AHL Milk Bacteriology In-Clinic Laboratory Proficiency Program

Comparative results        

Over the last few years, an increased number of veterinary practices in Ontario have been offering in-clinic milk bacteriology testing as part of their service to their dairy clients.  Generally no standardized culture method has been used and no quality program has been available to assess and monitor the quality of results generated.  Therefore, in 2016, AHL started the AHL milk bacteriology in-clinic laboratory proficiency program to provide education and self-assessment for in-clinic staff. The goal is to assist in producing accurate and appropriate bovine mastitis diagnoses.  More information about the program can be found at (https://www.uoguelph.ca/ahl/content/ahl-labnote-47-ahl-milk-bacteriology-clinic-laboratory-proficiency-program).  

At present, 13 clinics are participating in the in-clinic mastitis proficiency program and are at different stages.  At the initial meeting, the goals of the program are discussed, and together the clinic technician(s) and the AHL Client Outreach Technician (COT) go through a checklist that covers laboratory setup, supplies, types of media, sample handling, culture setup, timelines for incubation, disposal of materials, and the submission of samples for the project to AHL. 

AHL does not release culture results until we have received the clinic’s in-house findings.  Once 4 samples are submitted, the COT reports the in-clinic results and the AHL results to the clinic. Samples for submission need to be split promptly as possible after collection (same day) and kept chilled.  Ideally the sample the AHL receives for culture will be as close as possible to the sample quality setup by the participating clinic to prevent adverse influence on results.   If a sample is submitted to the clinic from a farm in suboptimal condition (warm, dirty, or a composite), the owner should re-sample and re-submit to ensure a clean, useful sample. 


In-clinic and AHL results were grouped into 4 categories for assessing comparative outcomes:   

1)    Major variance (unsatisfactory results) when a main mastitis pathogen was missed or misidentified, which may have resulted in spreading of mastitis throughout the herd, inappropriate treatment decisions, or culling (e.g., frequent misidentification of Staphylococcus haemolyticus as Staphylococcus aureus). 

2)    Moderate variance (improvement needed and some consistency) when environmental mastitis pathogens are missed or misidentified, which may have resulted in inappropriate treatment or lack of it (e.g., inability to differentiate between Streptococcus uberis and Enterococcus spp.) or when some environmental pathogens were detected and some were missed or misidentified (some consistency).  

3)    Minor variance (acceptable results) when misidentification, identification to the genus level, or lack of isolation of mastitis bacterial pathogen would not have changed any treatment or management decisions (e.g., Citrobacter spp. being misidentified as Enterobacter spp.)

4)  In agreement (meets expectation) when in-clinic results matched AHL results.

Recommendations based on project findings so far:

Best Practices:


1.    Culture only samples collected from quarters with clinical mastitis. 

When results for quarter milk samples were compared to results for composite milk samples, in-clinic results were more accurate for quarter milk samples. 

        2.    Split and deliver milk samples to AHL as soon as possible. The cold chain is  important! .

Any delay in culturing samples at the AHL may result in overgrowth of contaminants or non-target bacteria and cause discrepancies in results.  Samples must remain chilled until received at AHL. Milk is a good growth medium for bacteria. 

3.    Develop in-clinic Standard Operating Procedures (SOP) for all steps of milk bacteriology including sample acceptance criteria and sample collection procedure.

Remember ‘garbage in-garbage out’ is true.  A poor quality sample you will not give you results you can trust.  Your SOP should also contain minimum standards that are acceptable for bacterial identification.  Not all hemolytic colonies growing on blood agar plates or on Staph selective agar are S. aureus

4.    Invest in education (initially and continuing) and provide adequate resources to ensure that minimal standards for bacterial identification are met. 

At minimum, each clinic should have National Mastitis Council (NMC) Laboratory Handbook on Bovine Mastitis.  The latest version of this Handbook is available from AHL for a discounted price of $70 (regular price $129 US) for all clinics participating in this program. 

5.    Educate herd owners about proper sample collection, explaining the difference between quarter and composite milk samples. 

6.    Confirm in-clinics results in an accredited laboratory before final culling decisions are made.

7.    Dispose of samples and used plates and other biohazards as per Guideline C-4: The Management of Biomedical Waste in Ontario) https://www.ontario.ca/page/c-4-management-biomedical-waste-ontario


1.    Accept suboptimal quality samples for culturing. 

        Use aseptic technique to collect milk samples and refrigerate them immediately particularly during summer months.  If samples are collected by farm staff, teach them proper sampling technique and explain why it is important to follow it.  Do not accept samples collected in non-sterile containers (e.g., mason jars, baby food jars, etc.), submitted in vials that are visibly contaminated or that were not immediately cooled after collection and kept cold during transport. 

2.    Culture composite samples, high SCC cows selected off the DHI SCC report or whole herds.

        Bacterial growth in these types of samples is usually mixed and interpretation can be difficult considering the limited resources available in-clinic for full bacterial identification.  For additional information please see best practice  #1 above.    

3.    Identify bacteria beyond genus level or beyond levels recommended by the culture system you are using or the NMC Handbook. 

For certain bacteria (e.g., CNS), species identification can be difficult at the best of times even in clinical diagnostic laboratories.  There are also minimuml standards that need to be followed when speciation is attempted.  For example, it is highly recommended to confirm S. aureus identification using a coagulase test to differentiate it from other hemolytic CNS. 

4.  Make culling decisions or any major management changes without corroboration of your in-clinic results.            

The AHL supports clinics by providing laboratory quality assurance advice, performing mini-audits, and having clinics submit quarterly samples for proficiency testing. 

The AHL has the NMC Laboratory Handbook on Bovine Mastitis available at a discounted rate of $70.00 (regular price $129 USD).  This handbook covers many aspects of in-clinic milk culturing and is a wonderful resource.  If not already purchased, please consider getting one for your clinic to aid in the development of SOP’s and procedures.

As we have moved forward on this project, it has been difficult for the AHL to comment on some of the methods used as we are unfamiliar with them and their limitations.  We are beginning a small project where we will take milk samples coming into the AHL and culture them using some of the various rapid culturing systems. 

•             Minnesota Tri-plates

•             Sensor Health Quad Plates

•             Sensor Health Tri plates

•             Blood plates – singly and with MacConkey (and ancillary testing)

•             Check – up (East Gen)

•             Accumast triplate (New York)

Please note that we do have some experience with Petri film and will not be using this method. It does not provide enough range in detection for unknown mastitis samples.

This will allow us to compare the methods and develop experience in the different systems.

Josie Given

Jim Fairles

Durda Slavic

Ann Godkin