Antibiotic Selective Treatment System based on quarter level Petrifilm™ results at dry-off

From research project : Selective Dry-off Treatment based on quarter level using on-farm diagnosis.
Jean-Philippe Roy (Principal investigator, UMontréal), Fidèle Kabera (Ph.D. candidate, UMontréal),
Greg Keefe (collaborator, UPEI) and Simon Dufour (collaborator, UMontréal)

Herd Selection Criteria

• SCC < 250,000 cells/ml in bulk tank milk

Cow Selection Criteria
  • Dry-off duration: 45 – 75 days
  • Currently pregnant and in good shape
  • No antimicrobial treatment during the last 14 days before dry-off
The day before drying off, collect a sterile sample of milk per quarter
Milk culture per quarter on a Petrifilm AC

  • Petrifilm identification for each quartier

  • Dilution 1:10 of the quarter milk sample

  • Petrifilm inoculation for each quarter

Petrifilm Interpretation

  • 0 – 4 colonies : non-infected quarter

  • ≥ 5 colonies : infected quarter

Apply dry-off treatment to quarter accordingly to Petrifilm results

  • 0 – 4 colonies : use internal teat sealant only in that quarter

  • ≥ 5 colonies : use antibiotic treatment and internal teat sealant in that quarter

MATERIALS
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Ink marker

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Applicator

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Syringes 3ml, 10ml and 1 1/2 in. needles

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Petrifilm AC plates

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Sterile Water Bottle

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Disposable gloves

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Incubator

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Sterile sample vials

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70% alcohol soaked gauze pad

NOTES
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Repeat the same steps for the four quarters of the cow (a Petrifilm plate for each quarter).

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INOCULATE EACH PETRIFILMTM PLATE AND SPREAD THE SAMPLE ON CIRCULAR AREA BEFORE GOING NEXT

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SAMPLING, DILUTION AND INOCULATION MUST BE MADE WITH PRECAUTION AND STERILITY

  • Do not touch the inside of the tube and the cap with the syringe or fingers or remove the cap on the table when handling.
  • Change the syringe and needle for each quarter.
  • Disinfect the cap of the sterile water bottle each time the sample is taken.
  • After opening, write the date on the bottle and keep it in the fridge for a maximum of one month.

Petrifilm AC inoculation protocole

(Test for the counting of the total aerobic flora)

Step 1

Take 9 ml of sterile water and put in a sterile tube. Do not touch the inside of the tube and cap with the syringe or fingers.

Step 2

Take 1 ml of the milk sample.

Step 3

Put 1 ml of milk in a tube containing 9 ml of sterile water (dilution 1:10).

Step 4

Homogenize: Close the tube and shake the diluted milk tube by flipping it up and down at least 5 times.

Etape 5 Prélever 1 ml de lait dilué à l’aide de la seringue utilisée précédemment. Aspirer et remettre le lait dilué une fois, afin de rincer la seringue.

Step 5

Take 1 ml of diluted milk using the syringe used previously. Aspirate and return the diluted milk once, to rinse the syringe.

Step 6

Place the previously identified Petrifilm plate (cow, quarter, date and time) on a flat surface, lift the top film, then place 1 ml of diluted milk in the center of the lower film without touching it with the syringe or fingers.

Step 7

Release the top film. Place the applicator over the top film (circular side down) and lightly apply pressure to spread the sample over the circular area.
Image credits: 3M

Step 8

Put the Petrifilm in the incubator at 35 °C (95 °F) for 24 hours. Make sure it contains some water to maintain a slight humidity.

Step 9

Read the results on the Petrifilm. Each point represents a bacterial colony. Count the entire plate, not just a square. Record the results in your files.