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Specimen Guidelines


  1. Determine the type of culture bottles to utilize, as indicated per physician's order (aerobic and anaerobic or resin and anaerobic bottles), or other types as specified below. If necessary, discuss timing of cultures, sites, need for any special instructions on the lab slip, etc., with the physician.

  2. Gather necessary equipment.
    1. If patient is not on antibiotics, a routine culture set consists of an aerobic (blue cap) and anaerobic (burgundy cap) culture bottle.
    2. If patient is on IV or PO antibiotics or has been off antibiotics for less than 24 hours or for suspected Neisseria or other fastidious organisms, a resin aerobic (green cap) may be substituted for the aerobic (blue cap) bottle. An anaerobic (burgundy cap) culture bottle should also be sent.
  3. Explain procedure to patient and/or significant other.
    1. Verify the patient's identification by checking the identification wristband for patient name and history number. If the patient does not have an identification wrist band, as in some outpatients, ask the patient to state his or her name and birth date and verify that these are the same as the name and birth date on the order.

  4. Wash or sanitize hands before and after removing gloves. Follow Standard Precautions for all patients. Masks with face shields should be worn for drawing blood cultures.

  5. Assemble necessary equipment before preparation of the patient's skin.
    1. Remove dust caps from culture bottles.
    2. Clean surface with alcohol wipe.
    3. Leave the alcohol wipe on the bottle top during skin preparation.
    4. Remove alcohol wipe just prior to inoculating the bottles - do not use iodine.

  6. Apply tourniquet to the extremity and identify the phlebotomy site.

  7. Preparation of the phlebotomy site:
    1. Preferred: Use Chloraprep® One-Step Frepp(Cumitech recommends cleaning with 70% isopropyl alcohol for 30 seconds prior to Chloraprep).
      1. Open the Chloraprep® One-Step Frepp.
      2. Squeeze the handle to break the ampule and scrub the skin with the foam surface.
      3. Use a firm scrubbing motion for 30 seconds to disinfect the site.
      4. A 5 cm area of the skin should be disinfected.
      5. Allow the site to dry at least 30 seconds before venipuncture.
    2. If unable to utilize Chloraprep® One-Step Frepp, use 1% tincture of iodine frepp.
      1. Povidone-iodine swabs permit more contamination than either chlorhexidine or 1% iodine frepps.
      2. Using the 1% iodine frepp, cleanse a 5 cm area using circular motion starting at the site and working outward.
      3. Allow to dry for a at least 30 seconds to allow antiseptic effect
      4. If using iodine product, clean patient's skin with alcohol to remove excess iodine (to prevent iodine burns).
    3. If unable to use Chloraprep® One-Step Frepp or iodine (e.g., allergic):
      1. Use alcohol pad to cleanse the patient's skin, using a circular motion starting at the site and moving outward.
      2. Repeat times two.
      3. Allow to dry.
  8. Do not touch the venipuncture site after skin preparation. If palpation is absolutely necessary, sterile gloves must be applied immediately prior to palpation.

  9. Insert needle into vein and withdraw appropriate amount of blood (See 3.10). Draw blood cultures prior to drawing other blood samples.
    1. Syringe collection and transfer into culture bottles (preferred method because it allows for exact measurement of 10 cc of blood into each culture bottle): If collecting into a 20 cc syringe you will need to remove the Vacutainer Adapter at the end of the Vacutainer Safety Lok® Blood Collection Set. After collecting into syringe, distribute the blood volume evenly between the culture bottles using the Vacutainer Safety Lok® needle. For blood culture procedures, needles should not be changed before inoculating culture medium. It is not necessary or safe.
    2. Collection into culture bottle: If collecting blood through a Vacutainer Safety Lok® Blood Collection set (butterfly needle) and a Blood Culture Adapter Cap, use the 5 ml markings on the side of the blood culture bottle to determine when appropriate amount of blood has been added to each bottle. It may be helpful to make a mark on the bottle, indicating appropriate fill volume. Keep blood culture bottles upright during procedure (do not let media come in contact with needle, i.e., do not hold culture bottle upside down). Post procedure, engage safety lock and discard entire set up. Blood Culture Adapter Caps are for single use only.

  10. Appropriate amount of blood for culture draw:
    1. 10 ml blood per culture bottle is preferred. Do not overfill.
    2. If an inadequate amount of blood was obtained (less than 5 cc), and repeat phlebotomy cannot be performed; all blood should be preferentially inoculated in the aerobic culture bottle (or Resin bottle if on antibiotics).

  11. If absolutely necessary to draw from a central catheter site, utilize the site that has been most recently inserted (unless ruling out catheter sepsis). Use the following procedure for drawing blood out of a catheter.
    1. Open sterile gloves, place near needle less port of catheter to be used. Tear alcohol swab and lay swab near edge of sterile field. Open 10 cc syringe and place on sterile glove field.
    2. Attach a Vacutainer Luer Adapter (blue cap) to a Blood Culture Adapter Cap. Place on edge of sterile glove field.
    3. Clean catheter infusion port closest to the insertion site with alcohol. Scrub thoroughly with alcohol pad for 30 seconds. You do not have the sterile gloves on yet.
    4. Allow to dry by placing needleless port on the open sterile field from sterile glove package.
    5. Don sterile gloves.
    6. Using the sterile syringe, withdraw 6 cc of blood and discard.
    7. Using Vacutainer Luer Adapter (blue cap) to a Blood Culture Adapter Cap, withdraw 7-10 ml of blood in each bottle. It may be helpful to mark bottle at appropriate fill volume. Blood culture bottles must be upright during the procedure (do not let the media come in contact with the needle, i.e., do not hold bottle upside down).
    8. If withdrawing blood into a syringe, use a Vacutainer Luer Adapter Sample (blue cap), a Blood Culture Adapter Cap and Double Connector to allow for needleless transfer of blood. After transfer of blood, discard entire system. Blood Culture Adapter Caps should not be reused.
    9. Flush per the VAD protocol.

  12. Label culture bottles with patient's name and history number. Do not place label over bar-coded area of the bottle.

  13. Fill out Pathology 5 (Microbiology-routine) lab slip.
    1. Indicate site from which blood was collected using comment section. If using a VAD to draw culture, you must indicate type and site of VAD in the comments section (i.e., left subclavian triple lumen).
    2. Select appropriate test (i.e., Blood Culture - 7510).
    3. Indicate suspected diagnosis, if necessary (required for R/O endocarditis).
    4. Include date and time of collection.
    5. Document that cultures were obtained on appropriate nursing form (e.g., Eclipsys).

  14. Send specimens to the laboratory as soon as possible (utilize pneumatic tube). Never refrigerate blood culture specimens. Send specimens directly to Microbiology Lab.

  15. Send second set of blood cultures using the same procedure as above. If a different peripheral site is possible, the second set may be drawn immediately. If using the same site, wait at least 10 minutes for the second set, and if possible (i.e. not waiting to give antibiotics) draw second set 1-3 hours later.

  16. In order to rule out diagnoses, more specific blood culture procedures may be necessary. See below for recommendations.
    1. Suspected catheter sepsis
      1. Draw two culture sets.
      2. One set is obtained from a suspected site.
      3. Second set must be from a separate peripheral site.
      4. If catheter is removed, send tip using sterile procedure for cultures. Do no send catheter tip without sending concommitant blood cultures.
    2. Acute endocarditis
      1. Draw two culture sets from two separate sites during the first 1-2 hours of evaluation.
      2. Begin therapy.
    3. Subacute endocarditis
      1. Draw 2-3 blood culture sets on day 1.
      2. If all are negative additional sets can be drawn on days 2 and 3 (no more than 4 sets in a 24 hour period).
      3. Immediate antibiotics are less important than establishing a specific microbial diagnosis.
    4. Endocarditis patients on anti-microbial therapy
      1. Draw resin blood culture sets on each of three successive days.
      2. Indicate "R/O endocarditis" in special instructions on lab requisition.
    5. Mycobacterial blood cultures (AFB)
      1. Use one Mycobacterial blood culture bottle (BD Bactec Myco/F Lytic culture bottle), because the media is unstable, bottles must be obtained from the Mycobacteriology (AFB) Lab (Meyer B1-124, 5-6470) on dayshift and the Microbiology Lab (5-6510) during evening hours. Pneumatic tube can be used to obtain culture media.
      2. Prepare skin as for routine blood cultures.
      3. Inoculate 1-5 ml blood into culture bottle (do not exceed 5 ml of blood). 
      4. Deliver the blood bottle in the brown bag promptly to the Microbiology Lab. Pneumatic tube can be used as long as sample is sent directly to the Microbiology Lab.
      5. A physician order must specify this culture in the orders.
    6. Fungal Cultures
      1. Candida spp. - If physician orders fungal cultures, follow routine procedure for bacterial cultures as described above.
      2. Histoplasma, Cryptococcus, or other filamentous organisms - If the order is specific for Histoplasma, Cryptococcus, or other filamentous orgnanisms, obtain an isolator tube from Microbiology Lab (call 5-6510).
        1. Isolator media is light sensitive and is stored in brown paper bags.
        2. Inoculate isolator tube with 5cc of blood (minimum 3cc).
        3. Send culture in brown paper bag directly to Microbiology Lab.
    7. Viral blood cultures (heparin tube not acceptable for PCR please see Molecular testing guidelines)
      1. Obtain 3 ml green top tube (heparinized) after preparing the skin as above.
      2. Deliver the tube promptly to the Microbiology Lab. Pneumatic tube can be used as long as sample is sent directly to the Microbiology Lab.
      3. Indicate suspected virus on the requisition to optimize culture technique and reduce cost.
      4. Viral blood cultures must be specifically ordered by the physician.
    8. Malaria, Babesia or other blood parasites
          1.  Draw 3 ml of blood in a lavender top (EDTA) vacutainer tube.
          2.  Deliver the tube to the Microbiology lab within 2 hours of collection.
          3.  Indicate patient's foreign travel (if available).
    9. Antibiotic Activity (Serum)
      1. Requires authorization by Infectious Disease, Microbiology Faculty or Clinical Pathology Resident.
      2. Collect 2-5 ml of blood in a red top tube pre and one hour post infusion.
      3. Include: patient isolate, culture number, specimen collection time, dosage time, and antibiotic program.
      4. Process requirements: aliquot 2 ml of serum into a plastic vial and freeze.