The UMMC Microbiology Laboratory utilizes a variety of methods for identification of bacteria, mycobacteria and fungi. Methods include, but are not limited to, traditional biochemical and enzyme-based methods, serotyping, and microscopy. It is our goal to utilize the most current methods available to provide our patients with accurate and rapid results.
Specimen collection guidelines
Valid interpretation of results can be achieved only if the specimen obtained is appropriate for processing. As a result, care must be taken to collect only those specimens that may yield pathogens, rather than colonizing flora or contaminants. Specific rules for the collection of material vary, depending upon the source of the specimen, but several general principles apply:
- Make every effort to obtain specimens prior to the initiation of antimicrobial therapy.
- Wear appropriate PPE when collecting specimens from sterile sites. Use strict aseptic technique.
- Collect an adequate volume of specimen; send tissue or fluid whenever possible rather than submitting a specimen collected on a swab.
- If the specimen is collected in a syringe, remove the needle and replace it with a cap prior to transport. Specimens will NOT be accepted with needles attached.
- Label all specimen containers with identifying information about the patient (name and MRN) and the specimen source and date and time of collection.
- Complete all paper or electronic requisitions completely and precisely, including requested details on patient history, antimicrobial therapy, and specimen source, so that the laboratory can best determine the appropriate method for processing the specimen.
- Notify the laboratory in advance if special tests are requested or if unusual pathogens, including potential agents of bioterrorism, are suspected.
- Place warning labels on specimens from patients suspected of having highly contagious diseases and notify the laboratory supervisor.
- Prompt transport of specimens to the laboratory is essential in order to optimize the yield of cultures and the interpretation of results. Delays in processing may result in the overgrowth of some organisms or the death of more fastidious ones. Drying out of the sample must also be avoided.
- Always collect two swabs if smear is desired.
Aerobic cultures
Use of the aerobic culture swab is recommended for most sites for which a swab of such size is a suitable method to collect the specimen.
- Collect the specimen and replace the swab in the plastic cylinder.
- If the collection swab has an ampule, crunch it to ensure the transport fluid comes into contact with the swab.
- If unable to deliver immediately, refrigerate the following specimens only: Urine, Sputum, Feces, Gastric washings, Tracheal and Transtracheal aspirate. All other specimens must be maintained at room temperature and delivered within 24 hours of collection.
Anaerobic cultures
Anaerobic bacteria form a major part of the body's normal flora so extreme caution must be taken to avoid contamination by anaerobic normal flora and overgrowth by coexisting aerobic bacteria.
- Collect specimen from the active site of infection, taking precautions to exclude surface contamination and aeration of the sample. Whenever possible, specimens should be pus or fluid obtained by needle aspiration through intact skin or mucosa, which has been cleansed with an antiseptic.
- Swabs from abscess cavities that have been incised under aseptic conditions may be used if immediately placed in an anaerobic environment, however, tissue and aspirated material are the preferred specimens.
- Soft tissue infections may be cultured by injecting 1-2 mL of sterile saline into the infected site with withdrawal of saline and tissue juice into the syringe and immediately injecting the fluid into an anaerobic transport media.
- Specimens not routinely accepted for anaerobic culture:
- Throat, nasopharyngeal or gingival swabs
- Sputum obtained by nasotracheal or endotracheal suction, or expectorated sputum
- Bronchial washings
- Voided or catheterized urine
- Vaginal or cervical swabs
- Feces or any area with fecal contamination
- Do NOT refrigerate specimens for anaerobic culture. Deliver to laboratory as soon as possible. Must be received within 24 hours of collection.
- It is recommended that an aerobic culture be ordered in addition to the anaerobic culture.
Collection procedures
The collection procedures offered below are guidelines only to provide for the optimum specimen possible.
Blood culture
Chlorhexidine Blood Culture Prep kits must be used according to package insert. If kits are not available, the following procedure must be used: Swab skin over the site of venipuncture with 70% alcohol. Allow alcohol to dry completely. Swab the site in concentric circles with an iodophor solution (2% iodine or a povidone-iodine preparation). If the patient is known to be hypersensitive to iodine, the skin may be prepared exclusively with 70% alcohol. Do not touch venipuncture area after preparation. Gloves should be worn when performing the venipuncture. Do not recap needles. Decontaminate the diaphragm on the tops of the Bactec bottles with a 70% alcohol solution before injecting the blood into the bottle. Inoculate a Bactec Plus aerobic bottle with 8 - 10 mL and an anaerobic bottle with 5 - 7 mL. Inoculate Peds Plus with 1 - 3 mL. Three sets of cultures per twenty-four hours should pick up 97-99% of positive results. Blood cultures should not be drawn from indwelling intravenous or intra-arterial catheters because of the risk of contamination unless venipuncture cannot be obtained. In cases where cultures are drawn from indwelling catheters discard the first 5 mL.
- Adults: Bactec Plus Aerobic and Anaerobic; Two bottles = one set.
- Pediatrics: One Peds Plus bottle only per set. Pediatric bottles require a minimum of 1.0 mL of blood. Do Not Refrigerate.
- Acute febrile episode: Antibiotic to be started or changed immediately; collect 2 sets from different site within 10 minutes prior to starting or changing antibiotics.
- Non-acute disease: Antibiotic will not be started or changed immediately; collect 2-3 sets within 24 hours at 3 hour intervals before starting or changing antibiotics.
- Fever of unknown origin: 2-3 sets at least one hour apart in the first 24 hours; if negative after 48 hours collect 2-3 more sets.
- Acute bacterial endocarditis: 3 blood culture sets in the first 1-2 hours before starting antibiotics.
- Subacute bacterial endocarditis: 3 blood culture sets at least one hour apart within 24 hours; if negative at 24 hours collect 2-3 more sets.
Catheter
Use sterile scissors to cut off tip and/or 5-7 cm of intra-cutaneous segment. Submit in sterile container. Foley catheters are not acceptable specimens. Do Not Refrigerate. Must be received in lab within 24 hours.
Cerebrospinal fluid (CSF)
Collect CSF aseptically from a lumbar puncture into sterile tubes. Send tube #2 or #3 for Microbiology testing. A minimum of 1.0 mL is required for each culture needed; i.e., bacterial, fungal, etc. Do Not Refrigerate. Must be received in lab within 2 hours.
Dialysis fluid
Collect outflow fluid aseptically in a sterile container.
Ear
- Inner ear: For intact eardrum, clean ear canal with soap solution and collect fluid via syringe aspiration or swab. For ruptured eardrum, collect fluid on flexible shaft swab via an auditory speculum. 2 swabs are required for each culture needed; i.e., 2 for bacterial and 2 for fungal. Do Not Refrigerate. Must be delivered to lab within 24 hours.
- Outer ear: Cleanse external ear canal with soap solution. Obtain specimen from active margin, preferable including fresh secretion from deeper areas. 2 swabs are required for each culture needed; i.e., 2 for bacterial and 2 for fungal. Do Not Refrigerate. Must be delivered to lab within 24 hours.
Eye
- Inner eye: Needle aspiration of fluid. Place in sterile container, anaerobic transport system if anaerobes are suspected, or deliver in capped syringe with all air expressed. Do Not Refrigerate. Must be received in lab within 2 hours.
- Outer eye: Cleanse area around eye with mild antiseptic. Pass swab moistened with sterile saline twice over Conjunctiva. Avoid eyelid border and lashes. Must submit 2 swabs for each culture needed; i.e., 2 for bacterial, 2 for fungal, etc. Conjunctival or corneal scrapings should be collected by ophthalmologist and submitted in sterile container; cover with small amount of sterile saline to prevent drying out of specimen. Do Not Refrigerate. Must be received in lab within 24 hours.
Fluids (other than blood, urine or CSF)
Place 5-10 mL (0.5 mL minimum) in sterile container, or anaerobic transport system if anaerobes are suspected for each culture needed; i.e., bacterial, fungal, etc. Can add sterile heparin if specimen may clot. Do Not Refrigerate. Must be received in lab within 2 hours.
- Hematomas, pericardial, peritoneal, amniotic, ascitic, pleural, synovial: Aspirate using needle and syringe.
- Bile: Aspirate with syringe during surgery or from post-op drainage site, or via nasogastric tube from duodenum.
- Breast milk: Cleanse area around areola with mild antiseptic. Obtain through gentle manual expression.
Gastrointestinal tract
- Duodenal aspirate: Aspirate via tube. Place 2 - 5 mL in sterile container, or anaerobic transport system if anaerobes are suspected. Do Not Refrigerate.
- Feces: Collect directly into clean container/diaper. Avoid contamination with urine, soap, etc. Do not collect from toilet. Place in transport media and refrigerate if unable to deliver to lab within 1 hour. Only 2 specimens per admission are recommended. No specimens will be accepted if the patient has been in hospital more than 3 days without prior consultation.
- Gastric aspirate: Patient should have nothing by mouth for 5 hours before test. Specimens are to be collected via a nasogastric tube introduced orally or nasally to the stomach. Overlying and adjacent areas must be carefully prepared to eliminate surface (normal flora) anaerobes. Place 5 - 10 mL in sterile container and refrigerate if unable to deliver to lab immediately. Must be received in lab within 24 hours.
- Rectal swab: Insert swab 1 inch beyond anal sphincter and rotate over crypts once. Anoscopy may be helpful. Use for GC (Neisseria gonorrhoeae) screen, infants or patients with active diarrhea only. If unable to deliver to lab immediately, refrigerate unless for GC. Do not refrigerate swabs for GC.
Genital tract - female
- Amniotic fluid: Aspirate 5 - 10 mL with syringe and place in sterile container. Do Not Refrigerate. Transport to lab within 30 minutes if GC is suspected.
- Cervix: Clean cervix of vaginal secretions and mucus. Use speculum without lubricants. Gently compress cervix with blades of speculum and use a wringing motion with swabs. Obtain exudate from endocervical glands. Do Not Refrigerate. Transport to lab within 30 minutes if GC is suspected.
- Cul-de-sac: Aspirate with needle through posterior vaginal vault. Place a minimum of 1.0 mL in anaerobic container. Do Not Refrigerate.
- Endometrium: Collect as for cervix. If swabs are to be used, collection through a sterile tube sheath may avoid contamination with vaginal flora. Curettings or aspirations are the optimal specimens. If anaerobes are suspected, collect with anaerobic swab. Do Not Refrigerate.
- Intrauterine device (IUD): Remove device plus obtain swab of any secretions/pus. Must request anaerobic culture if Actinomyces/other anaerobe is suspected. Do Not Refrigerate.
- Products of conception (fetal, placenta, membranes): Select suspicious areas of tissues and collect tissue and/or aspirates. Place as much as possible is sterile container, and collect anaerobic swab. Do not send lochia. Do Not Refrigerate.
- Urethra: One or more hours after urinating, clean the area with sterile gauze or swab. “Milk” the urethra or use thin wire swabs to collect material from 2 cm inside urethra. Do Not Refrigerate.
- Uterus, fallopian tubes, ovaries: Usually collected surgically; obtain tissues, aspirates or swabs.
- Vagina: Collect as for cervix. Can collect through aspiration or swabbing. Transport to lab within 30 minutes if GC is suspected. Do Not Refrigerate. For Group B Strep testing, collect at 35-37 weeks gestation using swabs with Aimes or Stuart’s Media without charcoal. Rectal swab for Group B Strep may be placed in transport media with vaginal swab.
- Vulva (including labia, Bartholin's gland): Clean area with antiseptic. Do not use alcohol on mucous membranes. Collect with swab (2) or aspirate with needle and syringe. Include anaerobic swab for Bartholin's gland. Do Not Refrigerate. Must be received in lab within 30 minutes if GC is suspected.
Genital tract - male
- Penis: Clean area with antiseptic. Do not use alcohol on mucous membranes. Vesicles must be opened and secretion collected as aspirate, or on a swab.
- Prostate: Perform digital rectal prostate massage and collect 0.5 mL secretion in sterile container. Do Not Refrigerate.
- Urethra: Same as for female.
Hair
Examine with Wood's light. If Wood's light positive (green fluorescence) pluck fluorescing hairs with sterile forceps. If Wood's light negative, pluck dull gray hairs. Can also scrape the scalp with a scalpel. Hair stubs, contents of plugged follicles, and skin scales are also acceptable. For fungus detection only.
Nails
Clean skin around nail with 70% alcohol. Take deep scrapings of involved areas or clip generous amounts of nails. Remove surface nail and discard. Send clippings from inner nail. Store at room temperature and deliver to lab within 24 hours.
Oral cavity
- Mucosal surface: Rinse mouth with water prior to collection. Scrape or vigorously rub any suspicious lesions with a swab. Collect 2 swabs if fungal and bacterial cultures are needed.
- Dental abscess: Rinse mouth with water and prep with dry sterile gauze. Aspiration in syringe/sterile container is preferable to swab.
Respiratory tract
- Bronchoalveolar/Bronchotracheal secretions, washings or brushings: Collect 2 - 5 mL for each culture needed; i.e., bacterial, fungal, etc., via bronchoscopy and place in sterile container. 1 brush minimum, 2 if fungus or AFB is needed. Cover brushes in 1.0 mL sterile saline. Do not refrigerate unless transport will be > 2 hours. Must be received in lab within 24 hours.
- Epiglottis: Depress tongue with blade and swab epiglottis. Use caution if epiglottis is inflamed, as sampling may cause serious respiratory obstruction.
- Nasopharynx: Pass thin wire swab gently through nostril into Nasopharynx. Stay near septum and floor of nose.
- Nose: Insert swab into nasal passage and rotate against nasal wall. 2 swabs are required for each type of culture; i.e., 2 for bacterial, 2 for fungal, etc. Do Not Refrigerate.
- Sputum: Early morning specimens are preferred. Instruct patient to produce lung material, not saliva. Expectoration can be assisted by saline nebulization, postural drainage or physiotherapy. Collect 3 - 5 mL in a sterile container. Refrigerate if delivery to lab will be greater than 2 hours. AFB and fungal cultures require 3 specimens collected over 3 successive days. Do not pool specimens. Specimens can also be collected according to the Bronchoscopy Protocol by Respiratory Therapy. With induced sputum specimens, the quality of the sputum specimen for AFB cultures exceeds the usual specimen collected under conventional means. Therefore, 3 good specimens obtained in succession the same day by respiratory therapy techs will equal 1 good specimen a day obtained over 3 days. These specimens are pooled and treated as 1 sample. Label the samples 1 of 3, 2 of 3 and 3 of 3. Specimens received before 9am will be pooled, the specimens received after 9am will be handled as separate specimens.
- Throat/Pharynx: Depress tongue with blade. Swab areas of exudation, membrane formation or inflammation. Rub tonsillar crypts vigorously. Use caution if epiglottis is inflamed, as sampling may cause serious respiratory obstruction. Cultured for beta hemolytic Strep only; must order GC Screen if N. gonorrhoeae is suspected. Do Not Refrigerate.
- Tracheal/transtracheal aspirates: Collect through mouth or nose using sterile tubing. Tracheal aspirates are not routinely tested for anaerobes; place 3 - 5 mL in sterile container. Transtracheal aspirates are routinely tested for anaerobes; collect 3 - 5 mL in sterile capped syringe with all air expressed or anaerobic transport system.
Skin
Cleanse infected area with 70% alcohol. Using a sterile scalpel, scrape skin from the borders of the active lesion. Skin from the periphery of the lesion, as well as adjoining healthy areas is preferable. Separate swab needed for each type of culture needed; i.e., 1 for bacterial, 1 for fungal, etc. If stain is requested, submit additional swab. Do Not Refrigerate.
Tissue
Collect surgically or through needle biopsy. Place a 4 mm2 portion into a sterile container for each culture needed; i.e., bacterial, fungal, etc. Indicate which culture is most critical in case of insufficient quantity to perform all. If anaerobes are suspected, place specimen in the inner tube of anaerobic transport system. Do Not place in formalin. Do Not Refrigerate. Must be received in lab within 24 hours.
Urine
1 - 2 mL required for routine culture; 25 - 50 mL of urine is required for AFB or fungus testing. If unable to deliver to lab immediately, refrigerate specimen.
- Clean voided urine: Instruct patient to clean area well and void for 2-3 seconds into toilet then stop. They can begin voiding again into the sterile container until approximately half full. Females should hold labia apart, and uncircumcised males should hold foreskin back while collecting specimen. First morning urine is preferable specimen.
- Catheter urine: Disinfect the catheter tube’s sampling port with 70% alcohol or iodine. Aspirate urine through sampling port with needle, or syringe if using a needle-free system. First morning urine is preferable specimen.
- Suprapubic (bladder) urine: Cleanse site with 70% alcohol or iodine. Aspirate specimen with needle and syringe. First morning urine is preferable specimen.
Wounds/abscess
Collect 2 swabs for each culture needed; i.e., 2 for bacterial, 2 for fungal, etc. Must collect with anaerobic swab if anaerobes are suspected. Do Not Refrigerate. Deliver to lab within 24 hours.
- Open: Pass the swab deep into the base of the lesion to sample the fresh border.
- Closed: Aspirate with needle and syringe. Inoculate aerobic and/or anaerobic transport system with 0.5 mL each.