Microbiology Specimen Collection and Transport

Deliver all specimens as soon as possible to the Clinical Microbiology Laboratory 6004 BT. Collection devices are available from Hospital Stores.
Blood Culture Transport
Aerobic Blood Culture Bottle 924171
Anaerobic Blood Culture Bottle 924172
Isolator Tube 922848
Pediatric Isolator Tube 923003
  
Aerobic Transport
Aerobic Culturette 922349
MRSA PCR requires Copan Dual Swab 26200
Sterile Wide Mouth Screw-cap Container 907069
Sterile Lumbar Puncture Collection Tube for CSF 907131
Sterile Sputum Trap 907093
  
Anaerobic Transport
ACT II Tube System for Fluid and Swab Specimens 907464
BBL™ Port-A-Cul Jar for Tissue Specimens 907722
  
Specimen Kits
Respiratory virus (Flexible minitip flocked nasopharyngeal swab and transport media)-green sticker 33595
HSV/VZV lesion (Regular straight flocked swab and transport media)-beige sticker 33625
Bordetella pertussis (swab, transport media, mailer, req for UHL) 923012
UHL Stool Ova & Parasite Kit 923450
Chlamydia Transport Kit (Large cervical swab and small male urethral swab, transport media) 923740
Viral/Chlamydia/Mycoplasma transport media for large tissue sections - Available from Microbiology Laboratory (BT 6004 GH)
  1. Use universal precautions for collecting and handling all specimens.


  2. Whenever possible, collect all culture specimens prior to administration of any antimicrobial agents.


  3. Avoid contamination with indigenous flora.


  4. All specimens must be appropriately labeled with two patient identifiers. Identifiers used at University of Iowa Hospitals and Clinics (UIHC) include patient name, birthdate and/or hospital number. The requisition will include the patient name, hospital number, hospital service, date and time of collection, specimen type and tests requested. A requisition needs to accompany each different specimen type.


  5. Deliver all specimens to the laboratory as soon as possible after collection. Specimens for bacterial culture should be transported at room temperature. If transport is delayed the following specimens should be refrigerated: urines (within 30 min), stool (within 1 h), respiratory specimens. Specimens for viral culture must be transported to the laboratory immediately on ice. See specific specimen and culture type for detailed collection and transport guidelines.


  6. Specimens may be hand delivered to the laboratory or transported via the runners from Technical Services if the specimens are not indicated as deliver immediately. Specimens may be transported through the pneumatic tube system if approved by Pneumatic Tube Administration. This includes blood culture bottles, vacutainer tubes and swabs. See specific culture.


  7. Specimens should be in tightly sealed, leak proof containers and transported in sealable, leak-proof plastic bags. Specimens for TB should be double bagged. Specimens should not be externally contaminated. Specimens grossly contaminated or compromised may be rejected.


  8. If anaerobic organisms are suspected make certain to use proper anaerobic collection containers.


  9. Further questions may be referred to the Microbiology laboratory (356-2591) or pathology resident (pager 4903 weekdays; pager 3404 evenings and weekends).



Bacterial Cultures:  Transport at room temperature unless otherwise specified.
  1. 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 device (907464) with alcohol; 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.


  2. 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: BBL™ Port-A-Cul anaerobic collection jar (907722) for tissue or ACT II tube system for fluid and swab specimens (907464). A sterile screw-cap container (907069) may be used for tissue if transported to the microbiology lab immediately (add drops of sterile saline to keep small pieces of tissue moist). Deliver all specimens to the laboratory immediately after collection.


  3. Blood
    1. Adult – Cleanse skin with ChloraPrep® one-step 1.5 mL Frepp® Applicator (907672):
      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 inoculating.
      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 two initial sets of blood cultures sequentially from separate phlebotomy procedures followed by a third and a fourth set at 4-6 hour intervals (will detect >99% of BSIs). Three sets of blood cultures collected within a 24 hour period will detect 96.9 - 98.3% of BSIs. A single set of blood cultures to detect BSIs in adults is inadequate (only 73% sensitivity); two sets of blood cultures will allow detection of 87.7-89.7% of BSI episodes. (J Clin Microbiol 2007; 45:3546).
      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.
    2. Pediatric – Prepare skin and bottles as for adult.  Collect as much blood as possible up to 10 mL per bottle.

  4. Bone marrow aspirate – Prepare puncture site as for surgical incision.  Inoculate blood culture bottle or lysis centrifugation tube.  Transport time <2 hours.  Routine bacterial culture of bone marrow is rarely useful.


  5. Burn – Clean and debride burn. Place tissue in sterile screw-cap container (907069). 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.


  6. Catheter Tips – Only intravascular catheter tips from pediatric patients and peritoneal dialysis catheters are routinely accepted for culture. Send 5 cm of distal tip in sterile screw–cap container (907069). Transport time <15 min. Foley catheters are not accepted for culture since growth represents distal urethral flora.


  7. Cerebrospinal Fluid (CSF) – Aseptically collect CSF from a lumbar puncture into sterile tubes (907131). Send second tube (>3 mL) to the Microbiology Laboratory. Transport time <15 min. Cerebrospinal fluid for bacterial culture should never be refrigerated.


  8. 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 <2 hours.


  9. Ear
    1. 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.
    2. 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.

  10. Eye
    1. 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.
    2. 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.
    3. Vitreous fluid – Prepare eye for needle aspiration of fluid. Transfer fluid to sterile tube. Transport time <15 min.

  11. Feces - see stool.


  12. Fistula - see abscess.


  13. Fluids - see sterile body fluids.


  14. Genital –Cultures for Neisseria gonorrhoeae should be collected with a sterile swab and inoculated directly to a Jembec plate (obtain from laboratory; place white tablet in hole of Jembec plate to provide CO2 enriched atmosphere, close top of the plate tightly and place in ziploc bag provided, keep at room temperature, deliver to lab as soon as possible). If a Jembec plate is unavailable, an aerobic culturette swab (922349) may be used if transported to laboratory immediately.
    1. Endocervical - Remove cervical mucus with swab and discard. Insert a second swab into endocervical canal and rotate against walls. Allow time for organisms to absorb onto the swab surface.
    2. 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.

  15. Pilonidal cyst – see abscess.


  16. Respiratory, lower – Transport time <2 hours.
    1. Bronchoalveolar lavage or brush, endotracheal aspirate – Collect fluid in a sputum trap; place brush in sterile container with 1 mL saline.
    2. Sputum, expectorated - Patient should rinse mouth and gargle with water prior to collection; instruct patient to cough deeply.  Collect specimens in sterile transport containers (907069). 
    3. 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.

  17. Respiratory, upper  – Transport time <2 hours.
    1. 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 preferred.
    2. Nasal swabs (R/O MRSA) – Insert a sterile swab (use Copan dual swab 26200 for PCR; 922349 for culture) into the nose until resistance is met at the level of the turbinates (approximately 2-3 cm into the nose).  Rotate the swab against the nasal mucosa.  Repeat the process on the other side.
    3. Sinus aspirates – Aspirate with needle and syringe.  Cleanse rubber stopper of anaerobic transport device (907464) with alcohol; push needle through septum and inject specimen on top of agar. 
    4. Throat - Routine throat cultures will be processed only for growth of ß-hemolytic Streptococcus species.  Contact Microbiology Lab (6-2591) to arrange for provision of special media if culture for other organisms (Corynebacterium diphtheria, Neisseria gonorrhoeae) is desired.  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 sterile swab.

  18. Sterile body fluids (other than CSF) – Disinfect overlying skin with iodine or chlorhexidine preparation.  Obtain specimen with needle and syringe.  Transfer fluid to anaerobic transport system (907464) - cleanse rubber stopper with alcohol; push needle through septum and inject fluid on top of agar, sterile screw-cap container (907069), or aerobic blood culture bottle (923387). Amniotic and culdocentesis fluids should always be transported in an anaerobic system. Submit as much fluid as possible.  NEVER submit a swab dipped in fluid.  If blood culture bottle is inoculated, submit separate aliquot in sterile container for preparation of cytocentrifuged Gram stain.  Transport time <15 min.


  19. Stool – Submit 10-20 g in sterile container. Transport time ≤1 hour. Refrigerate if transport is delayed. Stools are cultured to isolate bacterial causative agents of diarrheal illness; Salmonella, Shigella, Campylobacter, and Shiga toxin producing E. coli. Routine stool culture includes EIA for Shiga toxin from E. coli. Cultures for Yersinia are performed by special request. Stools for C. difficile toxin detection must be transported to the laboratory immediately or refrigerated if transport is delayed. 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.


  20. Tissue – Submit in anaerobic collection jar (907722) or sterile screw-cap container (907069); add drops of sterile saline to keep small pieces of tissue moist.  Transport time <15 min.


  21. Urine – Collect 1-10 mL of urine in a sterile specimen container (907069). 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.
    1. Midstream clean catch method: Patients should be instructed to wash hands prior to collection and offered exam gloves.
      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.
    2. 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 urine for submission in a sterile container.
    3. 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 5-10 mL freshly voided urine though catheter port. Transfer to sterile container. Do not collect urine from collection bag.
    4. 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. Use of a double catheter helps to minimize contamination of the specimen with skin flora.

  22. Wound – See abscess.

Fungal Culture
  1. Deliver all specimens to the laboratory as soon as possible after collection.
    1. Blood: Cleanse skin with ChloraPrep® one-step 1.5 mL Frepp® Applicator (907672). Collect 8-10 mL of blood for adult (1.5 mL for child) and inoculate into an Isolator tube (Adult=922848; Pediatric=923003). Collect in addition to bacterial blood culture bottles.


    2. Skin: Using a scalpel blade, scrape the periphery of the lesion border and transport in a sterile container.


    3. See Bacterial Culture for collection and transport of all other specimen types.

Mycobacterial Culture (AFB Culture)
  1. Deliver all specimens to the laboratory as soon as possible after collection. Specimens for mycobacteria should be double bagged and sent sealed in leak-proof containers.
    1. Blood: Media and instructions available upon request from the Microbiology Lab. Test available for limited patient populations only.


    2. Sputum: Collect an early morning specimen on three consecutive days. Collect 5-15 mL in a sterile container.


    3. See Bacterial culture for collection and transport of all other specimen types.


    4. Swabs are suboptimal for recovery of mycobacteria due to limited material and the hydrophobicity of the mycobacterial cell envelope (often compromises a transfer from swabs onto media). Dry swabs are unacceptable. The lab only accepts culturette swabs for AFB culture when the ordering physician confirms that the swab is the only possible way to obtain the specimen.

Chlamydia Trachomatis Culture Isolation
  1. Amplified DNA (PCR) testing is recommended for endocervical or urethral specimens ("see Chlamydia trachomatis Detection by PCR"). Culture is recommended for Chlamydia trachomatis detection in other specimen types, in suspected sexual abuse, or for suspected failure of therapy. Specimens with a request for C. trachomatis culture will be sent to University Hygienic Laboratory (UHL).

  2. Transport media: Chlamydia transport kit available from Hospital Stores (923740). Specimens must be aseptically collected with some vigor (swabbing or scraping) to insure an adequate number of epithelial cells are collected. All specimens should be transported to the laboratory in viral transport media on ice (4°C) or refrigerated. After 2245, specimens should be sent to Specimen Control (6248 RCP). Do not freeze.
    1. Endocervical, urethral, rectal, and conjunctival swabs: Use swab supplied in kit (large swab for female cervical specimens and small swab with metal shaft to collect male urethral specimens). Swab area and place in tube containing transport medium. Do not submit dry swabs, wood swabs, or calcium alginate swabs.
    2. Nasopharyngeal wash/aspirate/swab: Acceptable for newborns (see viral culture instructions below).
Viral Culture

Collect specimens for culture early in illness when viral shedding is maximal. Place swabs in viral transport medium (33595, 33625, 923740). Collect bronchoalveolar lavage, tracheal aspirate, or nasopharyngeal wash/aspirate in sterile, leak-proof container or viral transport media (33595). Collect specimens from normally sterile sites in a sterile container. Transport the specimen to the Microbiology laboratory (6004 BT) immediately on ice or refrigerate. From 2245 to 0700, deliver specimen to Specimen Control (6240 RCP) on ice.

Viruses that may be isolated at UIHC include respiratory viruses (influenza A including H1N1; influenza B; RSV; parainfluenza 1, 2, 3; adenovirus), Herpes Simplex virus (HSV)/varicella-zoster virus (VZV), and cytomegalovirus (CMV). Specimens submitted for culture of other viruses (including enteroviruses and nonrespiratory specimens submitted for adenovirus culture) will be sent to the University Hygienic Laboratory (UHL). PCR assays for the detection of HSV (CSF), enterovirus (CSF), and CMV (blood or CSF) are available in the Molecular Pathology laboratory (see individual test listings). Requests for CMV PCR on amniotic fluid will be sent to a reference laboratory (see "CMV Quant PCR, Amniotic Fluid"). Requests for detection of enteric adenovirus antigens 40-41 in stool specimens will be sent to a reference laboratory for an ELISA test (see "Adenovirus 40-41 Antigens by EIA, Fecal").
  1. Biopsy or tissue: Keep moist with sterile saline or viral transport media [do not use viral transport media (VTM) if bacterial, AFB, or fungal cultures are also requested; VTM is available in kits from Hospital Stores (923740, 33595, 33625)].

  2. Blood: Collect 10 mL whole blood in a EDTA (lavender top) or heparinized (green top) tube. Must be received in the laboratory within 6 hours of collection.

  3. CSF: Collect 1-5 mL in a sterile container. HSV requests on CSF will be performed as PCR only.

  4. Nasopharyngeal swab: Collect specimen using the flexible minitip flocked swab (Hospital Stores #33595). Measure the distance from the patient’s nostril to the nasopharynx (half the distance from nostril to base of the ear) and hold the swab at that location. Do not advance the swab beyond that point. Gently insert the swab along the base of one nostril (straight back, not upwards) and continue along the floor of the nasal passage until reaching the nasopharynx. Rotate swab 2-3 times and hold in place for 5 seconds. Place swab in tube containing viral transport medium. Break off the excess length of swab at the score mark to permit capping of the tube.

  5. Nasopharyngeal wash/aspirate:
    • Assemble equipment:
    • Sterile specimen trap
    • Personal Protective Equipment (gloves, surgical mask, eye protection)
    • Appropriate size suction catheter (8 fr for infants/children, 10/12 fr for adults)
    • Normal saline vial
    • Wall suction
    • Bag or cup of ice for specimen transport to laboratory

    Place patient with the head tilted slightly back. With sterile gloved hand, insert suction catheter into the patient’s nose to the depth of the nasopharyngeal area (beyond the turbinates). Do not remove catheter until end of procedure (see picture below). With the non-sterile gloved hand, instill approximately 1-2 ml normal saline outside the catheter. Apply suction to aspirate nasopharyngeal secretions. Above steps may need to be repeated to obtain 1 ml sample in specimen trap. Remove catheter from patient. With specimen trap still in-line, rinse catheter with remaining saline to clear secretions. Specimens transported by tube system must be transferred from trap to a leak-proof sterile container (be sure the lid is tightly secured).


  6. Respiratory secretions: Collect specimens in a sterile, leak-proof container. Sputum is unacceptable for viral cultures.

  7. Sterile Body fluids: Use sterile leak-proof containers.

  8. Stool: Use leak-proof container with no preservatives.

  9. Swab: Only use swabs available in transport kits. For respiratory virus testing use nasopharyngeal swab in flexible minitip flocked swab kit (33595, green sticker). For skin lesions use regular flocked swab in HSV/VZV kit (33625, beige sticker). For Chlamydia testing use Chlamydia transport kit (923740). Do not use calcium alginate swabs or wooden swabs. Place swab in transport medium.

  10. Vesicle: Expose the base of the lesion by lifting the cap of the vesicle or ulcer. Using swab from Regular flocked swab collection kit (33625), swab the entire base of the lesion firmly to collect cellular material (without causing bleeding). Place swab in viral transport media and transport immediately on ice.

  11. Urine: Collect 1-10 mL of clean voided midstream morning urine in a sterile container without preservatives.

Parasitology
  1. Ova and parasite exam: Within 1 hour of collection, transfer a few grams of stool to each vial of UHL collection kit (Hospital Stores 923450). Order on EPIC as "UHL, Routine O+P with trichrome stain", complete the UHL requisition that is in the box, and deliver to Specimen Control for transport to UHL. A minimum of three stool specimens collected on alternate days is recommended. Onset of diarrhea in patients hospitalized for >3 days is usually not attributed to a parasitic infection. Requests to include Microsporidia or Cyclospora detection must be specified on UHL requisition.


  2. Giardia/Cryptosporidium antigen detection by DFA: Submit 2-5 mL of stool in sterile container. Detection of Giardia or Cryptosporidium by DFA may require more than one specimen. Transport time ≤1 hour. Refrigerate if transport is delayed. Onset of diarrhea in patients hospitalized for >3 days is usually not attributed to a parasitic infection.


  3. Pinworm exam: Submit scotch tape prep. Touch the perianal folds with clear scotch tape, then attach the tape to a clean glass slide and transport to the laboratory sealed in a ziplock bag. Clear tape must be used, not invisible tape.


  4. Scabies exam: Sterile mineral oil is available from Pharmacy (item 991565, 10 mL container). Collect skin scrapings as follows:
    1. Place a drop of mineral oil on a sterile scalpel blade.


    2. Allow some of the oil to flow onto the papule. Scrape vigorously six or seven times to remove the top of the papule. (Tiny flecks of blood should be seen in the oil.)


    3. Transfer the oil and scrapings onto a glass slide (an applicator stick can be used).


    4. Add 1-2 extra drops of mineral oil to the slide and mix well. Clumps can be crushed to expose hidden mites.


    5. Place a coverslip onto the slide and transport to the Microbiology Lab immediately.

  5. Malaria smear: Collect venous blood in EDTA collection tube and deliver immediately to lab. If clinical suspicion for malaria remains after one set of negative smears, additional specimens should be submitted at 12 hour intervals for the subsequent 36 hour period.
Serology
  1. Collect all specimens in a 5 or 10 mL red or serum separater top vacutainer tube and transport to the laboratory as soon as possible after collection.


  2. Serological testing performed in Microbiology includes Rubeola (measles), Mumps, Varicella Zoster IgG, Lyme IgG/IgM, Toxoplasma IgG, Toxoplasma IgM, CMV IgG, CMV IgM, and Helicobacter IgG.


  3. Lipemic, hemolyzed or icteric serum may interfere with testing.

Molecular Testing

HIV Viral Load by PCR and Hepatitis C Virus RNA by PCR: For each test collect at least 6mL whole blood in one pink top tube (EDTA sprayed) and deliver immediately to laboratory. Each test requires a dedicated collection tube and cannot be added onto a previously opened vacutainer tube. Record draw time. All collection tubes need to be processed within 6 hours of collection. Two lavender top tubes (3 mL) may be substituted for one pink top tube (6mL).