PRINCIPLE: The following procedure summarizes collection and transport procedures for all Microbiology/Serology tests.

MATERIALS: Collection containers, swabs and requisitions are available from Hospital Stores.

Deliver all specimens as soon as possible to the Clinical Microbiology Laboratory 6004 BT. Collection devices are available from Hospital Stores.

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.

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 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.  

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: 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. 

C. Blood

a. 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 923387 and 1 anaerobic 923388) 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.

b. Pediatric – Prepare skin and bottles as for adult.  Collect as much blood as possible up to 10 ml per bottle.

D. 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.

E. 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.

F. 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.

G. 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.

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 <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 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.

a. 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.
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 <2 hours. 

a. Bronchoalveolar lavage or brush, endotracheal aspirate – Collect fluid in a sputum trap; 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 (907069). 
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.

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 preferred.
b. Nasal swabs (R/O MRSA) – Insert a sterile swab (922349) 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.
c. 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. 
d. 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.

R. 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.

S. 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. Culture may also be requested to R/O Anthrax 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.

T. 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.

U. Urine – Collect 1-10 ml of urine in a sterile specimen container (907069) by catheterization, suprapubic aspiration, or the following midstream clean catch method: Patients should be instructed to wash hands prior to collection and offered exam gloves. 

a. 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.
b. 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.
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.

V. Wound – See abscess.


Fungal Culture

1. Deliver all specimens to the laboratory as soon as possible after collection.

A. Cleanse skin with ChloraPrep® one-step 1.5 ml Frepp® Applicator (907672). Collect 8-10 ml of blood and inoculate into an Isolator tube (922848). Collect in addition to bacterial blood culture bottles.

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

C. 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 leakproof containers.

A. Blood: Media and instructions available upon request from the Microbiology Lab. Test available for limited patient populations only.

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

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

D. Specimens received on swabs may be rejected. See M-20.09 for recommendations. Exceptions will be made if ruling out rapid growers. Questions may be referred to a Director or Lead Scientist.

Chlamydia Trachomatis Culture Isolation

1. Transport media: Viral/Chlamydia transport kit. Available from Hospital Stores (923740).

2. For maximum results, the specimens must be aseptically collected with some vigor (swabbing or scraping) to insure an adequate number of epithelial cells. All specimens should be transported to the laboratory in transport tube on ice (4°C). After 2245, specimens should be sent to Specimen control Lab (6248 RCP). If specimens cannot be processed immediately, they should be refrigerated (0°C) or frozen at -70°C if the delay is more than 48 hours. Never freeze -20°C).

A. Endocervical, urethral, rectal, and conjunctival swabs: Use swab supplied in kit. Avoid swabs with a wooden shaft as they may inhibit growth. Swab area, return swab to container.

B. Nasopharyngeal wash: Draw 3 ml phosphate buffered saline (PBS) into an infant bulb syringe, tilt head back and inoculate 1-2 ml into nostril. Aspirate fluid from nostril into bulb syringe. Place contents in sterile container.