Illinois Veterinary Diagnostic Laboratories - Laboratory User's Manual

 

Bacteriology

Used correctly, microbiologic cultures can identify etiologic agent(s) and so contribute key information towards a diagnosis. However, improperly used microbiologic cultures can identify contaminants or overgrowing organisms and lead to erroneous diagnoses. Actual etiologic agents can be lost due to improper transport medium, improper transport environment, or improper preservative 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.
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.
2. 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 or origin, different culture requirements may be necessary to isolate and identify specific pathogens.
3. Always specify the tests 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 are looking for, we may not inoculate the proper media to find it.
4. It is best to collect other samples before opening the gastrointestinal tract. Tissue samples (lung, liver, spleen, kidney, etc.) should be 2 cm or larger to allow surface searing in the laboratory to reduce contaminants. Fecal samples should not be submitted in stoppered tubes, as fermentation will dislodge the stoppers.
5. Place each sample in a separate container. If the intestine is to be cultured, tie off both ends of a segment and place in a separate container.
6. Fluids for culture (i.e., body cavities, pericardium, joints, etc.) are best collected with a culturette or submitted in a sealed sterile tube or blood culture bottle. Fetal fluids (thoracic or peritoneal fluids, heart blood) to be examined for leptospiras should be submitted in a sealed sterile tube as soon as possible. Never submit fluids or other specimens in EDTA blood tubes, as EDTA is highly toxic to bacteria.
7. Milk samples should be submitted in screw–top tubes and frozen or placed on ice packs. Less than 1 ml is required for culture, and larger volumes are undesirable, if the samples are frozen. If an evaluation of somatic cell count is made, larger volumes (10 ml) of milk is required.
8. Specimens for isolation of anaerobic pathogens require special care. Anaerobic bacteria die in the presence of oxygen and should be shipped in an reduced container, such as anaerobic swab or Port–a–Cul system. Cultures for Clostridium spp. in parenchymatous organs and intestines ordinarily provide little 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 spp. 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. > 15 milk samples for mastitis diagnosis or > 10 fecal samples for Johne’s
disease diagnosis), call the laboratory for scheduling. This permits the laboratory to have personnel and media available for prompt processing.
11. 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. 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. Submit to the laboratory immediate in an insulated container with ice packs.
13. Select samples judiciously! See Appendix A 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/sensitivity) 3 days
Anaerobic 7–10 days
Mycology 3 weeks
Mycoplasma 4 weeks
Mycobacterium paratuberculosis 13 weeks
Listeria spp. 1–2 months
Leptospira spp. 1–2 weeks
E. coli serotyping 3 days

Revised 1-3-00 website designer Lou Ann Miller