Bacterial Culture
BT 6004
356-2591 Bacteriology/Virology Section
Delivery Instructions:
Deliver to laboratory immediately after collection.
Gram stains are automatically performed on fluids and exudates 
other than blood and urine.  Do not send sterile body fluids in 
plastic red top tubes.  These tubes contain a clot activator which may 
make testing unreliable. 

Label transport tube with two patient identifiers, date and 
time of collection. Transport at room temperature unless otherwise 
A. Abscess - Tissue or aspirates are always superior to swab
   specimens.  Remove surface exudate by wiping with sterile saline or
   70% alcohol.  Aspirate with needle and syringe. Cleanse rubber
   stopper of anaerobic transport vial (59546) with alcohol; allow to
   dry 1 min before inoculating; push needle through septum and inject
   all abscess material on top of agar. If a swab must be used, pass
   the swab deep into the base of the lesion to firmly sample the 
   fresh border. Transport time ≤2 hours.

B. Anaerobic cultures - Tissue or aspirates are preferred 
   rather than swabs. Fluid collections should be aspirated through 
   disinfected tissue or skin. For superficial ulcers, collect material
   from below the surface (after surface debridement or use a needle
   and syringe).  Submit specimens using anaerobic transport media:
   a. Anaerobic transport vial (fluid specimen, 59546): Cleanse rubber
      stopper with alcohol; allow to dry 1 min before inoculation; 
      push needle through septum and inject specimen on top of agar.
   b. Anaerobic jar (tissue specimen, 59547). Place sample on top of
      agar.  Keep jar upright to maintain atmosphere in jar.
   c. A sterile container (37777) may be used for tissue if
      transported to the microbiology lab immediately (add drops of
      sterile saline to keep small pieces of tissue moist).
   d. Copan Liquid Amies Elution Swab (ESwab) (74541) - swab specimens
      are suboptimal, but will be accepted if no other sample can be
   e. Deliver all specimens to the laboratory immediately after 
   f. Anaerobic flora is prevalent on mucosal surfaces of the oral
      cavity, upper respiratory, gastrointestinal, and genital tracts;
      specimens collected from these sites should not ordinarily be
      cultured for anaerobic bacteria. The following is a list of
      specimens that are likely to be contaminated with anaerobic
      normal flora and are NOT routinely accepted for anaerobic
      1) Throat or nasopharyngeal swabs
      2) Gingival or other intraoral surface swabs
      3) Expectorated sputum
      4) Sputum obtained by nasotracheal or endotracheal suction
      5) Bronchial washings
      6) Voided or catheterized urine
      7) Vaginal or cervical swabs
      8) Gastric and small bowel contents (except for "blind loop" or
         bacterial overgrowth syndrome)
      9) Feces (except for specific etiologic agents such as
         C. difficile and C. botulinum)
     10) Rectal swabs-Surface swabs from ulcers and wounds(collect
         material from below the surface)
     11) Material adjacent to a mucous membrane that has not been
         adequately decontaminated

C. Blood
   a. Adult - Cleanse oil or visible dirt from site with
      alcohol pads before using ChloraPrep skin prep. Cleanse skin with
      ChloraPrep® one-step 1.5 mL Frepp®
      1) Holding the applicator sponge downward, pinch wings on
         applicator to break ampule and release the antiseptic.
      2) Use a side-to-side motion to scrub the site with the friction
         pad for a full 30 sec; allow site to dry completely (at least
         30 sec) before venipuncture. Do not touch site after prep.
      3) Remove overcaps from bottles (1 aerobic 924171 and 1 anaerobic
         924172) and cleanse each rubber septum with separate 70%
         alcohol swabs. Allow septum to dry for 1 min before
      4) Draw 20 mL of blood and inoculate each bottle with 10 mL of
         blood.  Do not vent or overfill bottles. Adding low (<8 mL)
         or high (>10 mL) volumes may adversely affect the recovery of
         organisms.  Transport time <2 h.
      5) For adults with a suspected bloodstream infection (BSI),
         collect three initial sets of blood cultures sequentially from
         separate phlebotomy procedures. Ideally, three venipunctures
         should be performed immediately but a third set of bottles
         can be drawn at a 4-6 hour delay without significant loss of
         yield. (will detect >98% of BSIs).  
      6) If patient is allergic to chlorhexidine, prep site with a
         povidone iodine swab stick (907172) applied in concentric
         circles (start at center). Allow to dry at least 1 min before
         venipuncture. If patient is allergic to iodine, cleanse site
         with 70% alcohol for 60 sec.
   b. Pediatric - Apart from NICU patients, the minimum volume
      drawn should be 1 mL per year of age per blood culture set. This
      volume should be split between an aerobic and anaerobic bottle.
      See pediatric blood culture order for more detail.

D. Bone marrow aspirate - Prepare puncture site as for surgical
   incision.  Inoculate yellow top tube (104184).  Transport time <2
E. Burn - Clean and debride burn. Place tissue in sterile screw-
   cap container (37777). Transfer aspirates to a sterile container.
   These are processed for aerobic culture only. Quantitative culture
   may or may not be valuable. A 3 to 4 mm punch biopsy specimen is
   optimum when quantitative cultures are ordered. Cultures of surface
   samples can be misleading.

F. Catheter Tips - Catheter tips are not routinely accepted for
   culture.  Consult Microbiology laboratory for approval (pager 4903
   weekdays; pager 3404 evenings and weekends). Foley catheters are not
   accepted for culture since growth represents distal urethral flora.

G. Cerebrospinal Fluid (CSF) - Obtain CSF for gram stain, cell
   count, protein, glucose and aerobic culture where able. Obtain kit 
   922257 (20G needle) or 922258 (22G needle) from Hospital Stores 
   356-1784). The kit should contain 4 pre-numbered tubes to be filled
   in chronological order. Avoid covering tube numbers with 
   stickers to ensure appropriate routing of samples.

   • With low volume, one-tube specimens not all testing may be
     possible and the clinician must determine which tests should be
     prioritized. If cultures are desired, Microbiology must receive
     the specimen first to ensure the culturing of a sterile specimen.

   • Transport time <15 minutes.  Do not refrigerate CSF
     for bacterial culture.

   Refer to the Microbiology Specimen Collection and Transport page
   for orders placed per tube.

H. Decubitus ulcer - A swab is not the specimen of choice.
   Cleanse surface with sterile saline. Submit tissue or aspirate 
   inflammatory material from the base of the ulcer in a sterile tube
   or anaerobic system.  Transport time is ≤2 hours.

I. Ear
   a. Inner ear - Tympanocentesis should be reserved for
      complicated, recurrent, or chronic persistent otitis media. For
      intact eardrum, clean ear canal with soap solution and collect
      fluid via syringe aspiration. Submit in sterile container. For
      ruptured eardrum, collect fluid on flexible shaft swab via an
      auditory speculum.  Transport time <2 hours.
   b. Outer ear - Use moistened swab to remove any debris or
      crust from ear canal. Obtain sample by firmly rotating swab in
      outer canal. For otitis externa, vigorous swabbing is required -
      surface swabbing may miss streptococcal cellulitis.

J. Eye
   a. Conjunctiva - Sample each eye with separate swabs 
      (premoistened with sterile saline) by rolling over conjunctiva. 
      When only one eye is infected, sampling both can help distinguish
      indigenous microflora from true pathogens.
   b. Corneal scrapings - Collected by ophthalmologist. Using
      sterile spatula, scrape ulcers and lesions; inoculate scraping 
      directly onto media (BHI with 10% sheep blood, chocolate, and 
      inhibitory mold agar). Prepare 2 smears by rubbing material onto 
      1-2 cm area of slide. Transport time ≤15 min.
   c. Vitreous fluid - Prepare eye for needle aspiration of
      fluid. Transfer fluid to sterile tube. Transport time 
      ≤15 min.

K. Feces - see stool.

L. Fistula - see abscess.

M. Fluids - see sterile body fluids.

N. Genital - Cultures for Neisseria gonorrhoeae should
   be collected using a Copan Liquid amies Elution Swab (ESwab).
   Transport to laboratory immediately.
   a. Endocervical - Remove cervical mucus with swab and
      Insert a second swab into endocervical canal and rotate against
      walls. Allow time for organisms to absorb onto the swab surface.
   b. Urethral - Collect urethral specimens at least 1 h after 
      patient has urinated. Insert small swab 2-4 cm into urethral 
      lumen, rotate, leave for 2s to facilitate absorption.

O. Pilonidal cyst - see abscess.

P. Respiratory, lower - Transport time is ≤2 hours.
   a. Bronchoalveolar lavage or brush, endotracheal aspirate -
      Collect fluid in a sputum trap (907093); transfer to leak-proof
      container (37777) for transport to microbiology laboratory; place
      brush in sterile container with 1 mL saline.
   b. Sputum, expectorated - Patient should rinse mouth and
      gargle with water prior to collection; instruct patient to cough
      deeply.  Collect specimens in sterile transport containers 
   c. Sputum, induced - Have patient brush gums and teeth, then
      rinse mouth thoroughly with water.  Using a nebulizer, have the 
      patient inhale 20-30 mL of 3 to 10% sterile saline.  Collect 
      sputum in sterile container.
   d. If Nocardia is suspected, culture for Nocardia should be
      requested as an add-on test as standard culture is inadequate
      for its recovery.

Q. Respiratory, upper - Transport time ≤2 hours.
   a. Oral - remove oral secretions and debris from surface of 
      lesion with a swab.  Use a second swab to vigorously sample 
      lesion, avoiding normal tissue.  Superficial swab specimens 
      should not be submitted.  Tissue or needle aspirates are 
   b. Nasal swabs (R/O SAPCR) - Insert a sterile swab (use Copan
      dual swab 26200) into the nose until resistance is met at the
      level of the turbinates (approximately 1-2 cm into one nostril).
      Rotate the swab against the nasal mucosa for 3 sec.  Apply slight
      pressure with a finger on the outside of the nose to ensure good
      contact between swab and inside of nose. Using the same swab,
      repeat for the other nostril.
   c. Sinus aspirates - Aspirate with needle and syringe. 
      Cleanse rubber stopper of anaerobic transport  vial (59546) with
      alcohol; push needle through septum and inject specimen on top
      of agar.
   d. Throat - Routine throat cultures will be processed only
      for growth of β-hemolytic Streptococcus species. 
      Do not obtain throat samples if epiglottis is inflamed, as
      sampling may cause serious respiratory obstruction.  Sample the
      posterior pharynx, tonsils, and inflamed areas with a Copan
      Liquid Amies Elution Swab (ESwab).

R. Sterile body fluids (other than CSF) - 
   a. Transport fluid to laboratory in sterile, leak-proof container
      (BD Vacutainer, no additive, yellow top, 924044) or anaerobic
      transport vial (Vial, 59546).
   b. Cleanse rubber septum of container with 70% alcohol. Allow
      septum to dry for 1 min before inoculating. 
   c. Disinfect overlying skin with iodine or chlorhexidine 
      preparation.  Obtain specimen with needle and syringe. Push
      needle through septum of transport container and inject fluid.
   d. Amniotic and culdocentesis fluids should always be transported 
      in an anaerobic transport vial (59546). Agar in anaerobic vial
      should be clear before inoculation; inject fluid on top of agar.
   e. Submit as much fluid as possible. NEVER submit a swab dipped in
      fluid. NEVER inject fluid into swab container.  
   f. One aerobic blood culture bottle (924171) inoculated at bedside
      (up to 10 mL) is highly recommended provided adequate sample is 
      available. If blood culture bottle is inoculated, submit separate
      aliquot in anaerobic transport vial (59546) or sterile container
      (924044) for preparation of cytocentrifuged Gram stain and
      inoculation of solid media (allows quantitation, aids in culture
   g. Transport time ≤15 min, room temperature.

S. Stool - Stools submitted on patients admitted for >3 days
   will be rejected without prior preapproval (pager 4903 weekdays,
   pager 3404 evenings and weekends).
   a. Please use FecalSwabs [Stores #105117]. 1) Obtain a stool
      specimen in a clean pan or container. Stool specimens should not
      contain urine or water. 2) Holding FecalSwab shaft above the red
      breakpoint mark, insert the entire tip of the FecalSwab into the
      stool sample and rotate. Do not use FecalSwab as a spoon;
      rather, coat swab with a visible layer. 3) If visible stool is
      not coating the FecalSwab tip, reinsert until swab is coated.
      4) Using swab and aiming tube away, mash and mix the stool
      sample against the side of the tube to suspend the sample. 
      5) Invert the tube several times to homogenize the sample and
      expose the sample to Cary Blair preservative fluid.
   b. The FilmArray Gastrointestinal Panel is a multiplex PCR
      test capable of qualitatively detecting DNA or RNA of 22
      pathogens (bacteria, parasites, and viruses). It requires a
      FecalSwab.  The panel is used to diagnose infection caused by
      Campylobacter species, Plesiomonas shigelloides,
      Salmonella species, Vibrio species, V.
      cholerae, Yersinia species, enteroaggregative E.
      coli, enteropathogenic E. coli, enterotoxigenic
      E. coli, Shiga toxin producing E. coli, E. coli
      O157, Shigella/Enteroinvasive E. coli,
      Cryptosporidium species, Cyclospora cayetanensis,
      Entamoeba histolytica, Giardia lamblia, Adenovirus F 40/41,
      Astrovirus, Norovirus, Rotavirus and Sapovirus.
   c. Stools for C. difficile toxin detection must be
      transported to the laboratory immediately or refrigerated if
      transport is delayed.  This test requires raw stool, not a
   d. Surveillance cultures may be ordered on Bone Marrow
      transplant and other immunocompromised patients to detect
      overgrowth of normal flora by Staph aureus, yeast or a
      gram negative bacillus.
   e. Test of Cure Stool Culture (Salmonella, Shigella, EHEC) is
      only for the listed organisms. For organisms other than these
      please contact the Microbiology laboratory for approval.
   f. Aeromonas Culture - Should be collected in FecalSwabs
      (Stores #105117). This test may be added onto the FilmArray
      Enteric Panel.

T. Tissue - Submit in anaerobic collection jar (59547) or
   sterile screw-cap container (37777); add drops of sterile saline to 
   keep small pieces of tissue moist.  Transport time ≤15 min.

U. Urine - Collect 4 mL of urine in a sterile specimen container
   (37777). Transfer urine to a gray top C&S urine container. Tubes 
   must be filled to 3 mL do prevent inhibition of bacterial growth.
   Transport to the microbiology laboratory. If unable to collect 3 mL
   of urine, collect in sterile specimen container (37777) and 
   transport urine specimens to the Microbiology Laboratory or 
   refrigerate within 30 minutes. Refrigerated 
   specimens should be delivered to the lab as soon as possible, and 
   may be rejected if not received within 24 hours of collection.

   Gray top C&S urine containers are not acceptable for urinalysis and
   urine chemistries because the preservative interferes with testing.

   Cultures can not be performed as an add-on test to urinalysis.  Send
   separate sample for urinalysis (random urine yellow top, round
   bottom tube (no additive)) and culture (as above).

   a. Midstream clean catch method: Patients should be 
      instructed to wash hands prior to collection and offered exam 
      1. Female patients should be instructed to sit 
         on toilet with legs apart and spread labia with one hand. 
         First void in toilet and then, continuing to void, hold 
         specimen container in "midstream" to collect sample.
      2. Male patients should be instructed to retract
         foreskin if uncircumcised. First void in toilet and then, 
         continuing to void, hold specimen container in "midstream" to 
         collect sample.
   b. Straight catheter: Thoroughly cleanse the urethral
      opening with soap and water. Rinse area with wet gauze pads. 
      Aseptically insert catheter into the bladder. After discarding
      initial 15 to 30 mL of urine, collect 4 mL of urine for 
      submission in a gray top C&S urine container.
   c. Indwelling catheter: Clamp catheter below port and allow 
      urine to collect in tubing. Disinfect the catheter collection 
      port with 70% alcohol. Use needle and syringe to aseptically 
      collect 4 mL freshly voided urine though catheter port. 
      Transfer to gray top C&S urine container. Do not collect urine
      from collection bag.
   d. Ileal conduit: Remove the external device and discard 
      urine within device. Gently cleanse the stoma with 70% alcohol
      followed by povidone-iodine swab stick (907172). Using sterile
      technique, insert a double catheter into the cleansed stoma, to
      a depth beyond the fascial level, and collect the urine into a
      sterile container. Transfer to gray top C&S urine container.  Use
      of a double catheter helps to minimize contamination of the
      specimen with skin flora.

V. Wound - See abscess.