1. Number and timing
   Most cases of bacteremia are detected by using three sets of separately collected blood cultures. More than three sets of blood cultures yield little additional information. Conversely, a single blood culture may miss intermittently occurring bacteremia and make it difficult to interpret the clinical significance of certain isolated organisms. Whenever possible, peripheral sites are preferred. If a central line draw is done, a peripheral draw must be done also. When other lab work is needed, blood culture should be done first. Aseptic technique is critical to obtain reliable results. If venipuncture site is to be palpated before needle insertion, sterile gloves must be worn.

   (1) Acute sepsis
   Collect two or three sets of blood cultures from separately prepared sites without delay.

   (2) Endocarditis
   

   1. Acute
      Obtain three sets of blood cultures with three separate venipunctures over 1 to 2 h, and begin therapy.
   2. Subacute
      Obtain three sets of blood cultures on day 1 (15 min or more apart). If all are negative 24 h later, obtain three more.
   3. Antimicrobial therapy 1 to 2 weeks before admission
      Obtain two separate sets of blood cultures on each of three successive days.

   (3) Fever of unknown origin
   Obtain two separate sets of blood cultures at least 1 hour apart prior to starting antibiotics. If these are negative, then 24 to 36 h later, obtain two more sets of blood cultures.

2. Volume of blood
   The volume of blood is critical because the concentration of organisms in most cases of bacteremia is low, especially if the patient is on antimicrobial therapy. In infants and children, the concentration of organisms during bacteremia is higher than in adults, so less blood is required for culture.

   (1) Children and infants: 1 to 4 ml of blood per venipuncture
   (2) Adults: 20 ml of blood per venipuncture divided into two bottles, aerobic and anaerobic

3. Culture Technique
   (1) The BacT/Alert system of liquid culture media should be used for the isolation of aerobic, facultative, and anaerobic microorganisms and yeast.
   (2) The Isolator Lysis-centrifugation system is reserved for the detection of dimorphic fungi and must be obtained from the Microbiology Lab.
   (3) The MB Bact Alert System should be used for detection of mycobacteria. Obtain from Microbiology Laboratory.

Supplies

Two BacT/ALERT blood cultures bottles; one (green top) aerobic bottle, one (orange top) OR 1 pedi bottle (pink top), winged needle set, luer adapter, gauze, gloves, alcohol pads, 1 Chloraprep® One-Step Frepp kit. Optional items: large blood culture adapter, syringes, 18 gauge Needles

Procedure

1. Remove plastic cap from each blood culture bottle and disinfect the top using an alcohol pad, discard pad and allow top of bottle to dry.
2. Remove Frepp (Chloraprep®) from kit. Hold in a horizontal position and pinch the handle once to break the ampoule. DO NOT continue to squeeze handle. Place sponge on selected venipuncture site, and depress once or twice to saturate. Scrub vigorously with back and forth motion for 30 seconds and allow to air dry..

METHOD A:

1. Attach multiple sample luer adapter to winged set. This may be used separately or a blood culture adapter may be attached to the winged set-up.
2. Insert needle into the venipuncture site.
3. While holding the adapter, insert adapter needle into the aerobic culture bottle and allow to fill (10ml).
4. Remove adapter needle from the aerobic bottle and insert it into the anaerobic bottle. Allow to fill (10ml).
5. If additional blood work is ordered, attach a vacutainer tube holder or use culture adapter insert to the luer of the syringe and proceed with the blood draw.

METHOD B:

1. Attach syringe to the winged needle set.
2. Insert needle into the venipuncture site.
3. Draw one syringe: 10ml of blood for each of the two culture bottles.
4. Attach 18 gauge needle to a syringe. Insert into the aerobic bottle and allow to fill. DO NOT FORCE BLOOD INTO BOTTLE. Remove needle and syringe from bottle and discard both into a Biohazard "sharps" container.
5. Repeat procedure with anaerobic bottle and 2^nd syringe.
6. If additional blood work is required, you have the option of drawing additional syringes of blood, replacing the syringe with a luer adapter and vacutainer holder, or re-sticking the patient.
7. Label the culture bottles with patient’s name and MRN, fill in appropriate areas on the requisition form, ie. Date/time drawn, location of venipuncture site, whether peripheral or line draw, and name or initials of person drawing the sample.
8. Send all culture bottles to Microbiology (Tube station 73) without delay.

Related Information:

• This procedure must be performed using strict aseptic technique. Attention should be paid to maintaining sterility of the blood culture bottle tops as well as any needles and syringe openings.
• Use the following guidelines to distribute blood collected into blood culture bottles:
  • Less than or equal to 4 ml blood: use one (yellow) pediatric bottle.
  • 4 - 10 ml's of blood: place in one (green) aerobic bottle.
  • 10 - 20 ml's of blood: divide evenly into both (green)aerobic and (orange) anaerobic bottles.
• Drawing 20ml of blood using one syringe for both bottles is not encouraged. There is the risk of aerosol production when the needle is removed from the first bottle as "spray back" may occur. A face shield must be worn if this technique is used.
• Care should be taken to avoid letting air into the anaerobic blood culture bottle. Expel any dead air space in syringe before injecting blood.

Mycobacterium Culture Procedure

If a blood culture for mycobacterial or AFB is to be drawn, one must call Microbiology to get the appropriate MB BacT Mycobacterial bottle sent to the respective floor.

NOTE: Up to three (3) specimens collected on successive days may be appropriate.

• The same aseptic procedure is used that is followed for Blood Cultures.
• Remove plastic top from the culture bottle and disinfect with an alcohol pad and allow to air dry.
• Collect the blood using a winged set without anticoagulant and inoculate directly into the MB BacT culture bottle (3-5 ml per bottle). To prevent over inoculation, monitor the blood volume intake into the bottle, using the 5ml incremental markings on the bottle label.
• Label the bottle with the appropriate patient information.
• Transport the inoculated culture bottle promptly to the Microbiology Laboratory.