First and foremost part of mastitis diagnosis is sample collection, let us have a look.
In order to collect samples aseptically, aseptic technique is required. Sample quality is critical in any diagnostic method; however, sample quality for mastitis diagnosis can be more difficult to achieve than for other infections. Organisms with the potential to cause mastitis are ubiquitous and prevalent throughout any dairy, and hence samples can readily become contaminated if adequate sampling technique is not used. Contaminated samples result in misdiagnosis, additional work, misunderstanding, and irritation.
Sample storage and handling are just as crucial as sample collection. The majority of mastitis-causing microbes can withstand chilling for several days or freezing for several weeks. Improper chilling, chemicals, and contaminating organisms in the sample can all affect diagnostic results by affecting pathogen development or, in the case of contaminating organisms, overgrowing the pathogen and obscuring the diagnostic results. Some species, such as E. coli, Nocardia spp., and Mycoplasma spp., may not be able to tolerate prolonged refrigeration or freezing.
Shortcuts in the initial phases of diagnosis can waste a lot of time, energy, and money. Some environmental factors may make aseptic sample collection impossible. Doctors must understand how to identify contaminated samples. Following the finding of a contaminated sample, the most prudent choice is to make no diagnostic of infection status and resample the cow.
Materials for Sampling
| Sterile tubes | Sterile vials or tubes, 5- to 15-ml capacities |
| Alcohol | 70% alcohol (ethyl or isopropyl) |
| Cotton or gauze wipes | Cotton balls, gauze squares, or pledgets soaked in 70% alcohol. or commercially prepared, individually packaged alcohol swabs |
| Gloves (latex or nitrile) | To prevent bacteria on hands from contaminating sample |
| Cooler with ice packs | Cooler with ice or freezer packs for storing samples |
| Markers | Means of identifying samples: permanent ink pen (with ink that is stable in both water and alcohol) or typed labels |
| Plastic wrap or sealable bag | To either wrap or place samples in, respectively, and provide secondary containment during transport |
| Racks | Racks for holding sample tubes or vials while sampling cows and for storage in the cooler |
| Teat dip | Disinfectant for cleaning teats (germicidal products used for pre-milking teat dipping are recommended) |
| Towels | Paper towels or individual cloth towels |
Sampling Technique
| Label tubes | Label tubes prior to sampling (date, farm, cow, and quarter, as applicable). |
| Gloves | Put on clean latex or nitrile gloves prior to starting sampling. Gloves should be kept clean and replaced if they become contaminated or ripped. |
| Clean teats | Using a hand or a dry paper towel, brush loose dirt, bedding, and hair from the gland and teats. Grossly dirty teats and udders should be washed and dried thoroughly before proceeding with sample collection. Udders should be washed as a last resort. |
| Forestrip | Discard a few streams of milk from the teat (strict foremilk) and observe milk and gland for signs of clinical mastitis. Record all observations of clinical signs. |
| Pre-dip | Pre-dip all quarters in an effective pre-dip product and allow 30 seconds contact time |
| Dry teats | Dry teats thoroughly with a paper towel or clean individual cloth towel. |
| Alcohol scrub | Beginning with teats on the far side ofthe udder, scrub teat ends vigorously (10 to 15 seconds) with cotton balls, gauze squares or wipes, moist (not dripping wet) with 70% alcohol. When cotton balls are saturated with alcohol, simply squeeze out excess alcohol prior to use. Use as many cotton balls, gauze squares or wipes, as necessary, to clean the teat ends. Teat ends should be scrubbed until no more dirt appears on the swab or is visible on the teat end. A single cotton ball, gauze square, or wipe should not be used on more than one teat. Care should be taken not to touch clean teat ends. Also, care should be taken to avoid clean teats coming into contact with dirtytail switches, feet, and legs. In herds where cows are not cooperative, begin by scrubbing the nearest teat until clean, obtain the sample, and move to the next teat. |
| Sample | To collect individual quarter milk samples, begin sample collection from the closest teat and move to teats on the far side of the udder – the reverse order from cleaning – to collect the sample, remove the cap from the tube or vial but do not set the cap down or touch the inner surface of the cap. Always keep the open end of the cap facing downward. Maintain the tube or vial at approximately a 45° angle while taking the sample. Do not allow the lip of the sample tube to touch the teat end. Strip one stream of milk outside the tube, then collect one to three streams of milk and immediately replace and tightly secure the cap. Make sure milk entering the tube does not touch fingers or hands. Two to 3 ml of milk is generally a sufficient sample size, and there is seldom need to collect >5 ml. Sample vials should never be filled more than three-fourths full. Large volume samples are not required and increase the risk of contamination. To collect a composite sample (milk from all four quarters in the same tube), begin sample collection with the nearest teats and progress to teats on the far side of the udder. A representative sample (1 to 2 ml) should be collected from each quarter of the udder. There is greater risk of contamination of composite samples because tubes are open for a longer period of time. |
| Teat dip | When samples are taken at the end of milking or between milkings, teats should be dipped in an effective germicidal teat dip following sample collection. |
| Store samples | Store samples immediately on ice or refrigerate. Samples to be cultured at a later date (after 24 to 48 hours) should be immediately frozen (-20°C). |
