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

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

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.
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Specimens for Bacteriological Culture

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

Antibiotics

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).

Mycobacteriology

Requisition: General Microbiology [pdf]

Culture & smear for Acid-fast bacilli (AFB)

Reporting

  • Positive smears and positive cultures are called to the ordering physician or laboratory followed by a written report.
  • Negative cultures are reported in 8 weeks.
  • 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

     

    Mycology

    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

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    Parasitology

    Specimen Collection

     

    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

     

    Serology

    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

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    Last updated: 5/2/2006