Beth Israel Deaconess Medical Center Laboratory Manual

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We live in a microbial world inhabited by bacteria, fungi, viruses, and parasites. This can make it difficult to obtain specimens that are free from contaminating organisms. For instance, many specimens are taken from areas of the body that have "normal" flora (e.g. sputum), or cultures from a sterile site are often collected through a contaminated or potentially contaminated area (e.g. blood, bronchoalveolar lavage, urine).

In order to get culture results that are interpretable, prior to sampling, the indigenous microbial flora in overlying and adjacent areas and surfaces must be decontaminated whenever possible. As appropriate, sterile supplies should be used and great care must be taken to avoid contact with areas and surfaces that have not been disinfected.

• Wash hands and put on sterile gloves.
• Prepare site with iodophor and/or alcohol solution using circular motions from the center to perimeter without returning to the previously cleaned site.
• Allow this solution to AIR DRY for at least 2 minutes. Antisepsis will be achieved only if the solution is allowed to dry.
• Repeat, if necessary, as for preparation of the skin prior to phlebotomy for blood culture collection.
• Loosen lid or cap of the specimen container or specialized transport device.
• Perform procedure
• If a syringe and needle have been used, use the "hands free" method to cap the needle or needle recapping device and REMOVE capped needle from syringe.
• Remove the lid without setting it down and put, express or inoculate the specimen into the container.
• Replace the lid of the container being careful not to touch surrounding surfaces.
• After discarding the sampling device in the appropriate discard container, if that is appropriate, tighten the lid of the specimen container.
• Label the specimen and requisition slip appropriately and deliver quickly to the Laboratory.

• Wash hands and put on sterile gloves
• Prepare site with tincture of iodine solution using circular motions from the center outward.
• Allow this solution to AIR DRY for at least 2 minutes. Antisepsis will be achieved only if the solution is allowed to dry.
• Aspirate material.
• Use "hands free" method to cap needle or needle recapping device and REMOVE capped needle from syringe.
• Quickly open the cap of an anaerobic transport tube, express up to 4.5 ml of material from the syringe into the tube, and quickly recap the tube and close TIGHTLY.
• If there is extra material, express the remaining culture material into a sterile collection container.
• Tightly close the cap of this container.

• SUBMIT THE MAXIMUM AMOUNT OF TISSUE POSSIBLE.
• Small samples of tissue may be placed in a small amount of non-bacteriostatic saline to prevent drying. Specimens in formalin are unacceptable for culture.
• Rapid transport is especially important for sensitive detection of anaerobes, especially for small pieces of tissue <10 mm. In this situation, hand carrying to the micro laboratory will be beneficial in ensuring optimal recovery of anaerobes and other fastidious organisms.
• Swabs should be used in the Operating Room ONLY in the RARE case when tissue (or fluid) are not obtainable. If a swab must be used, submit one anaerobic swab for EACH type of culture requested (e.g., three separate swabs would be needed for aerobic, anaerobic, and fungal cultures). Anaerobic swabs are provided, but aspirated fluids and tissue are better specimens for recovery of anaerobes.

• Prepare skin per kit instructions.
• Put on protective attire per hospital policy.
• Insert the needle and collect CSF into the 4 sterile tubes provided with the kit.
• Collect tubes 1 & 4 for Hematology testing, tube 2 for Chemistry testing, and 8 ml of fluid in the 3rd tube for microbiological analysis.
• This will provide enough fluid for culture of bacteria, fungi, AFB, viruses, for serologic tests, and a finite number of send out tests that may be necessary.
• At least 3 ml are necessary to culture for AFB.
• Note, that because of the low number of AFB present in the CSF during TB meningitis, smears are rarely positive. Further, due to the few organisms present and the fact that mycobacteria take a long time to grow (3-4 weeks minimum), culture results are not final for 8 weeks.