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Canadian Bovine Mastitis Research Network

All about Mastitis I Diagnosis

Equipment I  Time of Sample Collection I Preparing Udder and Teats I Collecting Samples I Handling and Storing Samples

Procedures for Collecting Milk Samples

Strict aseptic procedures must be used when collecting milk samples in order to prevent contamination with the many microorganisms present on the skin of cow's flanks, udder and teats, on the hands of the sampler, and in the barn environment. The following procedures help to reduce contamination during sample collection.

Sterile glass or disposable plastic vials with tight fitting push-on or screw caps are satisfactory. Vials of at least 15 ml capacity are usually most convenient to handle, but smaller vials may be used. Glass vials should be sterilized with the caps loosened during autoclaving, then tightened after the vials have cooled. Sample vials should be labelled with a waterproof marker to identify the cow and quarter. Suitable racks are necessary for convenient handling of the vials.

Time of Sample Collection
Samples for culture may be collected before or after milking or in the intermilking interval. The time chosen will frequently be the sampling time that is most convenient under the management conditions of the individual herd. However, if a somatic cell count or screening test that depends on the cell content of the milk is to be run, it is important to recognize that immediately after milking and for at least six hours thereafter, the cell count will be elevated above that present just before milking. Therefore, if cell counts or screening test results are to be compared, the sampling times relative to milking should be consistent.
Preparing Udders and Teats
Udders and especially teats must be clean and dry before sample collection is attempted. First remove and discard a few streams of milk to reduce the number of contaminating bacteria in the teat canal. Teats that are clean should not be washed, but dust, particles of bedding, and other soil should be removed by brushing the surface with a dry paper towel. Dirty teats must be washed, preferably with a clean paper towel and disinfectant solution, then dried with a second paper towel. As an additional procedure, especially in research trials, some workers prefer to dip the teats in a germicide, such as a 1% iodophor or 4% hypochlorite solution before sampling. The germicide is allowed to remain on the teats for 20 to 30 seconds and removed by wiping with a dry paper towel.
Each teat end is then scrubbed vigorously with a pledget of cotton or gauze sponge moistened (but not completely wet) with 70% ethyl or isopropyl alcohol. Alcohol is the preferred antiseptic, as it evaporates quickly and contributes no bactericidal residue to the milk sample. Use a separate pledget or sponge for each teat. Scrubbing should be continued until a new surface of the cotton or sponge remains clean. More than one pledget or sponge may be needed to clean a teat end properly. To prevent recontamination of teats during scrubbing, scrub the teats on the far side of the udder first, then those on the near side. When duplicate samples are to be collected, as is required by some research protocols, the teat end must be scrubbed a second time after the first sample has been collected and before the second is obtained.

Collecting Samples
To reduce contamination of the teat ends during sample collection, sample the near teats first, then the far ones. Remove the cap from the sample vial, and without touching its inner surface, hold the cap so that the inner surface faces down. Hold the vial as near the horizontal as possible, and by turning the teat to a near horizontal position, direct streams of milk into the vial. Do not allow the cap or the vial opening to touch the teat end. A sample size of 3 to 4 ml is usually adequate. To minimize the opportunity for contamination, collect each sample and replace the cap as quickly as possible.
Collection and culture of samples from individual quarters is usually preferred, but it may be advantageous in a large herd test to collect a composite sample from each cow. After teats are prepared and sanitized as above, draw approximately equal volumes from each of the quarters into a common vial. Collecting samples quickly to minimize the chance of contamination is especially important with composite samples.
Handling and Storing Samples
After samples have been collected and placed in racks for ease of handling, they should be held in an ice chest at 5°C. In the laboratory, samples should be cultured immediately, or stored at 4-5°C and preferably cultured within 24 hours. However, isolation of most staphylococci and streptococci is not affected greatly when samples are stored at 4°C for one week or for up to six weeks. Nocardia spp. are an exception to this general rule, as storage of samples for only a few hours or freezing can reduce the likelihood of isolating these organisms. The numbers of samples positive for Escherishia coli may decline after freezing. Samples that have been frozen cannot be used for somatic cell counts.
From: "Microbiological Procedures for the Diagnosis of Bovine Udder Infection", 3rd Edition, National Mastitis Council, 1990 Source : www.nmconline.org



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