Print | Need Larger Text? | Share

Isolation of Thermophilic Campylobacter from Food

Laboratory Procedure MFLP - 46
March 2002

Help on accessing alternative formats, such as Portable Document Format (PDF), Microsoft Word and PowerPoint (PPT) files, can be obtained in the alternate format help section.

(PDF Version - 120 K)

D. Medeiros and L. Hofmann
Microbiology Research Division
Bureau of Microbial Hazards, Food Directorate
Postal locator: 2204A2
Ottawa, K1A 0L2
E-mail: Lisa_Hofmann@hc-sc.gc.ca

1. Application

This method is applicable to the determination of the thermotolerant and microaerophilic bacteria of the genus Campylobacter in foods to determine compliance with the requirements of Section 4 and 7 of the Food and Drugs Act. This revised method replaces MFLP-46, dated July 1998.

2. Principle

The procedure consists of four stages. The initial handling of the food at the enrichment stage varies according to the type of food under investigation.

2.1 Selective Enrichment

The food is initially inoculated into a selective enrichment medium designed to slow or inhibit the growth of competing microorganisms while favouring the growth of campylobacters.

2.2 Colony Formation on Selective Agars

Selectively enriched cultures are streaked onto selective agars specifically developed for the isolation and identification of campylobacters. Presumptive colonies are further identified by examining for characteristic morphology and motility using phase contrast microscopy.

2.3 Purification

The isolates are purified on an appropriate selective blood or charcoal agar plate.

2.4 Identification

Presumptive campylobacters are identified by biochemical reactions, tolerance to some chemicals and antibiotics, and by growth temperatures. Several biotypes are determined by biochemical reactions. Serotyping schemes for identifying heat-labile antigens (Lior's scheme) and heat-stable antigens (Penner's scheme) have been developed, however the antisera required are not readily available. If serotyping is required cultures may be sent to Health Canada's Laboratory Center for Disease Control in Winnipeg, Manitoba. Various nucleic acid-based and antibody-based rapid methods for the identification of Campylobacter are commercially available (9.1, 9.2, 9.4)

3. Definition

See Appendix A of Volume 3.

4. Collection of Samples

4.1 Sampling

See Appendix B of Volume 3.

4.1.1 Holding and Shipping

For holding and shipping, place the sample units in sealed containers at 4°C until analysis. With 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. Sample units of frozen products shall be kept frozen.

4.1.2 For environmental samples, follow MFLP-41.

5. Materials and Special Equipment

The following media and reagents (1-10) are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 and reference 9.1 for the formula of individual media.

1. Brucella Broth
2. Brucella Semi-solid Agar
3. Preston Agar (Also known as Campylobacter Agar Base)
4. Mueller Hinton Agar
5. Mueller Hinton Agar with Blood
6. Wilkins Chalgren Anaerobic Blood Agar
7. Wilkins Chalgren Anaerobic Broth
8. Campylobacter Agar with Charcoal and Deoxycholate plus supplement (CCDA) (Oxoid)
9. SIM media
10. Nitrate reagents [N(1-naphthyl)-ethylene diamine@2 HCl and Sulfanilic Acid]
11. 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
12. Park and Sanders Enrichment Broth
13. Rapid H₂S Test Medium (for biotyping)
14. Oxidase Test strips or Tetramethylparaphenylenediamine@2 HCl reagent
15. Gas Mixture (10% CO₂, 5% O₂, 85% N₂) or CampyPak 2 envelope (BBL) or Gas Generating Kit for campylobacters (Oxoid) or Anaerocult C (Merck)
16. Nalidixic Acid (30Fg) disks, Cephalothin (30Fg) disks or E-test strips (Oxoid)
17. Sodium Hippurate
18. Liquid Nitrogen
19. Dimethyl Sulfoxide
20. Hydrogen peroxide
21. Lead acetate strips
22. Vacuum Leak Detector (Electro-Technic Products)
23. Cryotubes
24. Positive control (ATCC or equivalent)
25. Shaking Incubator (37°C and 42°C)
26. CO₂ Incubator (37°C and 42°C) (optional)
    
    Note: It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths are maintained at the recommended temperatures. Where 35 C is recommended in the text of the method, the incubator may be 35 +/-1.0 C. Similarly, lower temperatures of 30 or 25ºC may be +/- 1.0 C. However, where higher temperatures are recommended, such as 43 or 45.5 C, it is imperative that the incubators or waterbaths be maintained within 0.5 C due to the potential lethality of higher temperatures on the microorganism being isolated.
27. Glass slides and coverslips
28. Blender, stomacher or equivalent
29. Refrigerated centrifuge capable of 16,000 X g.
30. Deep Freezer, -70°C
31. Phase Contrast Microscope

6. Procedure

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

6.1 Handling of Samples

6.1.1 See 4.1.1 and 4.1.2.

6.1.2 Analyse samples as soon as possible after receipt in the laboratory because campylobacters are extremely sensitive to oxygen, particularly at room temperature. Campylobacters are also sensitive to freezing, thawing and drying conditions.

6.2 Enrichment

6.2.1 Fresh raw meat, pork and poultry samples: Cut sample into pieces (0.3-0.5 cm³) and add 25 g into a 250 mL flask containing 100 mL of Park and Sanders Enrichment Broth. Incubate under microaerobic conditions (6.4) at 37°C for 3-4 h. Transfer flask to a 42°C incubator under microaerobic atmosphere for 24 and 48 hours. Alternately, increase the incubator temperature or use a programmable incubator which automatically adjusts temperature after 4h. Shake the contents of the flask through all steps at 100-120 cycles/min. using a shaking incubator (required for gas exchange during enrichment).

6.2.2 Milk: Pour 100 mL of milk into a centrifuge bottle. Spin at 16,000 x 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 of Park and Sanders selective enrichment broth and follow the procedures for incubation and plating described in 6.2.1 and 6.3.

6.2.3 Shellfish: Weigh a 25 g sample into a blender or 400 mL stomacher bag, and stomach or blend at low speed for 30 sec. Transfer the contents of the blender jar or stomacher bag into a 250 mL flask containing 100 mL of Park and Sanders Enrichment Broth, and follow the procedures for incubation and plating described in 6.2.1 and 6.3.

6.2.4 Frozen foods: After thawing, cut sample into pieces (0.3-0.5 cm³)and add 25 g into a 250 mL flask containing 100 mL of Park and Sanders Enrichment Broth and follow the procedures for incubation and plating described in 6.2.1 and 6.3.

6.2.5 Swab samples: Transfer each swab into a 250 mL flask containing 100 mL of Park and Sanders Enrichment Broth and follow the procedures for incubation and plating described in 6.2.1 and 6.3.

6.3 Plating

After 24 + 2 h and 48 + 2 h incubation, streak two loopfuls/plate of the culture onto CCDA agar and Preston agar. Incubate the agar plates at 37°C for up to 72h under microaerobic conditions.

6.4 Procedures for Microaerobic Atmosphere

Thermophilic campylobacters are strict microaerophiles and this requirement must be taken into account in enrichment and plating procedures. A commercial gas mixture of 5% O₂, 10% CO₂, and 85% N₂ is generally used. One of the following procedures may be used depending upon availability of the equipment and materials for generating microaerobic conditions.

6.4.1 Replacement of air with a gas mixture of 5% O₂, 10% CO₂, and 85% N₂ or use of Gas generator ion envelope kits, such as GasPak (BBL) kits (commercially available)

1. Place flasks (up to three) or stomacher bags 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.
2. If anaerobic jars with gas generator envelopes are being used follow manufacture's instructions for use.
3. Plates can be incubated in a CO₂ incubator with a CO₂ level at 10%.

6.5 Identification

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

6.5.1 From each selective agar medium, select a portion of 3 suspect colonies (smooth, convex, translucent, colorless to cream-colored and pin-point to 2-4 mm in diameter or often spread colonies).

6.5.2 Examine a wet mount preparation of each colony using a phase contrast microscope. Young cells of Campylobacter are Gram-negative, small (0.2-0.8 µm wide by 1.5-5 µm long) and S-shaped They possess a characteristic corkscrew-like motility. Old cells of C. jejuni grown for more than 72 hours or cells exposed to air often change to coccoid forms. Carbol-fuchsin is more effective than safranin as a counter stain. If safranin is used, the exposure time may be extended to 3 minutes or longer. However, Gram staining may be omitted.

6.5.3 Pick a portion of the same suspect colonies using an inoculating loop or needle and streak onto oneCCDA and Mueller Hinton with 5% blood plate, with each colony occupying 1/5 of a plate. Incubate plates at 37°C microaerobically for 1 - 2 days and check for purity.

6.5.4 Tests for identification

1. Catalase test: With an inoculating needle, mix a portion of a colony from the recently incubated CCDA plate with a drop of 3% hydrogen peroxide (H₂0₂) on a clean glass slide. (Or drop H₂0₂ directly onto the culture, provided all other tests have been done first). Development of bubbles is indicative of a positive reaction.
2. Oxidase test: With an inoculating needle, 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.
3. Nalidixic Acid / Cephalothin Resistance / Sensitivity: Streak a MHB blood agar plate as a bacterial lawn. Place one disc each of nalidixic acid (30 Fg) and cephalothin (30 ug) on the plate. Incubate the plates at 42°C for 48 h under microaerophilic conditions. After the incubation period, determine if there is a transparent (non-growth) zone around either disc. Resistance to nalidixic acid and cepthalothin is characterized by a zone of clearing of #13 and 14 mm, respectively.

6.5.5 Additional tests for identification

1. Using the MHB plate from 6.5.3, prepare a fresh culture in 50 mL of Brucella broth contained in a 250 mL flask. 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 (6.5.6b).
2. For each isolate, prepare one tube each of at least 6 different Brucella Semi-solid media (see sec. 10): glucose, nitrate, SIM, cysteine, glycine, and NaCl.
3. Inoculate all tubes with a Pasteur pipette by transferring 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.
4. 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.
5. Incubate all tubes aerobically or microaerobically at 37°C for 3-5 days. The screw caps should not be tightened during the incubation period.

6.5.5.1 Reading of biochemical tests

1. Glucose fermentation: A positive reaction gives a yellow color (acid with phenol red indicator) in the Brucella Semi-solid Glucose medium.
2. Nitrate reduction test: 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. The absence of color with zinc powder is indicative of a positive reaction which results from a complete reduction of nitrate to ammonia. [Note: (a) Zinc powder may become inactive with long storage. Check the activity periodically with uninoculated nitrate medium. Add a pinch 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 prereduced medium without microbial growth. Check each batch of medium by adding the 2 test reagents to the uninoculated medium. Discard the medium if it gives a red color].
3. H₂S tests:
   1. SIM medium: development of a black color in the medium indicates strong H₂S production.
   2. Brucella Semi-solid 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.
4. Test for growth in Semi-solid Brucella Medium with 1% Glycine and in Semi-solid Brucella Medium with 3.5% NaCl: Observe the growth at the top zone (10 mm layer) of the medium. If the growth is not distinctive, record as "no growth".

6.5.6 Additional tests for differentiating C. jejuni from C. coli and C. lari

1. Hippurate hydrolysis test:
   1. Inoculate a loopful of a 24 h culture from Mueller Hinton Blood Agar into 0.5 mL of sterile sodium hippurate solution in a small screw-capped or corked tube.
   2. Incubate tubes, including an uninoculated control, aerobically at 37°C for a minimum of 3 h.
   3. Overlay 0.2 mL of ninhydrin solution. (Do not mix)
   4. Development of a deep purple color within 5 minutes indicates a positive result; development of a slight bluish color is regarded as negative.
2. Trimethylamine N-Oxide (TMAO) test:
   
   Test the nalidixic acid resistant strains (presumptive C. lari) in TMAO medium as follows:
   1. Add a couple of drops of semi-solid medium culture to the top of a TMAO medium tube and incubate microaerobically at 37°C for 3-7 days .
   2. Observe for growth spreading below the top of the TMAO media. Only C.lari will grow in the presence of TMAO.

6.5.7 Identification criteria

Pure cultures of suspected Campylobacter are Gram-negative, vibroid shaped rods that have one curve or are "S" shaped (occasionally spiral). They are motile with a typical corkscrew-like motion. They have a single polar flagellum at one or both ends of the cell. C. jejuni cells older than 72 h, or cells exposed to air often change to spheric forms. Campylobacter sp. are all oxidase positive, non-fermentative, and will also not oxidize carbohydrates.

The following are major steps for identifying C. jejuni, C. coli and C. lari from other species of Campylobacter (see Table 1).

1. Campylobacters can be divided into 2 groups on the basis of the catalase test: Discard catalase-negative cultures as they are C. sputorum (ss. sputorum, ss. bubulus, or ss. mucosalis).
2. Catalase-positive campylobacters can be further divided into 2 groups on the basis of growth temperature, and sensitivity to antimicrobial compounds (nalidixic acid and cephalothin). (1) 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. In TMAO medium, C. lari will grow but both C. jejuni and C. coli will not. (6.5.7). (2) The nonheat-tolerant catalase-positive species (C. fetus ss. fetus and C. fetus ss. venerialis) grow at 25°C but not at 42°C, and are resistant to nalidixic acid and sensitive to cephalothin.

6.5.8 Biotyping of C. jejuni, C. coli, and C. lari

A provisional scheme is as follows:

Test	C. jejuni				C. coli		C. lari
	Biotype
	I	II	III	IV	I	II	I	II
Positive Negative
Hippurate hydrolysis
Rapid H2S test
DNA hydrolysis

1. Hippurate hydrolysis test:
   See 6.5.6
2. Rapid H₂S test:
   1. Gently suspend a large ball-like inoculum (5 mm loopful) from a 24h culture into the upper third of the Rapid H₂S Test Medium.
   2. Do not mix.
   3. Incubate in a 37°C waterbath for 2 h.
   4. A blackening around the bacterial mass represents a positive reaction and usually begins to appear within 30-45 min.
   Note: Use only 24-h cultures.
3. DNA hydrolysis test:
   1. Inoculate a circular area (diameter 5 mm) on Toluidine Blue - DNA Agar with a 3 mm loopful of a 24-48 h culture by pressing the loop with inoculum into the agar.
   2. Incubate at 42°C for 24-48 h under microaerobic conditions.
   3. A clear-colorless or pinkish zone around the inoculum is considered a positive reaction.

6.5.9 Serotyping See section 2.4

Rapid tests

Some rapid immunological tests have been developed, including latex agglutination tests, enzyme linked fluorescent assay systems and DNA hybridization (9.1, 9.2, 9.4)

7. Maintenance of Cultures

Stock cultures may be prepared and stored as follows:

7.1 Semi-solid medium

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

7.2 Culture storage in freezer

• Glycerol peptone medium at -70ºC:

1. Using a 24-h culture, prepare a heavy suspension of the bacteria in 1-2 mL of Glycerol-peptone medium.
2. Mix well and dispense 0.3-0.5 mL into cryogenic vials.
3. Store the culture at -70°C (Viability is excellent after several years of storage).

7.3 Liquid nitrogen method:

1. Prepare a heavy cell suspension in Brucella Broth using a 24-48 h culture from a Mueller Hinton Blood Agar plate; place 0.5 mL of the suspension into a (12.5 x 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.
2. Put the Cryotubes on metal racks and submerge the racks in liquid nitrogen.
3. Do not seal the lid of the tank. Add vacliquid nitrogen regularly to fill the tank.

7.4 Lyophilization

1. 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.
2. Distribute 0.2 mL into lyophilizing ampules and lyophilize.
3. Store lyophilized ampules at 4°C.

8. Transporting Cultures Through 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 campylobacters for about 5 weeks at 4°C or 2 weeks at room temperature (100% recovery).

1. Inoculate the culture onto a Mueller Hinton Blood Agar plate and incubate under microaerophilic conditions for 24 h at 42°C.
2. Remove growth from the plate with a sterile swab, break off the handle of 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.
3. Leave the swab in the bottle, tighten cap, and incubate in air overnight at 42°C, after which the culture is ready for shipping.

9. References

9.1 American Association of Analytical Chemists (AOAC). 1998. Bacteriological Analytical Manual. Eighth edition. International, Gaithersburg, MD.

9.2 American Public Health Association. 2001. Compendium of Methods for the Microbiological Examination of Foods, Fourth Edition, F. P. Downes and K. Ito ( eds. ). America Public Health Association, Washington, D.C.

9.3 Atlas, R.M. 1997. Handbook of Microbiological Media. Second edition. L.C. Parks (editor). CRC Press Inc.

9.4 Marshall, S.M., P.L. Melito, D.L. Woodward, W.M. Johnson, F.G. Rodgers and M.R. Mulvey. 1999. Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-Restriction Fragment Length Polymorphism analysis of the 16S rRNA gene. J. Clin. Micro.37:4158-4160.

9.5 Post, D.E. 2000. Food-borne pathogens monograph No. 3: Campylobacter. Oxoid Technical Support Department, Hampshire England.

10. Semisolid Media for Growth and Biochemical Tests (Tubes)

For larger view of Table 1

Table 1. Key characteristics of catalase-positive Campylobacter

	Oxidase	Ferment Sugars	NO3 Reduction	NO2 Reduction	H2S (SIM)Table 3 footnote a	H2S (strip) Table 3 footnote b	Hippurate Hydrolysis	1% Glycine	3.5% NaCl	25°C	37°C	42°C	Aerobic (plate)	5% O2 (plate)	Nalidixic Acid(30 µg)	Cephalothin (30 µg)	TMAOTable 3 footnote f
Table 3 footnotes Table 3 footnote a SIM medium for strong H2S producers. Return to table 3 footnote a referrer Table 3 footnote 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. Return to table 3 footnote b referrer Table 3 footnote c V = results are variable Return to table 3 footnote c referrer Table 3 footnote d S = sensitive, please Note some C. jejuni strains are resistant Return to table 3 footnote d referrer Table 3 footnote e R = resistant Return to table 3 footnote e referrer Table 3 footnote f TMAO = trimethylamine-N-oxide Return to table 3 footnote f referrer Legend:: Positive Negative Variable Resistant Sensitive
C. jejuni															Table 3 footnote d	Table 3 footnote e
C. coli
C. lari
C. fetus ss. fetus
C. fetusss .venerialis						Table 3 footnote c