Used correctly, microbiologic cultures can identify etiologic agent(s) and contribute key information towards a diagnosis. However, improperly collected microbiologic cultures may identify contaminants or overgrow pathogens and lead to erroneous diagnoses. Etiologic agents can be missed because of improper transport medium, improper transport environment, or improper preservation techniques.
The value of microbiologic culture depends to a considerable degree on the care and skill with which cultures are taken, stored and shipped to the laboratory.
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We offer the following guidelines to optimize these procedures:
- Samples should be collected aseptically and placed in sterile plastic bags (e.g., Whirl-pak) or heat sterilized containers. Seal tightly. Do not use chemically disinfected containers, or plastic gloves or sleeves.
- Label all submissions with the location (tissue) and species of origin. The same bacterial species may be highly significant or a meaningless contaminant, depending on the tissue and/or species from which the sample was obtained. Also, depending on the tissue/species of origin, different culture conditions may be necessary to isolate and identify specific pathogens.
- If possible, specify the test(s) you want done, and the pathogens you suspect, particularly in the case of specimens with normal bacterial flora (feces, intestinal contents, skin, or oral mucus membranes). If we don't know what you're looking for, we may not inoculate the proper media to find it.
- It is best to collect other samples before opening the gastrointestinal tract. Tissue samples (lung, liver, spleen, kidney, etc.) should be 5 g or larger to allow surface searing in the laboratory to reduce contaminants. Use screw top containers for fecal samples. Fecal samples should not be submitted in stoppered tubes, as fermentation will dislodge the stoppers.
- Place each sample in a separate container to prevent cross-contamination. If the intestine is to be cultured, tie off both ends of a segment and place it in a separate container.
- Except in the case of abortions, please separate samples that are to be examined by different laboratory sections. If a specimen is to be examined by both the virology and bacteriology sections, the specimen should be divided, each piece placed in a separate container, and labeled with the source of the tissue and the desired laboratory service.
- Fluids for culture (e.g. body cavity fluids, pericardial fluid, joint aspirates) should be submitted in a sealed sterile tube, in as large a volume as is available (up to ~10 ml), since the concentration of organisms may be very low in these samples. Fluids may be submitted in blood culture bottles or Isolator tubes (keeping in mind that Isolator tubes must be processed in the lab within 24 hrs) for highest sensitivity. Fetal fluids (thoracic or peritoneal fluids, or heart blood) to be examined for Leptospira sp. by FA test are best submitted in a sealed sterile tube to which 10% buffered formalin is added at a rate of 1.5 ml per 20 ml fluid. Never submit fluid in syringes, which tend to leak in transit and contaminate packaging. Never submit fluids or other specimens for bacteriologic culture in EDTA (purple top) Vacutainer tubes, as EDTA is highly toxic to many bacterial species.
- Milk samples should be submitted in screw top tubes frozen or placed on ice packs. Less than 1 ml is required. Large volume milk samples submitted frozen may lead to delayed analysis.
- Specimens for isolation of anaerobic pathogens require special care. Anaerobic bacteria are very susceptible to oxygen exposure. Tissue or fluid specimens are preferred, and if swabs are the only practical sample, the Port-a-Cul system is preferred. Cultures for Clostridium sp. in parenchymatous organs ordinarily provide no significant information concerning the cause of death if the samples are taken more than one hour after death.
- Some specimens, such as porcine nasal swabs for Bordetella sp. isolation must be delivered to the laboratory within 12 hours of collection. Fastidious organisms such as Campylobacter spp. require special media for transport to the laboratory. Where there is any doubt as to what samples to collect and how to transport them - - CALL THE LABORATORY FIRST!
- When collecting large numbers of samples (e.g. >30 milk samples for mastitis diagnosis or fecal samples for Johne's disease diagnosis), please call the laboratory for scheduling. This permits the laboratory to have personnel and media available for prompt processing.
- In general, it is advised to keep specimens cold from the time they are collected until they arrive at the laboratory. Specimens should be shipped in insulated containers with a sufficient number of ice packs to last 48 hours.Specimens arriving in the laboratory in a decomposed state will not be processed, since processing and culture of these tissues lead to meaningless or erroneous results.
- For cases where bacteremia is suspected and blood culturing is requested, blood culture systems should be inoculated with the proper amount of blood collected aseptically. Single bottle blood culture systems are recommended.
- Select samples judiciously! See the following list for guidelines, and call the laboratory if questions remain.
- Different bacteriologic procedures take different times to complete; approximate culture times are as follows:
|Aerobic (culture and sensitivity)||3 days|
|Listeria spp.||7 days|
|Mycobacteria paratuberculosis (culture)|
*Note, direct PCR is recommended for this agent.
Specimen Selection For Bacterial Diseases And Suggested Ancillary TestingAll cases are charged a $10.00 accession fee. Available procedures and prices can be accessed in the WADDL Web Test Search Tool. Type in "bacteriology" and click on Search.
Remarks & Suggested Ancillary Testing
|Abortion||Dam: Placenta (cotyledon best in ruminants),
vaginal swab, cervical mucus.
Fetus: liver, lung kidney, stomach contents, thoracic fluid.
|Histopathology, Virology, Serology
|Abscess||Swab||Anaerobic See note 9|
|Actinobacillosis or Actinomycosis||Exudate or lesion (sulfur granule if present)||Anaerobic See note 9|
|Anthrax (B. anthracis)||Blood, spleen, or lymph node||Necropsy contraindicated|
|Arthritis||Carcass, affected joint, synovial tissue, or joint fluid||Mycoplasma, Anaerobic See note 9|
|Atrophic rhinitis (B. bronchiseptica)||Swabs, must arrive at the lab within 12 hours.||Histopathology|
|Black leg (Clostridial myositis)||Entire carcass, Affected muscle||Histopathology See note 9|
|Blood culture||Inoculated blood culture system.|
|Botulism||Call the laboratory|
|Special transport media required|
|Caseous lymphadenitis (C. pseudotuberculosis)||Affected lymph node or swab of contents|
|Colibacillosis||Entire carcass, loop of duodenum, mesenteric lymph node|
|Corynebacterium equi pneumonia||See Rhodococcus equi|
|Contagious equine metritis (Taylorella equigenitalium)||Female: Cervical secretions|
Male: Urethral fossa secretions
|Not offered by WADDL. Call for suggested laboratories.|
|Dermatophilosis (Dermatophilus congolensis)||Scabs, purulent exudate||Histopathology|
|Enteritis||Entire carcass, loop of affected intestine, mesenteric lymph node||Histopathology|
E. coli typed by PCR
|Enterotoxemia (Clostridium perfringens)||Intestines or intestinal contents. Must be collected within 4 hours of death for interpretation of results.||Histopathology
Typed by PCR
|Erysipelas (Erysipelothrix rhusiopathia)||Entire carcass, Liver, heart, spleen, kidney, lymph node, affected joint.||Histopathology|
|Glasser's disease (Haemophilus polyserositis)||Entire carcass|
Unopened joint and inflamed serosa
|Deliver immediately or freeze|
|Hemophilus pneumonia||Entire carcass|
Thoracic fluid, lung, nasal swab
|Hemorrhagic bowel syndrome (Lawsonia sp.)||Non-culturable agent||Histopathology, Serology, Molecular diagnostics|
|Johne's disease (Mycobacterium paratuberculosis)||Entire carcass|
Rectal biopsy or large intestine/ileocecal valve, regional lymph node, feces (2-10g)
|Recommended test for fecal samples is PCR. |
|Leptospirosis (See abortion)||Entire carcass|
|Serology, Darkfield examination, IHC, Molecular Diagnostics|
1/2 cerebellum, pons, medulla
Fetal liver, kidney, spleen
|Malignant Edema||Entire carcass, Lesion||See note 9, Histopathology|
|Mastitis||Milk. Use proper sample container half full or less. Call lab before submitting large (>30) numbers of samples.||Mycoplasma cultures by specific request only|
Entire carcass or affected lobe(s)
|Histopathology, Culture requires special media. Deliver immediately or freeze samples.|
|Nocardiosis||Entire carcass, Lesion||Histopathology|
|Pasteurellosis||Entire carcass, Affected lobe||Histopathology|
|Pneumonia||Entire carcass, Affected lobe||Histopathology, Virology, Serology, Mycoplasma culture|
|Polyarthritis||Entire carcass, Joint fluid||Histopathology, Deliver immediately or freeze.|
|Polyserositis||Entire carcass, Joint fluid and affected serosa||Histopathology, Deliver immediately or freeze.|
|Rhodococcus equi pneumonia||Affected tissue, swab, or trans-tracheal aspirate||Histopathology|
Typed by PCR
|Salmonellosis||Entire carcass, Loop of intestine/colon, feces, lymph nodes, spleen, and lungs||Serogroup typing at WADDL, forwarded to NVSL for serotype determination.|
|Salmonellosis - Reptiles||Fecal||Reptiles that test negative still represent high risk for salmonella exposure.|
|Strangles (Streptococcus equi)||Swab of exudate|
|Swine dysentery||Entire carcass, Colon||Histopathology|
|Thromboembolic meningoencephalitis (Haemophilus somnus)||Affected organs, and 1/2 brain||Histopathology, Serology|
|Trichomoniasis||Uterine exudate, preputial scrapings.||Diamond's medium, Do not refrigerate or ship with ice packs|
|Tuberculosis||Culture not performed at WADDL, tissues sent to NVSL||Histopathology|
|Tyzzer's Disease(Bacillus piliformis, now Clostridium piliformis)||Culture not rewarding||Histopathology, submit liver and gut in formalin|
|Ureaplasma||Vaginal swabs from affected cows||Contact lab before collecting to ensure lab has media|
|Vibriosis||See Campylobacter spp.|