Department of Laboratory Medicine Microbiology Division

Antibiotics
Mycobacteriology
Mycology
Parasitology
Serology

General Information

Any questions concerning specimen suitability, transportation, or handling should be directed to the Microbiology supervisor at either Harborview Medical Center, (206) 731-5858, or University of Washington Medical Center, (206) 598-6147. AFB (Acid-fast) culture and quantitative biopsy culture examinations are performed at HMC only. University Microbiology will perform most bacteriology, parasitology, mycology, antibiotics, and selected serological procedures.

Requisition

• Special requests: Diphtheria, leptospira, actinomyces, nocardia, etc., should be noted on the microbiology requisition.
• The following information must be present on each request form and specimen container:

Requisition	Specimen Container
Patient's full name Physician name Patient location (referring laboratory) Test requested (be specific) Tentative diagnosis Antibiotic regimen or other treatment Date and time of specimen collection	Patient name Specimen source (type and site) Date of specimen collection

Specimen Collection

• Requests for culture of Bartonella, B. pertussis, H. ducreyi, F. tularensis, or quantitative biopsy require prior notification of the laboratory.
• Collect sufficient material for the tests requested. One specimen per one type of culture is highly desirable.
• Avoid contamination with normal flora during specimen collection.
• If swabs are used, they must be placed in approved swab transport devices. See below for transport of anaerobic cultures.

Anaerobic Cultures	The proper selection of specimens is extremely important for the laboratory confirmation ofanaerobic infection. Since anaerobes are the predominant flora of the mouth, intestinal tract, and vaginal tract, specimens taken from these sites may produce results that may be misleading. Specimens should be collected in a closed system (syringe, transtracheal aspirates, needle punctures, etc.) from deep sites, bypassing normal flora. Do NOT send syringe-transfer aspirate to Port-A-Cul vial (BBL 21608), A.C.T. tube (Remel 12401/12402), or other anaerobic transport tube.
Small Tissues	Collect in anaerobic transport tube. Do not add any fluid. Replace stopper immediately.
Large Tissues	Collect in sterile jar or petri dish. Larger tissue pieces do not require anaerobic transport tubes, since anaerobiasis is maintained inside the specimen for a reasonable length of time. DO NOT ADD FLUID (saline, formalin, or alcohol).
Fluid	AspiratesCollect in a syringe without air; use a small syringe for a small amount of fluid. Expel aspirate into Port-A-Cul vial or other anaerobic transport tube.
Swabs	Return swab to the anaerobic transport tube which contains a reducing atmosphere and an indicator which is colorless in reducing conditions. Swabs are the least desirable specimens for anaerobic cultures. Avoid taking specimens with a swab if at all possible. Note: fungal and AFB cultures require additional samples in AEROBIC transport devices.
Bacterial	See Serology section.
Antigen Collection Blood	Avoid drawing blood from an in-dwelling catheter. If the specimen must be drawn through the catheter, indicate on the requisition. Clean the venipuncture site with 2% iodine followed by alcohol. Collect 20 mL into yellow top blood collection tube(s). Not more than 3 specimens need be drawn in the initial 24-hour period.
Body Fluids	Ascites Pleural fluid Joint fluid Thoracentesis fluid Peritoneal fluid Peritoneal lavage Fluids may be collected into any sterile container. Also see Anaerobic Cultures.
Bronchial Washings	Collect in a sterile, leakproof container.
Cannulas, IUDs,	Submit in sterile containers. Note: Foley catheter tips are not useful for diagnosis of Etc.urinary tract infections.
CSF	Submit cerebrospinal fluid in a sterile container. Do not refrigerate
Feces	Collect specimen on three consecutive days in enteric transport media, such as Cary-Blair medium, one specimen per container. Routine culture includes gram stain and culture for Salmonella spp., Shigella spp. and Campylobacter spp. Culture for enteric pathogens includes gram stain and culture for Salmonella spp., Shigella spp., Campylobacter spp., Yersinia enterocolitica, and E. coli O157. Other enteric pathogens will be cultured for upon request. For ova and parasite examination, see Parasitology.
Gastric	Obtain specimen from patient who has been fasting for 12 hours. For gastric lavage use sterile jar.
Legionella	Culture: Collect in a sterile container. Keep specimen refrigerated. Appropriate respiratory specimens: lung tissue, pleural fluid, bronchial brushing, tracheal aspirate, and other specimens by request. Polyvalent Direct Fluorescence (DF): Submit sputum, bronchial wash, pleural fluid or lung tissue. Swabs are unacceptable.
Mycoplasma Ureaplasma	Collect in Mycoplasma Transport Medium. M. pneumoniae is the slowest growing human pathogenic mycoplasma. Isolation and identification may require from 5 to 30 days. On the other hand, Ureaplasma urealyticum may grow and be identified within 2 days. Unless requested otherwise, our procedure is to culture respiratory specimens for M. pneumonia;. Genital specimens are cultured for Ureaplasma urealyticum and M. hominis. An exception to the above could be respiratory specimens from newborns. In such patients, one may be interested in culturing for Ureaplasmata. Our culture report "NO MYCOPLASMA ISOLATED" indicates that no M. pneumonia or M. hominis has been isolated from respiratory specimens OR that no Ureaplasma urealyticum or M hominis has been isolated from genital specimens. The general term Mycoplasma includes all the mycoplasmata among which are M. pneumonia, M. hominis and Ureaplasma urealyticum.
Organism ID and/or Sensitivity Testing	Send pure culture of isolate on appropriate medium. If organism is an anaerobe, insert plate in Ana Bag.
Special Specimens (tissue, bone,etc.)	Place in a sterile container and transport to the Microbiology Laboratory at the University of Washington Medical Center without delay to prevent overgrowth of aerobes and death of anaerobes. For quantitative biopsy cultures, call Harborview Microbiology, (206) 731-5858, to make arrangements.
Sputum	First morning specimen is best. Reduce irrelevant flora by rinsing mouth and/or cleansing tracheostomy site. Instruct patient on the need for sputum rather than saliva. Collect specimen in a sterile container. A good specimen will show purulent material. If the specimen is watery and almost clear, it is probably saliva and is therefore inadequate for culturing.
Swabs Non-wound	Use only swabs approved for bacteriological examination and culturing. Q-tips and similar cotton tipped applicators may be bacteriostatic or bacteriocidal. Specimens may include throat, nasal, endocervical, urethral, rectal or vaginal sites. Firmly swab the area with a sterile, approved, cotton-tipped applicator. Return swab to plastic sleeve or insert swab deep into the transport medium and break the applicator just below the point where it was held.
Swabs Wound	Remove debris around the wound by gently wiping the area with cotton soaked in sterile saline or water (do not moisten the swab with fluid containing bacteriostatic substances or antibiotics). Obtain specimen from the most active site of the wound. Take care not to swab the normal skin area. Return swab to plastic sleeve or anaerobic transport device or insert swab deep into transport medium and break off applicator just below the point where it was held. On request form, clearly indicate site of wound. For special anaerobic culture, see Anaerobic Cultures.
Urine	It is of utmost importance to follow proper cleansing procedures for obtaining a clean catch or catheterized specimen. Submit in an approved transport device (BD urine transport which contains boric acid as a preservative), or refrigerate.

Reporting

Smear Reports	STAT smears will be called to the physician as soon as they are available.
Culture Results	Positive cultures from blood, CSF, and body fluid will be called to the physician. Cultures positive for N. gonorrhoeae and Group A Beta Strep will be called to the physician.
Antibiotic Susceptibility Reports	Results may be available in a minimum of two days for aerobes and rapid-growing anaerobes. For fastidious anaerobes, a sensitivity test may require four days or longer. Also see Mycobacteriology section.
MIC Results	Results are called as soon as completed.

top

Specimen Type or Site of Collection	Volume/Method	Container for Transport
Bile	Fluid, or swab if necessary	Fluid in sterile tube Swab in transport medium*
Biopsy, or burn, quantitative	Make arrangements with lab, (206) 731-5858	Sterile container, do not add fluid
Body fluids	5 mL	Sterile container
Blood	20 mL	Yellow top blood collection tubes
Bronchoscopy or Bronchial washing	5 mL	Sterile container
Cyst fluid	As much as possible	Sterile container, Port-A-Cul vial or other anaerobic transport tube
Dialysis fluid	5 mL	Sterile tube
Ear or eye	Swab	Swab in transport medium*
Joint fluid	5 mL optimal	Sterile container
Nasopharynx	Swab	Swab in transport medium*
Nose	Swab	Swab in transport medium*
Organism for identification or sensitivity testing	Pure culture of isolate	Agar plate or tube (in an Ana Bag for anaerobic isolate)
Pericardial, Peritoneal, or Pleural fluid	5 mL	Sterile container, Port-A-Cul vial or other anaerobic transport tube
Pus	Aspirate if possible Swab	Port-A-Cul vial, or other anaerobic transport tube Swab in transport medium*
Rectal Swab	Swab	Swab in transport medium*
Sinus (wound or respiratory)	Swab	Swab in transport medium*
Spinal fluid	5 mL optimal	Sterile container
Sputum	5 mL	Sterile container
Stool	5 mL	Enteric transport medium
Throat	Swab	Swab in transport medium*
Tissue	Tissue	Sterile container
Tracheal aspirate	5 mL	Sterile container
Transtracheal aspirate	As much as possible	Port-A-Cul vial, sterile container
Urethral	Swab	Swab in transport medium*
Urine	10 mL	Preservative tube or sterile container
Uterine	Swab	Swab in transport medium*or anaerobic transport tube
Vaginal/Cervical	Swab	Swab in transport medium*or Transgrow /JEMBEC plates to screen for N. gonorrhoeae
Wound	Aspirate or swab	Transport medium or anaerobic transport tube

*Recommend: Amies Transport Medium

Requisition: General Microbiology [pdf]

Specimen Antibiotic susceptibilities are performed only on pure culture of bacterial or fungal isolates

Methods

Disk Diffusion	Antibiotic susceptibilities can be performed by the Kirby-Bauer method for rapidly growing aerobes and by the E-test methodology for anaerobes and rapidly growing anerobes.
MIC/MBC	MIC (Minimum Inhibitory Concentration) for bacteria, (tube or agar dilution), and MBC (Minimum Bactericidal Concentration) are offered Monday through Friday. Please indicate the antibiotic to be tested. Automated (Vitek) MIC is performed daily; only a limited spectrum of antibiotics can be tested.
MFC	MFC (Minimum Fungicidal Concentration) is offered Monday through Friday. Please indicate the antibiotic to be tested.
Antibiotic Levels	Submit 1 mL serum, CSF, or other body fluid. Clearly indicate dosage regimen, last dosage given, and other antibiotics being administered (previous 48 hours). For aminoglycosides (Gentamicin, Tobramycin, Amikacin) and Vancomycin, submit pre-dose and post-dose specimens. Collect pre-dose serum immediately prior to next dose. Infuse antibiotic over a 30-minute period, and collect serum for the post-dose level exactly 30 minutes after the infusion has concluded.
Serum Inhibition Assay	Submit to the laboratory a 2-3 mL aliquot of the patient's serum or CSF and a pure culture of patient's organism.
Synergism Study	Make prior arrangements with Antibiotic Lab, (206) 598-6147.

Reporting

Kirby-Bauer and Vitek sensitivity tests are available daily and are usually reported within 24 hours after pure culture of the organism has been received. Anaerobic susceptibilities are performed Monday through Friday. Due to the fastidious nature of most anaerobes, sensitivity tests may require 48 hours or longer to complete. Results of tube or agar dilution MIC, MBC, MFC, Schlichter tests, antibiotic levels and synergism studies are telephoned as soon as completed (24-72 hours).

Requisition: General Microbiology [pdf]

Culture & smear for Acid-fast bacilli (AFB)

• All specimens except blood are cultured by both conventional and rapid detection methods (BD MGIT", Mycobacteria Growth Indicator Tube, system or the BACTEC" radiometric system). Blood specimens are cultured in BACTEC" Myco/F Lytic medium.
• Specimens are processed 7 days a week and respiratory specimen AFB smear results are reported the same day. Specimens received at Harborview after 9:00 A.M. will be processed on the following day.
• Same day smear results for late arriving specimens are available by request for specimens received at Harborview by 2:00 P.M.
• Drug susceptibility testing is performed for M. tuberculosis isolates by the BACTEC" rapid radiometric method.
• Significant mycobacteria other than tuberculosis are sent out by request for susceptability testing against first and second order drugs.

Reporting

M. tuberculosis	GenProbe" MTD2‘ Mycobacterium Tuberculosis Direct assay detects M. tuberculosis complex
Direct Test: Nucleic Acid Amplification	rRNA and is equivalent to PCR (PCR detects DNA). Any non-bloody AFB smear-positive respiratory specimen may be tested. Test is performed Tuesday & Friday the day AFTER processing for AFB culture. Results are usually reported within 2 days. Charge for assay includes test for inhibition of amplification and any necessary repeat testing.
Organism Identification	Send pure culture of isolate on appropriate media in double-walled container. Specify: DNA probe for M. tuberculosis complex, M. avium-intracellulare, and M. gordonae. Expect results within one week for isolates received on solid media, and within 2-3 weeks if liquid culture. Identification by HPLC (high performance liquid chromatography) and/or GLC (gas liquid chromatography). Time to result varies with species.
Susceptibility Testing: M. tuberculosis Rapid Radiometric Method	Send pure culture of isolate on solid media. Expect results within 2 weeks. The following drug concentrations are tested: Streptomycin 6.0 ug/mL Isoniazid 0.1 ug/mL Rifampin 2.0 ug/mL Ethambutol 7.5 ug/mL
Rapidly growing Mycobacteria	Rapid growers such as M. abscessus, M. chelonae, and M. fortuitum are tested by a rapid modified disk diffusion method. In house validation data shows excellent correlation with standardized but more time consuming MIC's. Send pure culture of isolate on solid media. Expect results within one week.

Specimen Collection

*Container for transport must be sterile, leak proof, and double-wrapped; Refrigerate. Place a BIO-HAZARD warning label on the outside of the shipping container. When sending prepared smears, transport in a slide container that is taped closed.

Body Fluids	Use sterile container. Smears will be made from all body fluids except CSF and urine. Blood: Collect one 10 mL blood collection tube with SPS anticoagulant (yellow top).
Gastric	Gastric washing should be considered only if sputum production cannot be induced. Collect a fasting, early morning specimen on three consecutive days. Use sterile saline and sterile container. Neutralize 35-50 mL of gastric washings with sodium carbonate immediately following collection. Smears will not be made from gastric washings.
Sputum	Collect an early morning sputum on three consecutive days. Do not collect a 24-hour sputum. Collect specimens at weekly intervals for therapy follow-up of known tuberculosis patients.
Stool	Stool culture for AFB will only be performed if AFB are seen on direct smear.
Urine	Collect the first morning specimen (midstream or catheter) and send to the laboratory immediately. Minimum acceptable volume is 40 mL. It is recommended to collect 3 specimens on 3 consecutive days. No more than one specimen per day per patient will be accepted. Smears will be made by request.

Specimen Type or Site of Collection	Volume or Method	Container for Transport*
Biopsy (tissue)	1 g of tissue, if possible	Sterile container, do not add fluid
Blood	10 mL	1 10 mL blood collection tube with SPS anticoagulant (yellow top)
Bronchoscopy or bronchial washing	5 mL or more	Sterile container
Cerebrospinal fluid (CSF)	As much as possible	Sterile tube
Fluids (pleural,pericardial, peritoneal, etc.)	As much as possible	Sterile container or tube
Gastric lavage	5-10 mL (neutralize with sodium carbonate)	Sterile container or tube
Organism for identification or susceptibility testing	Pure culture on appropriate media	Double-walled container
Sputum	5-10 mL	Sterile container
Transtracheal aspirate	As much as possible	Syringe
Urine	50 mL, first morning void, midstream or catheterized	Sterile container
Wound	1 mL of aspirate or 1g of tissue. Swab in aerobic transport media is acceptable only if biopsy or aspirate is not obtainable.	Sterile container

Requisition: General Microbiology [pdf] Indicate on requisition which fungus is suspected.

Specimen Collection

Blood	Collect in SPS anticoagulant (yellow top).
Candida or Cryptococcus Ag	See Serology section
Organism Identification	Pure culture of mold or yeast on appropriate medium-slant or plate. Isolates from routine cultures will be sent to Mycology for identification if the fungus is a mold or if yeast is the predominant isolate.
Probe Testing for Histoplasma & Coccidiodes	This is a sensitive and very specific method for making a rapid (as few as two days) definitive identification of Coccidioides immitis and Histoplasma capsulatum isolates. Submit C. immitis and/or H. capsulatum isolates on Sabouraud's agar slants (tubed). Clearly label tubes as possible Coccidioides immitis isolate.
Skin or Nail Scrapings	Clean area with 70% alcohol. Obtain scraping from active margin. Submit in sterile container.
Stool	Stools are generally not cultured for fungus unless enterocolitis due to fungal overgrowth is suspected.
Swabs	Swabs are poor specimens for fungus culturing. If a specimen is to be collected on a swab, transport in sterile tube to laboratory.

Specimen Type or Site of Collection	Volume or Method	Container for Transport
Abscess	5 mL if possible	Sterile tube
Biopsy	1 g of tissue if possible	Sterile container with saline
Blood	7 mL	SPS anticoagulant (yellow top)
Bronchoscopy or Bronchial washing or Bronchial brush	5 mL	Sterile container
Bone marrow	0.5 mL	SPS anticoagulant (yellow top)
CSF	5 mL	Sterile tube
Hair	Hair shaft and base	Sterile container or petri dish
Nail	Scraping from nail and active margin	Sterile container or petri dish
Skin	Scraping from active margin or lesion	Sterile container or petri dish
Sputum	5-10 mL	Sterile container
Tissue	1 g if possible	Sterile tube or petri dish
Transtracheal aspirate	As much as possible	Sterile container
Urine	25-50 mL	Sterile container
Vaginal	Secretion	Sterile container
Wound	Aspirate or Swab	Transport medium

MIC/MFC Synergism

MIC testing and synergism studies are available for most approved fungicidal and fungistatic drugs. Make arrangements by calling (206) 598-6147. Also, see the Antibiotics section.

Reporting

• Fungal cultures are reported as negative after 4 weeks.
• Positive cultures are reported at completion with interim reports to inform the physician of the progress of the culture.
• Blood cultures positive for fungus or yeast are telephoned to the physician or ordering location.
• Any positive wet mount or stain result is telephoned to the ordering physician or location, if requested.

Specimen Collection

• Patients should be supplied with a fecal specimen collection kit, available on request from the Microbiology Laboratory. The specimen can then be refrigerated up to one week. (Indicate date and time of collection of specimen).
• Swabs will not be accepted for parasite examination.
• Specimen should be in clean containers with a secure lid. Stool specimen should not be collected within two weeks after Barium X-ray procedure nor after laxatives.
• Generally, one specimen will be accepted for each patient per day.

Blood or Bone Marrow Parasites	At fever spike, draw blood in EDTA-containing tube (lavender top). At time of blood draw, make five thin and three thick smears (A thick smear is made by placing a dime-size drop of blood on a slide; let air-dry). Draw an additional EDTA-tube of blood 12 hours after the fever spike. Smears will be examined for the presence of Plasmodium, Hemoflagellates and microfilaria.
Cryptosporidium Cyclospora or Microsporidia	Send stool unpreserved or in formalin.
Echinococcosis	Send cyst fluid for examination.
Gastric Specimens	Deliver gastric aspirates for Giardia lamblia or Strongyloides stercoralis to the laboratory immediately after collection.
Giardia Antigen	Single stool specimen-freshly passed, refrigerated, frozen, formalinized or in S.A.F. preservative. Purged or enema-induced specimens or specimens containing medications such as antibiotics, oils or barium are unsuitable for testing.
Liver Abscess	Take specimen from margin of abscess. Indicate source of specimen on requisition. Deliver to lab within one hour.
Organism Identification	Send organism or appropriate specimen.
Ova & Parasite, Routine	Normally passed stool, submitted in a preservative kit such as formalin/PVA. Only one specimen (first morning specimen) accepted per day. A series of three separate specimens may be collected. If preservative kit is not available, deliver stool to lab within one hour of collection for detection of ameobic trophozoites.
Pneumocystis	Submit lung biopsy, bronchial biopsy, bronchial washings or induced sputum. Expectorated sputum is undesirable because of low sensitivity.
Sigmoidoscopy	Collect in clean tube (via aspiration, not swab), and add a drop of saline to prevent dessication. Transport to laboratory immediately.
String Test	String test for Giardia lamblia or Strongyloides stercoralis. Follow instructions on package insert. Pre-arrange with laboratory and deliver immediately after collection.
Urine	For detection of Trichomonas vaginalis, collect a urine specimen voided in the morning including the last few drops voided. For detection of Schistosoma haematobium, multiple specimens collected between noon and 3 PM are recommended.

Reporting

• Reports for the direct and concentrate test are available within 24 hours, Monday through Friday. On weekends, specimens will be received and preserved but not processed.
• Positive results are called to the physician or ordering location.
• Trichrome stain results are available within 48 to 72 hours after receipt of specimen.
  

The following tests are offered through the Serology section of the Clinical Microbiology Laboratory.

Antibody Detection:	Fungal antibody detection:	Bacterial Antigen detection:
H. Pylori (EIA)	Aspergillus	Beta-Streptococcus Group A
Toxoplasma IgG/lgM (EIA)	Blastomyces	Beta-Streptococcus Group B
	Coccidioides	Clostridium difficile
Syphilis:	Histoplasma	Streptococcus pneumoniae
RPR		Hemophilus influenzae
VDRL	Fungal Antigen detection:	Neisseria meningitidis
	Cryptococcus
Parasitic Antigen detection:
Giardia (EIA)	Streptococcal Antibodies:
	ASO
	Streptozyme

Other serologies are done at the Seattle-King County Health Department Laboratory, the Washington State DSHS Laboratory, the Center for Disease Control, or reputable reference laboratories. For all work referred to these laboratories, a complete patient history, as well as paired (acute and convalescent) sera, is required. It usually will be to the advantage of a laboratory or physician's office to send out their own serology requests. However, for a fee, the University Microbiology Laboratory will handle these referrals. Microbiology also has the forms required by these laboratories. They are available, on request, from the Microbiology laboratory at (206) 598-6147.

Requisition: General Microbiology [pdf]

Specimen Collection

Paired sera are recommended for optimal results of antibody tests.

Test	Amount of Specimen	Other Instructions
Antibody
ASO	1 mL	Serum
Fungal		Serum or CSF.
Aspergillus	1 mL
Blastomyces	1 mL	Precipitin tests for
Coccidioides	1 mL	Histoplasma, Blastomyces,
Histoplasma	1 mL	Coccidioides, and Aspergillus.
H. pylori	1 mL	Serum
RPR	0.5 mL	Serum
Streptozyme	1 mL	Serum
Toxoplasma (IgG & IgM or IgG only)	1 mL	Serum
VDRL/TP	0.5 mL	Serum/CSF

Antigen
Bacterial
Beta-Streptococcus Group A	Swab	Culturette or other Non-gel transport medium
Beta-Streptococcus Group B	1 mL	Urine or CSF (neonates only)
Clostridium difficile	5g or 10mL	Stool, fresh
S. pneumoniae	1 mL	Urine, CSF or Serum
H. influenzae	1 mL	Urine, CSF or Serum
N. meningitidis	1 mL	Urine, CSF or Serum
Fungal
Cryptococcus neoformans	0.5 mL	Serum or CSF
Parasitic
Giardia	5 g or 10 mL	Stool - fresh or preserved in 10% formalin or SAF

Reporting

• VDRL and RPR as well as TP are reported Monday through Friday.
• ASO, Streptozyme, and Toxoplasma test results are reported within 72 hours of receipt of specimen.
• Cryptococcus neoformans antigen test results are reported within 72 hours of receipt of specimen, Monday through Friday.
• Allow 72 to 96 hours for the completion of precipitin tests for deep mycosis.
• Bacterial antigen tests for Group A and Group B beta-hemolytic streptococcus are performed daily.
• Bacterial antigen tests for Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis are performed weekdays.
• Giardia results are reported 3 times a week, and Helicobacter pylori results twice per week.

top

Last updated: 5/2/2006

© 2008 University of Washington. All rights reserved.
Notice of Privacy Practices | Copyright and Disclaimer