United States Department of Agriculture 

Food Safety and Inspection Service (FSIS), Office of Public Health Science (OPHS) 

Detection and enumeration method for Campylobacter jejuni/coli from poultry

rinses and sponge samples

Introduction 

This direct plating procedure is a rapid and effective alternative to the MPN method for 

the enumeration of Campylobacter jejuni, Campylobacter coli and other possible

Campylobacter spp. The procedure was developed by the Agricultural Research Service, 

USDA and recommended by the National Advisory Committee on Microbiological 

Criteria for Foods (NACMCF) (1). This guidance protocol is based on methodology used 

for the current FSIS Young Chicken Baseline Study (YCBS) and Young Turkey Baseline 

Study (YTBS) for whole chicken rinses and turkey sponge samples. 

Campylobacter spp. are microaerophilic and are vulnerable to environmental stresses 

such as exposure to air, drying, low pH, and prolonged storage, all of which can impact 

their survival. Use of oxygen-quenching agents, a microaerobic atmosphere, and 

antibiotics that suppress competitors, significantly improve Campylobacter recovery. 

Furthermore Campylobacter spp are very sensitive to freezing and can die off at room 

temperature as well. Sample analysis should be initiated as soon as possible after 

receipt in the laboratory. Only samples received within the temperature range of 0-15°C 

should be analyzed. Upon receipt in the lab, 1 ml of the sample is dispensed across 4 

Campy-Cefex plates (0.25ml/plate) and 0.1 ml is dispensed onto each of 2 additional 

plates (duplicates). These plates are incubated in a microaerobic atmosphere at 42°C. 

Campylobacter colonies are enumerated after 48 hours of incubation. 

An enrichment step with Blood-Free Bolton's enrichment broth, (BF-BEB) may be used 

to supplement the direct plating for increased sensitivity of qualitative detection for low 

levels of potentially injured cells. After BF-BEB is incubated at 42°C under 

microaerobic conditions for 24-48 h, the broth is streaked onto Campy-Cefex agar for 

isolation of Campylobacter colonies. The plates are incubated as above and examined 

for typical Campylobacter growth. Typical colonies are confirmed by microscopy and 

serology. 

Although the numerical values given in this method may be expressed as exact values, 

such as those given for weight, volume, pH, time and temperature to achieve optimum 

results, it should be clearly understood that an acceptable range exists within which the 

optimum results can be expected. The following allowable ranges for the given 

parameters are considered to be acceptable and applicable: 

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• Weight and volume measures: ± 1% (± 5% for serological pipettes and graduated 

cylinders) 

• pH: ± 0.2 units 

• Time: hours ± 2 hour; minutes ± 1% 

• Temperature: ± 1°C 

The microbial density of air in the working area should be monitored, measured in fallout 

pour plates taken during plating. It should not exceed 15 colonies/plate during a 15 

minute exposure period. 

Facility, Equipment and Materials 

1. Work area, level table with ample surface in room that is clean, well-lighted, wellventilated, and reasonably free of dust and drafts. 

2. Storage space, free of dust and insects and adequate for protection of equipment and 

supplies. 

3. Petri dishes, glass or plastic (15 x 100 mm) 

4. Rainin P100-P200 and P1000, or equivalent, microliter pipette 

5. Equipment and materials for ensuring proper atmospheric conditions for 

Campylobacter growth; options include: 

• 42 ± 1.0°C Tri-gas incubator (static) charged with 5% O2, 10% CO2, and 85% N2

(Contact incubator manufacturer to assist you with the initial setup, including an 

alarm system for improper gas mixture). 

• Gas cylinders containing appropriate gas mixtures (certified by the supplier) to 

achieve 5% O2, 10% CO2, and 85% N2 with regulators compatible with 

Compressed Gas Association (CGA) connection on the cylinder. 

• Commercially available gas packs intended for Campylobacter testing. 

• Bags or other containers capable of maintaining the atmosphere during 

incubation. 

6. Glass slides with cover slips for wet mount preparations 

7. Sterile culture tubes, 16 x 150 mm 

8. Sterile bent glass rods, flexible plastic hockey sticks, or equivalent 

9. Vortex Mixer 

10. Autoclave bags, ~ 24” x 36” 

11. Microscope (phase contrast capability recommended) 

12. DrySpot Campylobacter test-Oxoid LTD., Basingstoke, UK, INDX Campy (jcl) 

[Integrated Diagnostics, Inc., Baltimore, MD), or equivalent validated procedure. 

13. Refrigerator, to maintain samples at 2-8°C. 

14. Vented, t-75cm2

 tissue culture flasks for incubating BF-BEB under microaerophilic 

conditions. 

15. Thermometers, appropriate range, accuracy checked with a thermometer certified by 

 NIST (National Institute of Standards and Technology). 

Media and Reagents: 

1. Buffered Peptone Water (BPW) 

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2. Campy-Cefex agar- Appendix I 

3. BF-BEB (2X)- Appendix I 

Prior Preparations for Campylobacter Method Implementation 

Drying plating media: 

Drying Campy-Cefex plates is critical for preventing spreading of colonies. Care should 

be taken to ensure that agar media are dried appropriately prior to use. Plates should be 

dried in an area that minimizes light exposure because light can possibly affect the 

growth and selection of Campylobacter. 

Atmospheric requirements: Campylobacter spp. is microaerophilic and, in order to grow 

requires an atmosphere of 5% O2, 10% CO2, and 85% N2. Add 4 drops of a humectant 

such as glycerol to a filter paper and place it in the bag or jar to diminish possible 

confluent and swarming growth of Campylobacter.

Use of control cultures: After media preparation, a positive control culture is streaked 

onto Campy-Cefex agar to determine whether the batch of medium supports growth and 

typical colonial morphology (media control). In addition, a positive control culture (such 

as C. jejuni ATCC 49943 or 29428) and an uninoculated media control should be 

analyzed concurrently with each group of daily samples to document proper performance 

of the media during the course of sample analysis. 

Procedure 

Sanitation 

All instruments must be sterilized in a manner consistent with the specific instrument to 

render the instruments free of viable organisms. All work surfaces must be cleaned with 

appropriate disinfectants both prior to and after sample collection, to include sanitizing 

between samples. The outside surfaces of sample containers must be carefully disinfected 

prior to sampling being careful not to introduce disinfectants to the sample. Prior to 

opening containers, aseptically remove residual disinfectant. Bare hands should never 

touch the sample and the interior of the sample container. 

Sample preparation, plating and enrichment 

Chicken rinsate sample:

Under the FSIS baseline-sampling program, a chicken carcass is rinsed with 400 ml of 

Buffered Peptone Water (BPW). For qualitative testing, a 30 ml test portion of this 400 

ml is enriched as described below with 30 ml of 2X BF-BEB. For quantitative testing, 1 

ml of the 400 ml is distributed across four plates to represent this 100 dilution, and 

subsequent dilutions are prepared as described below. 

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Turkey sponge sample: 

Under the FSIS baseline-sampling program a single sponge is used to sample 50cm2 back 

and 50cm2 thigh representing 100cm2 on one turkey carcass. The sponge sample is 

hydrated with BPW to a total volume of 25 ml. For quantitative testing, 1 ml of the 25 ml 

is distributed across four plates to represent this 100 dilution, and subsequent dilutions are 

prepared as described below. For qualitative testing, the entire remaining sponge and 

diluent is enriched as described below in an equivalent volume of 2X BF-BEB. 

 

To ensure even distribution, mix the test samples thoroughly by gentle shaking of the 

collected carcass rinsate for ~1 minute or by squeezing the sponge several times before 

taking the analytical unit from the sample. 

Aseptically dispense 250 ± 10 μl of the sample using a Rainin P1000 or equivalent 

microliter pipette onto each of four Campy-Cefex plates representing 1 ml sample or 100 

dilution of the sample. Dispense 100 ± 4 μl sample aliquot onto each of two CampyCefex plates representing 0.1 ml or 10-1 dilution. Prepare a 1:10 dilution using 1000 ± 40 

μl in 9.0 mL of BPW. Using the P100 pipette with a fresh tip, dispense 100 ± 4 μl of the 

1:10 dilution of the sample onto each of two Campy-Cefex plates. These are duplicate 

plates representing 0.01 ml or 10-2 dilution of the sample. Similarly any additional 

dilutions (10-3, 10-4, etcetera) are dispensed using a fresh tip on duplicate plates. 

Beginning with the highest diluted plates and working back through the more 

concentrated dilutions, use a single sterile hockey stick, or equivalent, to spread the 

inoculum evenly over the entire surface of the agar avoiding contact with the plate. 

Incubate both enrichment broth and plates at 42°C for 48 hours using a tri-gas incubator 

flushed with 5% O2, 10% CO2, and 85% N2, or equivalent. 

 

Plate Examination: 

After incubation, examine all plates to determine the relative proportion of various 

suspect colony types that are present in the sample. Often there is a single colony type, 

but there may be multiple colony types. Suspect colonies are translucent and wet, flat or 

slightly raised, and may vary significantly in size. 

Selecting Dilution for Confirmatory Testing 

1. When suspected colonies are found the following criteria are used to select the 

appropriate dilution for colony confirmation. Pick five colonies (if available) 

proportionally representative of all suspect colony types from one or more plates 

for confirmation: Pick from the dilution that averages 15-150 suspect colony 

forming units (CFU) per plate. 

2. If there are no dilutions that average 15-150 suspect CFU per plate, pick from the 

dilution that averages < 15 CFU per plate. 

3. If there are no dilutions meeting the above criteria (i.e., counts exceed 150 CFU) 

select well isolated colonies from the dilution that contain counts up to 300 CFU. 

4. If all dilutions are > 300 CFU then select well isolated colonies from the highest 

dilution. 

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If there are mixed confirmation results among the colonies of one perceived colony 

type (i.e., the colonies look the same but some confirm and others do not) pick several 

more colonies for a total of 10 colonies representing that type. 

If the last dilution has an average of > 50 suspect colonies per plate, pick 10% of the 

average number of colonies up to a maximum of 10 colonies from that dilution for 

confirmatory testing. 

Confirmation 

For any suspect or typical growth perform the following confirmatory steps: 

1. Microscopy: Touch a portion of the suspect colony and suspend in a drop of 

sterile 0.85% saline on a microscope slide. Cover with a glass cover slip and 

examine using oil immersion phase contrast microscopy. Suspensions 

demonstrating typical Campylobacter corkscrew morphology and darting motility 

are regarded as presumptively positive. 

2. Latex agglutination immunoassay: Each presumptively positive colony is 

confirmed by testing an additional portion of the colony using the DrySpot 

Campylobacter Test (Oxoid Ltd., Basingstoke, UK) or INDX Campy (jcl) 

[Integrated Diagnostics, Inc., Baltimore, MD), or equivalent validated procedure. 

Calculating: 

Count all colonies representing types that have been confirmed above on all plates for 

each dilution with an average count of 15-150 CFU. Perform additional counts for plates 

that are not in the countable range by following the criteria listed below: 

1. If there are plates with > 150 colonies in the same dilution with plates < 150, 

count plates with up to 300 CFU. 

2. If plates with > 150 colonies represent the final dilution where counts from all 

other dilutions are > 300 CFU, count plates with up to 300 CFU. 

3. If all plates and all dilutions appear to be > 300 CFU, estimate the count of the 

final dilution using the “4 blocks” method on a colony counter (i.e., average count 

from 4 blocks multiplied by total number of blocks per plate). 

Computing and Recording Counts: 

1. If only one dilution averages 15-150 colonies per plate, the CFU/ml will be the 

mean of all plates of that dilution multiplied by the dilution factor or the sum of 

the 4 plates from the 100 dilution whichever applies. 

2. If two separate dilutions average 15-150 colonies per plate the CFU/ml will be 

determined by calculating the average for each dilution as above, then the 

CFU/ml for each relevant dilution will be averaged together. 

3. For the plates representing the final dilution, count up to 300 colonies/plate for an 

estimated CFU/ml. 

4. If the final dilution is > 300 CFU, it may be necessary to report “TNTC” (Too 

Numerous To Count). Future analyses should employ additional dilutions to 

prevent TNTC results. 

If all colonies of specific morphology were confirmed, 100% of colonies with that 

morphology are included in the count. If there are mixed confirmation results among 

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colonies representing one specific colony type and 10 colonies representing that type 

were picked for confirmation the total count for that colony type must be multiplied 

by the percentage of colonies of that type that confirmed. 

Qualitative Enrichment for Post-Chill Rinsates: 

Qualitative assessment for Campylobacter spp. is reported as either positive or negative 

based on the presence or absence of confirmed Campylobacter spp., respectively. 

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Appendix I

Campylobacter Media

NOTE: The instructions below apply to media preparation situations where individual 

ingredients are used. These media are also available from commercial sources; in these 

circumstances, follow the manufacturer’s instructions for media preparation and storage. 

1) Campy-Cefex Agar 

Note: Considering the data of Oyarzabal et al. (2) and NACMCF recommendations (1) 

to employ a cost-effective modified Campy-Cefex formulation, the USDA-ARS semimodified formulation of Campy-Cefex specified below is both cost-effective and proven. 

Ingredient Amount 

1. Brucella agar 43 g/L 

2. Ferrous sulfate 0.5 g/L 

3. Sodium bisulfite 0.2 g/L 

4. Sodium pyruvate 0.5 g/L 

5. Deionized (DI) water 950 ml 

Supplements Amount 

Lysed horse blood 50 ml/L 

Cefoperazone 33 mg/L 

Cycloheximide 0.2 g/L 

Directions: Mix ingredients 1, 2, 3, 4 and 5 in a large flask and heat to boiling. 

Autoclave for 15 minutes at 121oC and cool it to 50oC. Add supplements after cooling to 

50oC. Dispense 20-23 ml per plate. Final pH = 6.8-7.2. Store and dry as mentioned 

above in the ‘Prior preparations for Campylobacter method implementation’ section. 

2) Blood-free Bolton’s enrichment broth (2X BF-BEB) 

Ingredient Amount 

1) Meat peptone 20 g

2) Lactalbumin hydrolysate 10g

3) Yeast extract 10 g

4) Sodium chloride 10 g

5) Sodium pyruvate 1.0 g

6) α Ketoglutamic acid 2.0 g

7) Sodium metabisulfite 1.0 g

8) Sodium carbonate 1.2 g

9) Haemin 0.02 g 

Distilled or DI water 950 mL

Supplement Amount 

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Cefoperazone 40 mg 

Vancomycin 40 mg 

Trimethoprim 40 mg 

Cycloheximide 50 mg 

Directions: Add basal ingredients 1-8 to water for a 2X broth solution and bring to a boil 

to dissolve completely. Autoclave for 15 minutes at 121-124°C. Cool to 50°C. Add 

supplements. Each above mentioned supplement comes in a vial that is sufficient for 

500ml. Therefore for 1000 ml 2X BF-BEB add four vials of each supplement. After 

supplement addition, medium is stable for approximately 48 hours. 

Note: This 2X BF-BEB formulation is twice as concentrated as the traditional BF-BEB 

formulation to meet the specific needs for diluting 1:2 with a 30 ml test portion; i.e., 30 

ml of 2X BF-BEB plus 30 ml sample makes 60 ml of 1X BF-BEB sample enrichment for 

incubation, as was used for FSIS baseline testing. 

Appendix II

Campylobacter Storage and Transport Media

Storage and Maintenance of Campylobacter Culture 

1) Wang’s Storage Medium

Ingredient Amount 

1. Brucella broth powder 28 g 

2. Glycerol 200 ml 

3. DI water 750 ml 

Supplement Amount 

Lysed horse blood 5 ml/L 

Directions: Add Brucella broth powder to water and bring to a boil to dissolve 

completely. Add 200 mL of glycerol to the homogenous mixture and mix well. 

Dispense 95 mL of the mixture into individual 100 mL bottles. Autoclave for 15 minutes 

at 121oC and cool it to 50oC. Add 5 mL lysed horse blood into each bottle and mix 

thoroughly. Dispense 1 mL Wang’s storage medium into a 2 ml cryovial. 

Note: Once prepared, Wang’s storage medium’s shelf life is 2-3 weeks at 2-8o

C 

regardless if maintained in a flask or dispensed into cryovials. 

Directions for Campylobacter spp. cultures storage: 

1. When a pure culture is obtained, streak the culture onto three to four trypticase 

soy agar with 5% sheep blood (SBA) plates. Place plates in a tri-gas incubator, or 

equivalent, to flush with gaseous mix of 5% O2, 10% CO2, and 85% N2. Incubate 

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plates at 42 ± 1.0oC for 24 hours. Do not streak for isolation; streak the plate 

whereby the culture completely covers the plate with a lawn of growth. 

2. Using a sterile cotton swab, plastic loop or equivalent, dispense the entire lawn of 

bacteria from the three-four SBA plates into a single cryovial. 

3. Vortex the mixture until bacterial cells have dispersed. This mixture will be thick 

due to high bacterial cell numbers in the cryovial. 

4. Initially, place the cryovials in a 2-8oC refrigerator for 20 minutes, and then 

transfer to the ≤ minus 70oC freezer for permanent storage. Note: Isolates are 

initially placed in the refrigerator to avoid sudden shock to the ≤ minus 70oC 

freezer temperature. 

Directions for recovering the Campylobacter spp cultures from ≤ minus 70oC 

freezer:

1. After removing the cryovial from ≤ minus 70oC freezer, immediately scrape a 

small amount of the ice crystals from the inoculum, transfer and streak to the 

appropriate plating media for isolation. 

2. Immediately return the cryovial to the ≤ minus 70oC freezer. Do not allow the 

culture to thaw. 

3. These streaked plates should be placed in a tri-gas incubator or equivalent, 

flushed with the gaseous mixture of 5% O2, 10% CO2, and 85% N2, and placed in 

a 42 ± 1.0oC incubator for 24 – 48 hours. 

Transport of Campylobacter Culture

2) Wang’s Transport Medium (Semisolid)

Ingredient Amount 

1. Purified grade agar 4 g 

2. Brucella broth powder 28 g 

3. DI water 950 ml 

Supplement Amount 

Lysed horse blood 5 ml/L 

Directions: Add Brucella broth powder and purified grade agar to water and bring to a 

boil to dissolve completely. Dispense 95 mL of the mixture into individual 100 mL 

bottles. Autoclave for 15 minutes at 121oC and cool it to 50oC. Add 5 mL lysed horse 

blood into each bottle and mix thoroughly. Dispense 1 mL Wang’s transport medium 

into a 2 ml cryovial. 

Note: Once prepared, Wang’s transport medium’s shelf life is 2-3 weeks at 2-8oC 

regardless if maintained in a flask or dispensed into cryovials. 

Directions for Campylobacter spp. cultures transport: 

1. When preparing for shipping, streak the culture onto three to four SBA plates. Do 

not streak for isolation; streak the plate whereby the culture completely covers the 

plate. These plates should be placed in a tri-gas incubator or equivalent, flushed 

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with the gaseous mixture of 5% O2, 10% CO2, and 85% N2, and placed in a 42 ± 

1.0oC incubator for 24 hours. 

2. On the day of shipping, remove the transport medium from the 2-8oC refrigerator 

and allow the cryovial to come to room temperature. 

3. Using a sterile cotton swab, plastic loop or equivalent, dispense the entire lawn of 

bacteria from the three-four SBA plates into a single, appropriately labeled 

cryovial. 

4. Ship isolates to destination with ice packs to keep cool within 24–48 hours of 

packing. Shipping shall comply with Department of Transportation (DOT) 

regulations. Typically, isolates shipped in Wang’s transport medium can last 

seven days. 

5. The receiver should immediately recover the cultures, store and freeze following 

the procedures for Storage and Maintenance of Campylobacter Culture as 

previously described. 

References

1. National Advisory Committee on Microbiological Criteria for Foods. 2007. 

Analytical utility of Campylobacter methodologies. J. Food Prot 70: 241-250.

2. Oyarzabal, O., K. Macklin, J. Barbaree, and R. Miller. 2005. Evaluation of agar 

plates for direct enumeration of Campylobacter spp. from poultry carcass rinses. 

Appl. Environ. Microbiol. 71:3351–3354.

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