Bacterial Culture
BT 6004
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 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 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 obtained. e. Deliver all specimens to the laboratory immediately after collection. 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 culture. 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 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 set at 4-6 hour intervals (will detect >98% 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 - 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 hours. 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 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 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 (37777). 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 preferred. 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 interpretation). 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 FecalSwab. 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 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. 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.