Geisinger Medical Laboratories Microbiology Specimen Collection Instructions


    1. Blood cultures are indicated for a sudden relative increase in patient's pulse rate and temperature, change in sensorium or blood pressure, chills, or prostration.
    2. Prolonged or intermittent and mild fever in association with heart murmur is also an appropriate indication.
    3. In general, any time bacterial, fungal, or mycobacterial sepsis is suspected, with the possible exception of minor mucocutaneous infections or of lower urinary tract infections.


  1. Obtain cultures prior to initiation of antimicrobial therapy if possible.
  2. For bacterial or fungal sepsis, two cultures per patient collected by separate venipuncture is the standard in the Geisinger system for all adults and children, except in unusual circumstances (e.g., neonates). For mycobacterial sepsis, three cultures collected on separate days is recommended.
  3. Numerous studies have been published regarding both the appropriate volume and numbers of blood cultures. The consensus of experts is that, except in very unusual cases, no more than four sets of blood cultures should be collected in one 24-hour period. If all four sets are negative after 24 hours and sepsis is still suspected, more cultures may be collected.
  4. No more than four sets of blood cultures are to be accepted for culture by the laboratory each 24 hours (calendar day) unless approved by the Microbiology doctoral director or Pathology resident on call.
  5. Separate venipunctures are required for each set of cultures. Exceptions may occur, such as in the case of a patient having poor veins or a bleeding problem. In these cases, the blood cultures may be collected with a single venipuncture; however, this should be avoided if at all possible. In addition, for young pediatric patients, two cultures may be collected from one venipuncture if specifically requested by the attending clinician. The two separate volumes should still be collected as for two separate venipunctures.
  6. Severe life-threatening septicemia: Two cultures, taken by separate venipuncture, should be collected immediately before starting treatment. There is no benefit for delaying a second venipuncture by 30 minutes when taking two blood cultures. We require that the blood cultures be obtained in duplicate from two separate venipunctures at the same sitting; however, as previously noted, no more than four total cultures should be collected in a 24-hour period.
  7. Suspected SBE or low-grade intravascular infection: Four cultures should be taken within the first 24 hours at intervals. Timing is not critical. In other situations, timing is difficult because bacteremia may precede the onset of fever or chills.
  8. A larger number of cultures may have to be collected from persons already receiving antimicrobics. Cultures should be taken immediately before the next dose of parenteral antimicrobial agent.
  9. Infants and small children: Two blood cultures usually are sufficient (one may suffice in the neonate).


  1. One blood culture consists of a FAN (Fastidious Antibiotic Neutralization) aerobic and a FAN anaerobic bottle. For patients < 13 kg, either one FAN aerobic bottle or one Pediatric FAN bottle is used (see table).
  2. FAN aerobic bottle = 30 mL BacT Alert bottle (green cap).
    FAN anaerobic bottle = 40 mL BacT Alert bottle (orange cap).
    FAN pediatric bottle = 20 mL BacT Alert bottle (yellow cap)
  3. Routine blood culture: This is used for culture of both bacteria and yeast: Two BacT Alert bottles (FAN aerobic and FAN anaerobic).
  4. AFB is cultured using a 10 mL green-top sodium heparin blood colleciton tube.
  5. Viral blood cultures: Lavender-top tube containing 7-10 mL of blood (EDTA anticoagulant).


  1. Materials needed:
  1. BacT Alert Blood Culture Bottles – consists of a FAN (Fastidious Antibiotic Neutralizations) aerobic (green) and a FAN anaerobic bottle  (orange). For patients <13 kg, one FAN aerobic bottle is used (yellow).
  2. Chloraprep One-Step Frepp Applicator
  3. A butterfly apparatus and one Saf-T holder Blood Culture device (CUP)
       for each set of cultures or one butterfly and one 20m> syringe for each  set.
  4. ChloraPrep Sepp applicator for cleaning the bottle tops.
  5. Gauze.

  B.  Procedure for the collection of routine blood cultures:

  1. Patient Identification - two forms of identification as per proper identification procedure.  (If using Collection Manager scan bracelet and print labels at bedside)
  2. Wash hands and apply gloves
  3. Apply a tourniquet and palpate arm for suitable vein.
  4. Release tourniquet.
  5. Using the Chloraprep One-Step Frepp Applicator, clean the patient's skin by scrubbing up and down and side-to-side.  Scrub for 30 seconds.  Allow to air dry.
  6. While drying, prepare the blood culture bottle(s) by cleaning the top of the bottles with Chloraprep Sepp applicator and marking a fill line on the blood culture bottles (10 mL’s each).
  7. Do not touch the venipuncture site unless your finger has been similarly disinfected.
  8. Venipuncture using Saf-T HOLDER Blood Culture Device with a Male luer adapter:
    1. Attach butterfly, perform venipuncture.
    2. Insert blood culture bottles green (aerobic) first, orange (anaerobic)
    3. Remove bottles, and insert adaptor for venipuncture tube back in  cup; continue drawing tubes with correct order of draw.
    4. Remove needle, activate safety device and apply pressure to venipuncture site.
  9. Venipuncture using butterfly and syringe.    
    1. Remove syringe from package.  Holding empty syringe, move plunger barrel out and then in as far as possible.  This will serve to make it
      easier to draw plunger back when actually drawing blood, and remove any excess air from syringe.    
    2. Place butterfly apparatus needle on syringe.
    3. For routine blood cultures, collect 20 mL of blood (if pediatric patient, 
      volume of blood is determined by patient weight; refer to table.
    4. Remove needle, activating safety device and apply pressure to 
      venipuncture site.
    5. Without allowing needle to become potentially contaminated by 
      touching bedding, remove the butterfly needle and replace with the 18 gauge transfer needle, place syringe directly into one blood culture bottle.  For routine cultures, distribute 10 mL into each bottle.  Inoculate the anaerobic (orange) first, aerobic (green) bottle second.      

C. Procedure for blood culture collection from Mediport and Broviac.

  1. Mediport: Clean and disinfect the skin as for a venipuncture and
       proceed to draw blood accordingly.
  2. Broviac:
    1. Clean the port with a ChloraPrep One-Step FREPP applicator.
    2. Disinfect the port using 2% tincture of iodine applicator. Do not allow iodine to pool in the stopper.
    3. Allow iodine to dry for one minute and collect the specimen.
  3. Write the time of collection, date, type of stick (straight or butterfly) and
       your tech code
    on the requisition form. Blood cultures will not be
       accepted by the Microbiology laboratory unless all of this information is

D.  Volume of blood to be collected for routine blood cultures:

  1.    Adults: 20 mL of blood should be collected by syringe and equally
       divided between each BacT Alert bottle. If this amount cannot be
       obtained, a lesser amount may be used and equally divided between
       the bottles. (If 5 mL or less is collected, place the entire amount in
       the green aerobic bottle.)
    Notify the laboratory if this occurs by writing
       amount collected on the laboratory requisition form.
  2.    Pediatrics: Collect as follows:
kg (lb)
Total Volume to Collect
Divide Among the Following

FAN BacT   Alert
(Aerobic) or
Pediatric Bottle (GMC Only)

FAN BacT Alert (Aerobic)

FAN BacT Alert (Anaerobic)

< 1.5 (3.3)



< 4.0 (8.8)



4-13 (8.8-28.6)



13-25 (28.6-55.0)





> 25.0 (55)





Pediatric guidelines were developed in conjunction with pediatric physicians at GMC, following guidelines developed at Mayo Clinic.

  1. Deliver bottles to the laboratory immediately so that incubation can be initiated. Be sure that the tech code of the phlebotomist is properly located on the label or requisition form. NOTE: Due to the critical importance of these culture results, every effort should be made to deliver inoculated blood culture bottles to Microbiology on the same day they are collected; however, if this is not possible, bottles must be kept at room temperature and delivered to the lab within 24 hours of collection.

Test Requested

May Be Requested By

Please Collect

Order and Chart As

Blood Culture

Any physician

BacT Alert Set 1

Blood Culture (BLC) or Blood Culture 2 (BLC)

Fungus, Blood, for Histoplasmosis

Infectious Disease Service

One 10 mL green-top sodium heparin blood collection tube

Fungus Culture- Blood (FCB)

AFB Blood Culture

Any physician

One 10 mL green-top sodium heparin blood collection tube

AFB Culture (AFBC)

1For pediatric patient weighing less than 13 kg (28.6 lb), collect single aerobic BacT Alert.


  1. Storage of blood culture bottles:
    1. Nursing units, clinics, and GML clients may store limited amounts of blood culture bottles.
    2. Store the blood culture bottles in a cabinet in a secure, low-traffic area separate from storage of other nursing items. The outside of the cabinet must be labeled with identification of contents.
  2.  All BacT Alert bottles must be incubated at 35°C upon receipt in the testing
     laboratory (the Microbiology Laboratory at GMC).
  3. Blood cultures are usually incubated for five days. Some organisms such as Brucella and certain streptococci may take longer than five days to grow. Brucella blood cultures are incubated for 21 days. The physician must obtain clearance from the Infectious Disease Department and notify the laboratory if he wants cultures held longer than five days. If approved by the doctoral director of Microbiology, any culture held longer than five days will be held for 21 days.


  1. Refer to hazardous clean-up procedure (Safety Manual).
  2. Fill out Employee Accident/Exposure Form.

Baron, Ellen, et al, 2005. Cumitech IC: Blood Cultures IV. American Society for Microbiology, Washington DC.

Biomerieux. Durham, NC. BacT Alert Culture Bottle product insert, September 2003.

Biomerieux. Durham, NC. BacT Alert PF bottle product insert, October 2004.

Washington, JA: 1985, p 27. Laboratory Procedures in Clinical Microbiology, 2nd ed. Springer Verlag, Princeton.

Revised: 2/27/2015

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