Bacteriology

BacteriologyBacteriology

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:

  1. 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.
  1. 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. 
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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. 
  8. 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.
  9. 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!
  10. 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.
  11. 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.
  12. 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.
  13. Select samples judiciously! See the following list for guidelines, and call the laboratory if questions remain.
  14. Different bacteriologic procedures take different times to complete; approximate culture times are as follows:
Aerobic (culture and sensitivity)3 days
Anaerobic7-10 days
Listeria spp.7 days
Mycology>2 weeks
Mycoplasma 2 weeks
Mycobacteria paratuberculosis  (culture)
*Note, direct PCR is recommended for this agent.
13 weeks


Specimen Selection For Bacterial Diseases And Suggested Ancillary Testing

  All 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.

Disease

Specimen

Remarks & Suggested Ancillary Testing

AbortionDam: Placenta (cotyledon best in ruminants), vaginal swab, cervical mucus.

Fetus: liver, lung kidney, stomach contents, thoracic fluid.

Histopathology, Virology, Serology

Histopathology, Virology

AbscessSwabAnaerobic See note 9
Actinobacillosis or ActinomycosisExudate or lesion (sulfur granule if present)Anaerobic See note 9
Anthrax (B. anthracis)Blood, spleen, or lymph nodeNecropsy contraindicated
ArthritisCarcass, affected joint, synovial tissue, or joint fluidMycoplasma, 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 muscleHistopathology See note 9
Blood cultureInoculated blood culture system. 
Botulism Call the laboratory
BrucellosisSee abortionSerology

Campylobacteriosis

Enteric: 

Reproductive:

Feces

See abortion

Special transport media required
Caseous lymphadenitis (C. pseudotuberculosis)Affected lymph node or swab of contents 
ColibacillosisEntire carcass, loop of duodenum, mesenteric lymph node 
Corynebacterium equi pneumoniaSee 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 exudateHistopathology

EnteritisEntire carcass, loop of affected intestine, mesenteric lymph nodeHistopathology
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 pneumoniaEntire carcass
Thoracic fluid, lung, nasal swab
Histopathology, Serology
Hemorrhagic bowel syndrome (Lawsonia sp.)Non-culturable agentHistopathology, 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. 
Histopathology, Serology
Leptospirosis (See abortion)Entire carcass
Kidney, Urine
Serology, Darkfield examination, IHC, Molecular Diagnostics
Listeriosis
CNS
Abortion
Entire carcass
1/2 cerebellum, pons, medulla
Fetal liver, kidney, spleen
Histopathology
Malignant EdemaEntire carcass, LesionSee note 9, Histopathology
MastitisMilk. Use proper sample container half full or less. Call lab before submitting large (>30) numbers of samples.Mycoplasma cultures by specific request only
Mycoplasma
Arthritis
Mastitis
Pneumonia
Entire carcass
Joint fluid
Milk
Entire carcass or affected lobe(s)
Histopathology, Culture requires special media. Deliver immediately or freeze samples.
NocardiosisEntire carcass, LesionHistopathology
PasteurellosisEntire carcass, Affected lobeHistopathology
PneumoniaEntire carcass, Affected lobeHistopathology, Virology, Serology, Mycoplasma culture
PolyarthritisEntire carcass, Joint fluidHistopathology, Deliver immediately or freeze.
PolyserositisEntire carcass, Joint fluid and affected serosaHistopathology, Deliver immediately or freeze.
Rhodococcus equi pneumoniaAffected tissue, swab, or trans-tracheal aspirateHistopathology
Typed by PCR
SalmonellosisEntire carcass, Loop of intestine/colon, feces, lymph nodes, spleen, and lungsSerogroup typing at WADDL, forwarded to NVSL for serotype determination.
Salmonellosis - ReptilesFecalReptiles that test negative still represent high risk for salmonella exposure.
Strangles (Streptococcus equi)Swab of exudate 
Swine dysenteryEntire carcass, ColonHistopathology
Thromboembolic meningoencephalitis  (Haemophilus somnus)Affected organs, and 1/2 brainHistopathology, Serology
TrichomoniasisUterine exudate, preputial scrapings.Diamond's medium, Do not refrigerate or ship with ice packs
TuberculosisCulture not performed at WADDL, tissues sent to NVSLHistopathology
Tyzzer's Disease(Bacillus piliformis, now Clostridium piliformis)Culture not rewardingHistopathology, submit liver and gut in formalin
UreaplasmaVaginal swabs from affected cowsContact lab before collecting to ensure lab has media
VibriosisSee Campylobacter spp.