The Bacteriology section of the Animal Health Centre offers extensive microbiological services for the isolation and identification of a wide range of bacterial and fungal pathogens from avian, mammalian, aquatic, reptile, feed and environmental specimens.
The laboratory workup may include aerobic, anaerobic and microaerophilic culture, as well as enrichment culture for a number of specific pathogens including Salmonella, Campylobacter, Listeria and many others. Identification of bacterial and fungal organisms is performed using standard microbiological culturing techniques, various biochemical testing methods and in some cases DNA sequencing.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing (AST) is performed using the Kirby-Bauer disk diffusion assay and follows Clinical and Laboratory Standards Institute (CLSI) guidelines. AST testing is performed on organisms that are deemed clinically significant. We may not perform AST testing in cases where organisms are recovered from environmental sources, are common contaminants or are considered normal flora as these do not provide useful information and may promote unnecessary antibiotic usage.
Specimens for bacterial and fungal culture - submission requirements
Proper collection and handling of diagnostic specimens are critical for the success of bacterial and fungal culture. Diagnostic specimens for bacterial and fungal culture should be collected immediately after the animal first develops clinical signs and prior to any antimicrobial treatment. Collection of samples must be performed aseptically to prevent microbial contamination and overgrowth of primary pathogens. Improper handling and transport of specimens will hamper the recovery and identification of significant bacterial or fungal pathogens. Samples should be sealed and transported in a secure container to prevent leakage during shipment.
Collection and storage
Samples must be aseptically collected, individually labelled and refrigerated immediately after collection. Samples that cannot be shipped the same day to the laboratory should be stored at 4°C for a maximum of two days. Depending on type of submission, samples must be kept chilled or frozen. The use of ice pack refrigerants to keep specimens chilled during transit is extremely important. If specimens are frozen, they must remain frozen in transport and not allowed to thaw. For further information, please refer to the Submission Guidelines.
Environmental Samples: (dust, sawdust, bedding material): For culture of environmental samples please submit approximately 100gm (about 1 cup) of a representative sample in a securely sealed container or Ziploc bag. Do not submit environmental samples in specimen gloves. Please note that environmental samples submitted to the AHC are for culture only, no antibiotic sensitivities will be performed on environmental samples.
Environmental Sponges/Drag swabs/Booties: Environmental sponges, drag swabs, or booties may be submitted for Salmonella spp. culture. Please ensure specimens are double bagged and identifications are written clearly using permanent marker on each specimen bag. The exterior of the bag must be clean and dry.
- Commercially available environmental sponges may be utilized for environmental Salmonella testing. Sponges should be submitted in a Whirl-Pak bag with the top tightly rolled over multiple times and then the metal tabs folded over to ensure no leakage of specimen.
- Drag swabs (or gauze swabs) may be used for environmental Salmonella testing. The swabs must be submitted in a Whirl-Pak bag with the top tightly rolled over multiple times and then the metal tabs folded over to ensure no leakage of specimen. DO NOT submit more than 4-5 pieces of gauze per specimen bag. If using a liquid (such as buffered peptone water) to moisten swab then use only enough liquid to moisten the swab, do not saturate it.
- Booties may be used for environmental Salmonella testing. Submit booties in a sealed and labelled bag (large Whirl-Pak or Ziploc bag).
Feed (fresh or dry): For feed testing submit approximately 50-100 gm (about 1 cup) of a representative sample. For dry samples submit in a securely closed container or Ziploc bag. For moist or liquid feed samples submit in a securely closed container. Do not use Whirl-Pak or other bags for moist or liquid specimens. Please note that feed samples submitted to the AHC are for culture only, no antibiotic sensitivities will be performed on feed samples.
Fluff: Fluff samples may be submitted for the detection of Salmonella spp. Please submit fluff in a securely closed specimen cup or Whirl-Pak bag. Do not overfill the specimen cup or bag; the fluff samples should fill no more than ¼ of the specimen cup or bag.
Fresh tissues: Whenever possible, submit fresh tissues in a sterile, leak proof container for bacterial and fungal culture. Tissue samples should be kept separate and if possible, the ends of intestinal specimens should be ligated and intestinal samples separated from other tissues. Whenever possible, submit a 2-5cm piece of tissue with any lesions present. Autolyzed tissues are not suitable for culture.
Feces: Submit approximately 10gm (10-20ml volume) in a securely sealed sterile container. Outer surfaces of the container must be clean and dry. Do not submit feces in plastic bags or gloves.
Fluids (aspirates, pus, exudate etc.): All fluid or semi-fluid specimens should be collected aseptically in a sterile, leak-proof specimen container or vial. DO NOT submit syringes with needles attached.
Swabs: When fresh tissues, fluids or feces are not available, specimen swabs may be submitted for bacterial and/or fungal culture. Only use swabs with appropriate bacterial (aerobic or anaerobic) transport media for collection and shipment to the laboratory.
DO NOT submit dry swabs to the Bacteriology laboratory. Swabs for anaerobic culture must be submitted in an anaerobic culture transport media to ensure recovery of anaerobic organisms.
DO NOT FREEZE TRANSPORT SWABS FOR BACTERIAL CULTURE
Milk: Proper collection of milk samples is of paramount importance for identification of mastitis associated pathogens. Aseptic technique is necessary when collecting milk samples to prevent contamination by commensal organisms found on the cows' skin, udder, and teats; hands of the sampler; and in the barn environment. Contaminated samples result in misdiagnosis, increased work and expense and misinterpretation of results. . Contamination can be avoided by following the procedures described below.
Materials for Sampling Milk:
- Sterile vials or tubes – do not use plastic or Whirl-Pak bags for milk sampling
- 70% alcohol (ethyl or isopropyl)
- Cotton balls or gauze soaked in 70% alcohol, or commercially prepared, individually packaged alcohol swabs
- Examination gloves
- Cooler with ice or freezer packs for storing samples
- Racks for holding sample tubes or vials while sampling cows, and for cooler storage
- Disinfectant for cleaning teats (effective germicidal products used for premilking teat disinfection are recommended)
- Paper towels or individual cloth towels
- Means of identifying samples: permanent ink pen (with ink that is stable in both water and alcohol) or typed labels
- Label tubes prior to sampling (date, farm, cow, quarter).
- Brush loose dirt, bedding, and hair from the udder and teats. Thoroughly wash with germicidal product and towel dry dirty teats and udders before proceeding with sample collection. Udders should be washed as a last resort.
- Discard several streams of milk from the teat (strict foremilk) and observe milk and mammary quarters for changes in consistency or appearance of milk that may indicate clinical mastitis. Record all observations of clinical signs.
- Dip all quarters in an effective premilking teat disinfectant and allow at least 30 seconds contact time.
- Dry teats thoroughly with an individual paper or cloth towel.
- Beginning with teats on the far side of the udder, scrub teat ends vigorously (10 to 15 seconds) with cotton balls or gauze moistened (not dripping wet) with 70% alcohol. Teat ends should be scrubbed until no more dirt appears on the swab or is visible on the teat end. A single cotton ball or alcohol swab should not be used on more than one teat. Take care not to touch clean teat ends. Avoid clean teats coming into contact with dirty tail 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.
- Begin sample collection from the closest teat and move to teats on the far side of the udder. 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 degree angle while taking the sample. Do not allow the lip of the sample tube to touch the teat end. Collect one to three streams of milk and immediately replace and tightly secure the cap. Do not overfill tubes, especially if samples are to be frozen.
- To collect a composite sample (milk from all four quarters in the same tube), begin sample collection with the nearest teats and progress to the teats on the far side of the udder. One to 2 ml of milk should be collected from each quarter of the udder.
- When samples are taken at the end of milking or between milkings, teats should be dipped in an effective germicidal teat disinfectant following sample collection.
- Store samples immediately on ice or in some form of refrigeration. Samples to be cultured at a later date (more than 48 hours) should be frozen immediately.
Source: Microbiological Procedures for the Diagnosis of Bovine Udder Infection and Determination of Milk Quality. [NMC publication, 2004