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.
- Use universal precautions for collecting and handling all specimens.
- Whenever possible, collect all culture specimens prior to administration of any antimicrobial agents.
- Avoid contamination with indigenous flora.
- Swabs are convenient but inferior to tissue and fluid. Tissue and fluid are essential for fungal and mycobacterial culture.
- All specimens must be appropriately labeled with two patient identifiers. Identifiers used at University of Iowa Hospitals and Clinics (UI Hospitals and Clinics) 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.
- 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.
- 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 (if placed in plastic carrier), vacutainer tubes and swabs.
- 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.
- If anaerobic culture is requested, make certain to use proper anaerobic collection containers (fluid: 59546, tissue: 59547, or ESwab, 74541).
- Further questions may be referred to the Microbiology laboratory (356-2591) or pathology resident (pager 4903 weekdays; pager 3404 evenings and weekends).
Transport at room temperature unless otherwise specified.
- 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.
- Anaerobic cultures - 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:
- 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
- Anaerobic jar (tissue specimen, 59547). Place sample on top of agar. Keep jar upright to maintain atmosphere in jar.
- A sterile container (37778) may be used for tissue if transported to the microbiology lab immediately (add drops of sterile saline to keep small pieces of tissue moist).
- Copan Liquid Amies Elution Swab (ESwab) (74541) – swab specimens are suboptimal, but will be accepted if no other sample can be obtained.
- Deliver all specimens to the laboratory immediately after collection.
- 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 culture.
- Throat or nasopharyngeal swabs
- Gingival or other intraoral surface swabs
- Expectorated sputum
- Sputum obtained by nasotracheal or endotracheal suction
- Bronchial washings
- Voided or catheterized urine
- Vaginal or cervical swabs
- Gastric and small bowel contents (except for "blind loop" or bacterial overgrowth syndrome)
- Feces (except for specific etiologic agents such as C. difficile and C. botulinum)
- Rectal swabs - Surface swabs from ulcers and wounds (collect material from below the surface)
- Material adjacent to a mucous membrane that has not been adequately decontaminated
- Adult Cleanse skin with ChloraPrep® one-step 1.5 mL Frepp® Applicator (907672):
- Holding the applicator sponge downward, pinch wings on applicator to break ampule and release the antiseptic.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- Bone marrow aspirate Prepare puncture site as for surgical incision. Inoculate blood culture (924171) or Isolator (lysis-centrifugation) tube (922848). Transport time <2 hours. Routine bacterial culture of bone marrow is rarely useful.
- Burn Clean and debride burn. Place tissue in sterile screw-cap container (37778). 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Vitreous fluid Prepare eye for needle aspiration of fluid. Transfer fluid to sterile tube. Transport time <15 min.
- Feces - see stool.
- Fistula - see abscess.
- Fluids - see sterile body fluids.
- Genital Cultures for Neisseria gonorrhoeae should be collected using an Copan Liquid Amies Elution Swab (ESwab). Transport to the laboratory immediately.
- 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.
- 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.
- Pilonidal cyst see abscess.
- Respiratory, lower Transport time <2 hours.
- Bronchoalveolar lavage or brush, endotracheal aspirate Collect fluid in a sputum trap (907093); transfer to leak-proof container (37778) for transport in pneumatic tube system); place brush in sterile container with 1 mL sterile saline.
- Sputum, expectorated - Patient should rinse mouth and gargle with sterile water prior to collection; instruct patient to cough deeply. Collect specimen in sterile transport containers (37778).
- Sputum, induced Have patient brush gums and teeth, then rinse mouth thoroughly with sterile water. Using a nebulizer, have the patient inhale 20-30 mL of 3 to 10% sterile saline. Collect sputum in sterile container.
- If Nocardia is suspected, culture for Nocardia should be requested as an add-on test as standard culture is inadequate for its recovery.
- Respiratory, upper Transport time ≤2 hours.
- 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.
- 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.
- 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.
- Throat - Routine throat cultures will be processed only for growth of ß-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 using a Copan Liquid Amies Elution Swab (ESwab).
- Sterile body fluids (other than CSF)
- Transport fluid to laboratory in sterile, leak-proof container (BD Vacutainer, no additive, yellow top, 924044) or anaerobic transport vial (Vial, 59546).
- Cleanse rubber septum of container with 70% alcohol. Allow septum to dry for 1 min before inoculating.
- Disinfect overlying skin with iodine or chlorhexidine preparation. Obtain specimen with needle and syringe. Push needle through septum of transport container and inject fluid.
- 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.
- Submit as much fluid as possible. NEVER submit a swab dipped in fluid. NEVER inject fluid into swab container.
- 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 (37778) for preparation of cytocentrifuged Gram stain and inoculation of solid media (allows quantitation, aids in culture interpretation).
- Transport time ≤15 min, room temperature.
- Stool Stools submitted on patients admitted for >3 days will be rejected without prior preapproval (pager 4903 weekdays; pager 3404 evenings and weekends). 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. C. difficile PCR is restricted to 1 sample/week if negative and a 10-day interval between a positive result and the next test. C. difficile PCR is not a test of cure. Solid stools that do not take the shape of their container are rejected as the toxin PCR will detect asymptomatic carriage of C. difficile and a positive test is meaningless in this context, and because the test cannot physically be run on solid stool.
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.
- Tissue Submit in anaerobic collection jar (59547) or sterile screw-cap container (37778); add drops of sterile saline to keep small pieces of tissue moist. Transport time <15 min.
- Urine Collect 4 mL of urine in a sterile specimen container (37778). 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 (37778) 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, conical bottom tube (no additive)) and culture (as above).
- Midstream clean catch method: Patients should be instructed to wash hands prior to collection and offered exam gloves.
- 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.
- 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.
- Straight 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.
- 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 20 mL freshly voided urine though catheter port. Transfer to gray top C&S urine container. Do not collect urine from collection bag.
- 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.
- Wound See abscess.
- Deliver all specimens to the laboratory as soon as possible after collection.
- 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. Isolator tubes are for molds, Histoplasma, Blastomyces, and Malassezia spp.; for bloodstream infection by Candida spp., inoculate aerobic blood culture bottles instead.
- Skin: Using a scalpel blade, scrape the periphery of the lesion border and transport in a sterile container.
- See Bacterial Culture for collection and transport of all other specimen types.
Viral/Molecular Infectious Disease PCR Testing
- 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.
- Blood: Media and instructions available upon request from the Microbiology Lab. Test available for limited patient populations only.
- Sputum: Collect an early morning specimen on three consecutive days. Collect 5-15 mL in a sterile container.
- See Bacterial culture for collection and transport of all other specimen types.
- 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 Copan Liquid Amies Elution Swab (ESwab) for AFB culture when the ordering physician confirms that the swab is the only possible way to obtain the specimen.
Collect specimens for PCR testing early in illness when viral shedding is maximal. Place swabs in viral transport medium (33595
). Collect bronchoalveolar lavage, tracheal aspirate, or nasopharyngeal wash/aspirate and specimens from normally sterile sites in a sterile, leak-proof container
(37778). Transport the specimen to the Microbiology laboratory (6004 BT) immediately.
Respiratory Virus PCR
PCR assay includes the detection of respiratory viruses (influenza A including H1N1; influenza B; RSV; parainfluenza 1, 2, 3; adenovirus; human metapneumovirus).
- 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 (33595, 33625).
- CSF: Collect 0.5 mL in a sterile container. Transport immediately to laboratory.
- 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.
- 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).
- Respiratory secretions: Collect specimens in a sterile, leak-proof container. Sputum is unacceptable for viral cultures.
PCR Assays are available for the following viruses: HSV1&2, VZV, enterovirus (CSF), EBV (Blood and CSF) and CMV (blood, CSF, or BAL). PCR testing requires a dedicated collection tube and cannot be added onto a previously opened vacutainer tube.
- HSV 1,2 or VZV PCR: submit CSF in sterile container. Submit vesicle fluid, surface swab, or BAL (sputum and tracheal aspirates are unacceptable) in UTM media. Transport to laboratory immediately.
- Enterovirus PCR: submit CSF in sterile container. Keep on ice and deliver to laboratory immediately.
- EBV PCR: Collect one 5 mL pink (EDTA) top tube. For CSF collect a minimum of 1.0 mL in a sterile container. Deliver to laboratory immediately after collection. EBV PCR is useful only for diagnosis and monitoring of posttransplant lymphoproliferative disorder and similar disorders and is not appropriate for the diagnosis of mononucleosis or meningitis/encephalitis in immunocompetent patients.
- CMV Quantitative PCR: Collect one 5 mL pink (EDTA) top tube. For CSF collect a minimum of 0.5 mL in a sterile container. Deliver to laboratory immediately.
- CMV Qualitative PCR: Submit a minimum of 2.0 mL BAL or 1.0 mL of amniotic fluid in a sterile container. Transport to laboratory immediately.
- HIV Viral Load by PCR, Hepatitis C Virus RNA by PCR and Hepatitis B Virus DNA by PCR: For each test collect at least 6 mL whole bold in one pink (EDTA) top tube. Deliver immediately to laboratory. Each test requires a dedicated collection tube and cannot be added onto a previously opened vacutainer tube. All collection tubes need to be processed within 6 hours of collection.
- Neisseria gonorrhoeae & Chlamydia trachomatis Detection by PCR: Amplified DNA (PCR) testing is recommended for urine, endocervical, urethral, oral or pharyngeal and rectal swab. Culture is recommended for suspected failure of therapy.
- Endocervical, urethral, oral or pharyngeal, rectal swab: Use multicollect specimen kit available from hospital stores (46161). Specimens must be aseptically collected with the orange shaft swab provided with the kit. After collection, specimens may be stored and transported at 2 to 30 ºC for up to 14 days. Do not freeze.
- Urine: The patient should not have urinated for at least one hour prior to sample collection. Collect urine in a typical collection cup (not provided in multi-collect kit). Using plastic transfer pipette provided in multi-collect specimen kit available from Hospital stores (46161), transfer urine from collection cup into the transport tube until the liquid level in the tube falls within the clear fill window of the transport tube label. Do not overfill. Slightly more than one full squeeze of the transfer pipette bulb may be required to transfer the necessary volume of urine specimen. After collection, specimens may be stored and transported at 2 to 30 ºC for up to 14 days. Do not freeze.
- Ova and parasite exam: Within 1 hour of collection, transfer a few grams of stool to each vial of SHL collection kit (Hospital Stores 923450). Order on EPIC as "SHL, Routine O+P with trichrome stain", complete the SHL requisition that is in the box, and deliver to Specimen Control for transport to SHL. 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 the SHL requisition.
- Cryptosporidium/Giardia: Submit 2-5 mL of stool in sterile container. Detection of Giardia or Cryptosporidium 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.
- 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.
- Parasite exam: For direct examination of parasites (worms), arthropods (insects, spiders), and suspect material passed in stool. This is not a stool ova and parasite (O&P) exam. An O&P can be ordered in EPIC as "SHL, Routine O+P with trichrome stain" and uses SHL collection kit (Hospital Stores 923450). Instructions:
- Submit whole worms, worm segments or other objects in 70% alcohol or 10% formalin.
- Submit arthropods in a clean, dry container.
- Scabies exam: Sterile mineral oil is available from Pharmacy (item 991565, 10 mL container). Collect skin scrapings as follows:
- Place a drop of mineral oil on a sterile scalpel blade.
- 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.)
- Transfer the oil and scrapings onto a glass slide (an applicator stick can be used).
- Add 1-2 extra drops of mineral oil to the slide and mix well. Clumps can be crushed to expose hidden mites.
- Place a coverslip onto the slide and transport to the Microbiology Lab immediately.
- Blood Parasite EXAM (R/O Malaria/Blood Parasites): Collect venous blood in EDTA collection tube and deliver immediately to lab. Malaria antigen testing is available 24 hrs/day, 7 days a week. Antigen results will be available within one hour of specimen arrival. Preliminary slide results will be available within 90 minutes if specimen received between 0700-1900 or by 0930 if after 1900. 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. Note on request if parasite infection other than malaria is suspected.
- Wet Prep for Trichomonas, Yeast or Gardnerella: Collect vaginal specimen in Affirm VPIII Collection and Transport System. Deliver to laboratory within 24 hours of collection.