On-Farm Mastitis Bacteria Identification Tool

Mastitis Treatment Decision System

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Why is this tool valuable?

Mastitis is the most frequent cause of antibiotic use on dairy farms. Various bacteria can cause udder infections, some of which have higher rates of self-curing or respond poorly to treatment. Correct identification is crucial to allow dairy producers and veterinarians to select, if needed, targeted treatments without compromising or delaying recovery.

How does it work?
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Bacteria are identified through milk culturing on plates containing nutrients that promote bacterial growth. An approximately 24-hour incubation period is required for the presence of bacterial colonies to be revealed. Various plates with specific nutrients can be used to select for appropriate bacteria present in milk. The category of bacteria is based on growth and colony numbers on specific plates.
Advantages and limitations
Advantages
  • Allows identification and targeting of cases caused by Gram + bacteria (requiring treatment)
  • Quicker identification of bacteria compared to bacterial cultures in laboratory (not replace it)
  • More rational use of antibiotics
  • Decreased milk losses
  • Decreased risk of antibiotic residues
Limitations

  • Care required in technique to avoid contamination

  • Interpretation influenced by the experience and training of the user

  • Cannot identify mycoplasma and yeasts (treatment not recommended)

In laboratory, staining procedures are used to classify most bacteria into two categories:
Gram positive bacteria
  • Mainly staphylococci and streptococci
  • Thin cell walls

Exemples of Gram positive

  • Staphylococcus aureus

  • Staphylococcus spp. 

  • Streptococcus uberis

  • Streptococcus dysgalactiae

  • Streptococcus agalactiae

  • Corynebacterium bovis

Gram negative bacteria
  • Mainly coliforms
  • Thick cell walls which protect against the effects of most antibiotics, detergents and chemicals
  • Contain toxic lipopolysaccharides
  • Do not respond well to antimicrobial treatment

Exemples of Gram negative

  • Escherichia coli
  • Klebsiella spp.
  • Enterobacter spp.
  • Serratia spp.
  • Pasteurella spp.

Necessary Materials

At the farm, home or veterinary clinic, set up the “laboratory” in a clean and dry area to avoid contamination.
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PetrifilmTM Coliform Count Plates (GRAM –) red circle with polystyrene foam, marked CC
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PetrifilmTM Aerobic Count Plates (GRAM +) white without polystyrene foam, marked AC
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PetrifilmTM Staph Express (staphylococci) Count Plates: white circle with polystyrene foam, marked STX and purple Staph Express disk (optional for Staph. aureus detection)
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Plaques PetrifilmMC Staph Express STX
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Spreader (ridged and flat sides)
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Spreader for Staph Express (round and flat)
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3 ml syringes
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Sterile sampling vials
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Vials of diluent (0.9% NaCl saline or PBS)
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Alcohol swabs
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Incubator
(The recommended incubator is the Turbofan Hova-Bator 1602, available from your local agricultural retailer; consult internet to locate the dealer nearest you.)

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Nitrile gloves
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Marker pen

Storage and Handling of Plates of MQM Kit

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Avoid parlours and dairies (high humidity)
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Can be stored for 1 year at room temperature. Keep the plates in their original packaging until use. Plates not sold as part of MQM kit and not sealed, should be kept frozen.
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Do not expose to sunlight and discard any discoloured plates
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Do not touch plate surface

CAUTION

  • The on-farm identification system should be used in mild or moderate cases of mastitis (grade 1 or 2) without fever (< 39.5 °C) and in the absence of other severe diseases.
  • Its critical to develop a sampling protocol and a treatment protocol based on the use of this system in collaboration with your veterinarian.
  • If clinical symptoms worsen, take immédiate action and consult your veterinarian.

To Order

The MQM Mastitis Treatment Decision System uses 3MTM PetrifilmTM enumeration plates to identify mastitis bacteria in milk. To order an on-farm MQM kit, please contact:

To order PetrifilmTM products directly or to consult a representative, please contact innovation Diagnostics, Inc.

Inoculation of the AC Petrifilm TM Plates 

Step 1

Take 9 ml of sterile water and place in a sterile tube. The syringe should not come in contact with the inside of tube or cap, nor should they be touched.

Step 4

Homogenize : Once cap is in place, rotate the tube containing the diluted milk back and forth at least 5 times.

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 credit: 3M

Step 2

Draw 1 ml of the milk sample.

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

Draw 1 ml of diluted milk using the same syringe and release back into the tube to rinse the syringe.

Step 8

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

Step 3

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

Step 6

Place the pre-identified Petrifilm plate (cow, quarter, date, time) on an even surface, lift the top film, then place 1 ml of diluted milk in the enter of the lower film without touching it with neither syringe nor fingers.

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.

 

Inoculation of the Petrifilm CC

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 4

Homogenize : Once cap is in place, rotate the tube containing the diluted milk back and forth at least 5 times.

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 2

Take 1 ml of the milk sample.

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 8

Place the Petrifilm in the incubator at 35 °C (95 °F) for 24 hours. Make sure it contains some water to maintain slightly moist conditions.

Step 3

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

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 9

Read the results on the Petrifilm. Each point represents a bacterial colony. Count the entire plate. Record the results in your files.

Inoculation of the Petrifilm STX

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 4

Homogenize : Once cap is in place, rotate the tube containing the diluted milk back and forth at least 5 times.

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 2

Take 1 ml of the milk sample.

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 8

Put the Petrifilm in the incubator at 35 °C (95 °F) for 24 hours. Insert purple disc between the plate and the film and incubate for an additional 3 hours at 35 °C (95 °F). Make sure the incubator contains some water to maintain slightly moist conditions.

Step 3

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

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 9

Read the results on the Petrifilm. Each point represents a bacterial colony. Count the entire plate, not just a square. Pink around the colonies confirms the presence of Staph aureus. Record the results in your files.

Interpreting the results (AC and STX)

Some veterinary assistance may be required for result interpretation.

With the help of your veterinarian, establish a treatment protocol according to the Petrifilm results.

Gram categorization (Gram positive, negative or absence of growth) can be obtained with AC an CC plates. An interpretation guide is available at www.milkquality.ca

For cases with positive Staph Express cultures, confirmed with the purple disc, treat as directed by your veterinarian.