Fresh or Frozen Milk Samples for Cultures

Dr. Allen Young
USU Extension Dairy Specialist

Collecting milk samples for culturing so that problem organisms can be identified is critical to proper treatment of cows with mastitis. It is common practice on many dairies to collect the sample, then store it in the refrigerator or freeze it until it can either be taken or shipped to the laboratory. Most laboratories have protocols for collection and handling of samples, but the question sometimes is what is appropriate handling for a sample if I am interested in bacteria X, Y or Z? A recent article attempted to address this question for Mycoplasma and its findings are relevant to other bacterial species.

Mycoplasma is a microorganism that causes mastitis which is resistant to treatment, can go through a herd rapidly, and results in a significant reduction in milk production. Cows generally don�t develop systemic symptoms. Aggressive management, including culling, is required to control and eradicate this problem organism.

Therefore, it is important for the dairy farmer to quickly and correctly determine if this organism is in the herd. Failure to culture the organism, which requires a specific set of procedures, can lead to unnecessary economic hardships. Identification requires sample collection that can then be cultured for the organism. The question for the farmer is how to best handle the sample so that an accurate and reproducible result is obtained.

Researchers at Washington State University asked that question.1 They collected milk samples from animals with known Mycoplasma infection and either cultured them immediately or froze a sample for 2, 3 or 4 weeks before culturing. They also froze a sample, then rethawed it, then refroze the sample again before culturing for Mycoplasma. The bottom line was that freezing the samples caused fewer of them to culture positive for Mycoplasma. Freezing, thawing, then refreezing caused even fewer cultures to be positive. For example, culturing samples that had been frozen for 2 to 3 weeks caused 24 to 27% of the samples to be negative, which increased to 45% by 4 weeks. Thawing and refreezing caused another 2 to 14% increase in negative cultures. They found that if frozen samples were to be used, thawing the sample under ambient temperature conditions resulted in fewer negative cultures than thawing in a 37o C water bath.

The take-home message is that if you suspect Mycoplasma in your herd, don�t freeze the sample. Put it in the refrigerator and then ship the sample as quickly as possible. Freezing the sample and holding it decreases the odds of correctly culturing the organism. If you have a self-defrosting freezer, you may be putting the sample into a thawing and refreezing situation that could further decrease your chances of success. This is an organism that you want to identify quickly and correctly.

How about other organisms? It is generally accepted that Staph aureus can handle freezing, and this condition generally does not contribute to misdiagnosis. However, organisms such as E. coli and Strep non-agalactiae do not handle routine freezing and thawing very well, and this may result in a 29 to 36% loss of the organism in the sample. If you suspect one of these organisms you probably should put your samples in the refrigerator and get them to the laboratory as soon as possible for a correct diagnosis.

Correct milk sample collection is important to obtain a correct diagnosis of the mastitis-causing organism. This research also suggests that how you handle the sample AFTER collection can be just as important as using proper collection techniques, and that if you do it correctly you can maximize the chances of determining the cause of your problems.
1Biddle, M.K., L.K. Fox, D.D. Hancock, C.T. Gaskins and M.A. Evans. 2004. Effects of storage time and thawing methods on the recovery of Mycoplasma species in milk samples from cows with intramammary infection. J. Dairy Sci. 87(4): 933. ©