to generate a bacterial lawn; place one disc each of nalidixic acid (30 µg) and cephalothin (30 µg) on the plate; incubate the plates at 42 °C for 48 ± 2 h under microaerophilic conditions; after the incubation period, determine if there is a transparent (non-growth) zone around either disc — sensitivity to nalidixic acid and cephalothin is characterized by a zone of clearing surrounding the antibiotic disc
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Growth temperature: streak a colony from the purified cultures onto two MHB plates; incubate microaerobically, one plate at 25 °C and the other plate at 42 °C for 24 to 72 h

Note: Conventional hippurate hydrolysis can be replaced by hippurate hydrolysis included in rapid identification kits. After microscopy and the hippurate hydrolysis test, identification may be done using rapid identification systems, replacing the Gram stain, catalase, oxidase and nalidixic acid / cephalothin steps above. These include validated systems such as VITEK 2, API system, latex agglutination tests, enzyme linked fluorescent assays and molecular based methods (e.g., DNA hybridization and/or PCR).

Additional biochemical tests for identification (optional)

Using the MHB plate from the colony purification step above, prepare a fresh culture in 50 mL of Brucella broth
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Incubate at 37 °C for 24–48 h under microaerobic atmosphere; the culture is used for inoculation of the following 6 tubes for biochemical and growth tests
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For each isolate, prepare one tube each of at least 6 different Brucella Semi-solid media: glucose, nitrate, SIM, cysteine, glycine, and NaCl
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For all tubes, transfer several drops of the fresh broth culture to the surface zone (10 mm layer) of the media; do not inoculate the entire tube from top to bottom
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Place a lead acetate strip into the cysteine tube; crimp the top of the strip over the lip of the tube and hold it in place with the cap
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Incubate all tubes aerobically or microaerobically at 37 °C for 3–5 days; the screw caps should not be tightened during the incubation period
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Interpret the tubes:
– Glucose fermentation: a positive reaction gives a yellow color (acid with phenol red indicator) in the Brucella Semi-solid Glucose medium
– Nitrate reduction: add an equal volume (0.5–1.0 mL) of the 2 nitrate reagents to a semi-solid nitrate tube, and mix; development of a red color in one minute indicates a positive test for nitrite production; if no red color develops, add a pinch of zinc dust — a red color developed with zinc dust indicates a negative test, while the absence of color with zinc powder is indicative of a positive reaction which results from a complete reduction of nitrate to ammonia
– H₂S production — SIM medium: development of a black color in the medium indicates strong H₂S production; Cysteine Medium with lead acetate strip: blackening of the lead acetate strip indicates H₂S production — since the reaction in the semi-solid Brucella Cysteine Medium is more sensitive than in the SIM medium, weak H₂S producers may give a positive test with the lead acetate strip but not with the SIM medium
– Growth in 1% Glycine and 3.5% NaCl: observe the growth at the top zone (10 mm layer) in the semi-solid Brucella medium with 1% Glycine and in the tube with 3.5% NaCl; if the growth is not apparent, record as “no growth”

Note: (a) Zinc powder may become inactive with long storage. Check the activity periodically with uninoculated nitrate medium: add a small amount of zinc to the medium, mix, and add the 2 nitrate solutions — if the zinc powder is still in an active form, a red color will develop. (b) Nitrate may be chemically reduced to nitrite in pre-reduced medium without microbial growth. Check each batch of medium by adding the 2 test reagents to the uninoculated medium, and discard the medium if it gives a red color.

Additional tests for distinguishing C. lari from C. coli and C. jejuni (optional)

Test the nalidixic acid resistant strains (presumptive C. lari) using one or both of the following tests:

• Indoxyl acetate test: test pure colonies after 18–72 h growth; using forceps, place an indoxyl acetate disk on a clean glass microscope slide or in a petri dish; rehydrate the disk with approx. 20 to 40 µL of demineralised (distilled) water — do not over-saturate; using an applicator (wooden or equivalent), inoculate the disk with several test colonies (heavy inoculum); incubate the disk at room temperature for 20 min; the development of a blue, blue-green, or indigo color within a few minutes is indicative of a positive reaction — the test is considered negative if no color change is observed within the 20 min incubation; C. lari will not hydrolyze indoxyl acetate, while C. jejuni and C. coli will hydrolyze the indoxyl acetate; commercial test kits can be used following the manufacturer’s instructions
• Trimethylamine N-Oxide (TMAO) test: add a couple of drops of Brucella semi-solid medium culture to the top of a TMAO medium tube and incubate microaerobically at 37 °C for 3–7 days; observe for growth spreading below the top of the TMAO media — only C. lari will grow in the presence of TMAO; other thermophilic Campylobacter species will not grow

Identification criteria

Pure cultures of Campylobacter are Gram-negative, vibrioid shaped rods that have one curve or are spiral shaped. They are motile with a typical corkscrew-like motion. C. jejuni cells older than 72 h or cells exposed to air often change to coccoid forms. Campylobacter spp. are all oxidase positive, non-fermentative, and will also not oxidize carbohydrates.

For distinguishing C. jejuni, C. coli and C. lari from other species of Campylobacter (optional), Campylobacter can be divided into 2 groups on the basis of the catalase test:

• Catalase-negative cultures are C. sputorum (ss. sputorum, ss. bubulus, or ss. mucosalis)
• Catalase-positive Campylobacter (see Table 1) can be further divided into 2 groups on the basis of growth temperature, and sensitivity to antimicrobial compounds (nalidixic acid and cephalothin):
• The heat-tolerant catalase-positive members (C. jejuni, C. coli and C. lari) grow at 42 °C but not at 25 °C; C. jejuni and C. coli are sensitive to nalidixic acid but resistant to cephalothin, and C. lari is resistant to both antibiotics
• The non-thermophilic catalase-positive species (C. fetus ss. fetus and C. fetus ss. venerealis) grow at 25 °C but not at 42 °C, and are resistant to nalidixic acid and sensitive to cephelothin

Table 1. Key characteristics of catalase-positive Campylobacter

• ᵃ SIM medium for strong H₂S producers.
• ᵇ Base medium (Brucella semisolid medium) with cysteine for detecting weak H₂S producers. Lead acetate impregnated strips of filter paper hung over the top of the culture tube.
• ᶜ V = results are variable
• ᵈ S = sensitive, note that some C. jejuni strains are resistant
• ᵉ R = resistant
• ᶠ TMAO = trimethylamine-N-oxide

Maintenance of cultures

Semi-solid medium

Inoculate the culture (heavy inoculum) at the top (10 mm) portion of Semi-solid Brucella Medium and incubate aerobically or microaerobically at 37 °C
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Keep in the incubator at 37 °C, and transfer weekly or when most of the medium turns yellow

Culture storage in the ultra-low temperature freezer

Viability is excellent after several years of storage.
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Glycerol peptone medium: using a 24 h culture on MHB agar, prepare a heavy suspension of the bacteria in 1–2 mL of Glycerol-peptone medium; mix well and dispense 0.3–0.5 mL into cryogenic vials
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Alternatively, bacteria can be added to cryobead vials (e.g., Microbank) according to the manufacturer’s instructions

Culture storage in liquid nitrogen

The major advantage of this method is eliminating accidents due to electrical power failure.
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Prepare a heavy cell suspension in Brucella Broth using a 24–48 h culture from a MHB plate; place 0.5 mL of the suspension into a (12.5 × 43 mm) tube designed for immersion into liquid nitrogen, such as a Cryotube containing 2 drops of dimethyl sulfoxide (DMSO); mix and let stand for 5–10 min before storage
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Put the Cryotubes on metal racks and submerge the racks in liquid nitrogen
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Do not seal the lid of the tank; add liquid nitrogen regularly to fill the tank

Lyophilization

Make a heavy suspension of a 24 h culture in 1–2 mL of Serum-inositol medium and incubate microaerophilically at 42 °C for 24 h
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Distribute 0.2 mL into lyophilizing ampoules and lyophilize; store lyophilized ampoules at 4 °C

Transporting cultures by mail

Special attention should be paid when sending Campylobacter strains through the mail, because the cells grown on agar plates or agar slopes are killed within 3 days in air at room temperature. The following is a procedure which will allow for survival of Campylobacter for about 5 weeks at 4 °C or 2 weeks at room temperature (100% recovery).
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Inoculate the culture onto a MHB plate and incubate under microaerophilic conditions for 24 h at 42 °C
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Remove growth from the plate with a sterile swab, break off the handle of the swab using sterile forceps; place the swab (heavily inoculated) at the top layer of Wilkins Chalgren Anaerobic Blood Agar transport medium which has been dispensed in 6 mL quantities into sterile bottles
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Leave the swab in the bottle, tighten the cap, and incubate in air overnight at 42 °C, after which the culture is ready for shipping

Precautions

• The laboratory Supervisor must ensure that completion of the analysis described in this method is done in accordance with the International Standards reference “ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories”
• It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made from scratch)
• It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures: where 35 °C is recommended in the text of the method, the incubator may be at 35 °C ± 1.0 °C, and similarly lower temperatures of 30 °C or 25 °C may be ± 1.0 °C; however, where higher temperatures are recommended, such as 43 °C or 45.5 °C, it is imperative that the incubators or water baths be maintained within 0.5 °C due to potential lethality of the higher temperatures on the microorganism(s) being isolated

References

• Health Canada, Health Products and Food Branch. Isolation of thermophilic Campylobacter from food. Compendium of Analytical Methods, Volume 3, Laboratory Procedure MFLP-46, December 2014.
• American Association of Analytical Chemists (AOAC). 1998. Bacteriological Analytical Manual. Eighth edition. International, Gaithersburg, MD.
• American Public Health Association. 2001. Compendium of Methods for the Microbiological Examination of Foods, Fourth Edition, F. P. Downes and K. Ito (eds.). American Public Health Association, Washington, D.C.
• Atlas, R. M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.
• Bell, C. and A. Kyriakides. 2009. Campylobacter: A practical approach to the organism and its control in foods. Wiley-Blackwell. Chichester, West Sussex.