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«Detection and enumeration method for Campylobacter jejuni/coli from poultry rinses and sponge samples Introduction This direct plating procedure is a ...»

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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:

• 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 microbialdensity 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)

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.

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.

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 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.

–  –  –

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.



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