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Mastitis Case Studies

Bacterial Culture


This resource describes bacterial culture of mastitis-causing pathogens.

Identification of the Pathogen Causing Mastitis

Collecting sterile milk samples for bacteriological culture is the only way to definitively diagnose infectious mastitis and identify the causative organism. Proper technique in collecting the sterile milk sample is essential! Samples usually are collected from each quarter. Remove dirt and water from the teats and udder; wash and dry hands; strip out the foremilk; disinfect the teat ends with alcohol and/or teat dip (do the far teats first); remove the cap from the sterile sample tube taking care to prevent contamination inside the tube; collect sample with tube at 45 degree angle to teat (collect the near teats first); replace cap on the tube and dip the teats.

Preparing teat to collect sterile sample. Preparing teat to collect sterile sample. Preparing teat to collect sterile sample.
Teat end is disinfected with an alcohol pad. Tube is held at a 45 degree angle to prevent dirt from getting into the tube. Milk is squirted into the tube.

Proper sample handling : Refrigerate or keep the samples on ice (less than 24 hours) or freeze.


Plating the sample : Plating the samples requires special bacteriological medium, conditions of a laboratory and personnel with good technique in microbiology. Agitate 0.01 ml milk/quarter of blood agar plate using a loop or pipette. For coliforms plate 0.1 ml milk/half of plates of Blood agar and MacConkey agar plates. Incubate at 37 C for 48 hours.

E. coli on blood agar plate.
Staph aureus on blood agar plate.
Esherichia coli on a blood agar plate. Staphylococcus aureus bacteria on a blood agar plate.
Klebsiella pneumoniae on blood agar plate.
Klebsiella oxytoca on blood agar plate.
Klebsiella pneumoniae on a blood agar plate. Klebsiella oxytoca on a blood agar plate.

Pathogens vs Contaminants : How do you tell if growth on the plate is a pathogen or a contaminant? These criteria assume you are using good technique in collecting the sterile sample and in plating the samples. Greater than or equal to 5 identical colonies in 0.01 ml milk (pure culture) = significant. Greater than or equal to 5 identical colonies in 0.01 ml milk (mixed culture) = significant, but questionable. Less than 5 identical colonies in 0.01 ml milk = a contaminant.

Intrepretation : Streptococcus agalactiae and Staphylococcus aureus are usually significant (causative agent) in any number. Bacillus is usually contaminant in any number.

Antibiotic Sensitivity Tests : are often used to determine what antibiotics may affect the bacterium isolated from the udder. However, these take time and additional microbiological techniques. These test use either disc diffusion or minimal inhibitory concentration assays. The in vitro results may not reflect in vivo sensitivity of the bacterium.

Antibiotic sensitivity palte.
Antibiotic sensitivity plate.

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