Bacteriology, Mycology, and Mycobacteriology Specimen Collection Guidelines

 

Specimen Type Collection Time and Temp

Replica  limits

Comment(s)
Guidelines Device and/or minimum vol Local Transportb Courier or local storage

Abscess

Remove surface exudate by wiping with sterile saline or 70% ETOHa.

ESwab transport system, sterile tube

2 h or less, RTa

24 h or less, RTa

 1/day/source

Tissue or fluid is always superior to a swab specimen. If swabs must be used, collect two: one for culture and one for Gram staining. Preserve them with Stuart’s or Amies medium.

    Open

Aspirate if possible, or pass a swab deep into the lesion and firmly sample the lesion’s advancing edge.

ESwab transport system

2 h or less, RTa

24 h or less, RTa

1/day/ source

A sample from the base of the lesion and a sample from the abscess wall are most productive.

    Closed

Aspirate abscess wall material with needle and syringe. Aseptically transfer all material into anaerobic transport device or sterile tube.

Transport Tube, 1 ml or more, or sterile tube.

2 h or less, RTa

24 h or less, RTa

1/day/ source

Sampling of the surface area can introduce colonizing bacteria not involved in the infectious process.

Bone marrow

Prepare puncture site as for surgical incision

Inoculate a pediatric 1.5 ml. lysis centrifugation tube.

16 h or less, RTa if in culture bottle or tube.

16 h or less, RTa

1/day

Small volumes of bone marrow may be inoculated directly onto culture media.

Burn

Clean and debride the wound prior to specimen collection.

Tissue placed in a screw-cap container. Aerobic culture. Swab exudate.

2 h or less, RTa

24 h or less, RTa

1/day/ source

A 3 to 4 mm punch biopsy is optimum when quantitative cultures are ordered. Process for aerobic culture only. Quantitative culture may or may not be valuable. Surface cultures of burns may be misleading.

Catheter:
    I.V.

1. Cleanse the skin around the catheter site with alcohol.
2. Aseptically remove catheter and clip a 5 cm distal tip of the catheter directly into a sterile tube.
3. Transport directly to microbiology to prevent drying.

Sterile screw-cap tube or cup

15 minutes or less, RTa

24 h or less, 4oC

None

Acceptable I.V. catheters for semiquantitative culture (Maki method): central, CVP, Hickman, Broviac, peripheral, arterial, umbilical, hyperalimentation, Swan-Ganz.

    Foley

Do not culture since growth represents distal urethral flora.

 

 

 

 

Not acceptable for culture.

Cellulitis

1. Cleanse site by wiping with sterile saline or 70% alcohol.
2. Aspirate the area of maximum inflammation (commonly the center rather than the leading edge) with a fine needle and syringe.
3. Draw small amount of sterile saline into syringe and aspirate into sterile screw-cap tube.

Sterile tube (syringe transport not recommended)

15 minutes or less, RTa

24 h or less, RTa

None

Yield of potential pathogens is only 25-35%.

CSFa

1. Disinfect site with 2% iodine tincture.
2. Insert a needle with stylet at L3-L4, L4-L5, or L5-S1 interspace.
3. On reaching the subarachnoid space, remove the stylet and collect 1-2 ml of fluid in each of three leakproof tubes.

Sterile screw-cap tube
Minimum amount required:
bacteria, 1 ml or greater; fungi, 2 ml or greater;
AFB, 2 ml or greater;
virus, 1 ml or greater.

Bacteria: never refrigerate; 15 min or less, RT

Virus: transport on ice; 15 min or less, 4oC

24 h or less, RT

 

72 h or less, 4oC

None

Obtain blood cultures also. If only 1 tube of CSF is collected, it should be submitted to microbiology first; otherwise submit tube 2.
Aspirate of brain abscess or a biopsy may be necessary to detect anaerobic bacteria or parasites.

Decubitus ulcer

See comment: A swab specimen is not the specimen of choice.
1. Cleanse surface with sterile saline.
2. If a sample biopsy is not available, vigorously swab the base of the lesion.
3. Place the swab in appropriate transport system.

ESwab or Transport Tube, or sterile tube (for tissue)

2 h or less, RTa

24 h or less, RTa

1/day/ source

A decubitus swab provides little clinical information; discourage collection of it. A tissue biopsy sample or a needle aspirate is the specimen of choice.

Ear:
     Inner

Tympanocentesis should be reserved for complicated, recurrent, or chronic persistent otitis media.
1. For an intact ear drum, clean the ear canal with soap solution and collect fluid via the syringe aspiration technique
2. For a ruptured ear drum, collect fluid on a flexible-shaft swab via an auditory speculum.

Sterile tube, or Transport Tube.

2 h or less, RTa

24 h or less, RTa

1/day/ source

Throat or nasopharyngeal cultures are not predictive of agents responsible for otitis media and should not be submitted for that purpose.

      Outer

1. Use a moistened swab to remove any debris or crust from the ear canal.
2. Obtain a sample by firmly rotating the swab in the outer canal.

ESwab transport system.

2 h or less, RTa

24 h or less, 4oC

1/day/ source

For otitis externa, vigorous swabbing is required since surface swabbing may miss streptococcal cellulitis.

Eye:
     Conjunctiva

1. Sample both eyes using separate swabs (premoistened with sterile saline) by rolling over each conjunctiva.
2. Inoculate media at time of collection.
3. Smear swabs onto 2 slides for staining.

Direct culture inoculation: BAPa and CHOCa or ESwab transport system.

Plates: 15 min or less, RTa

Swabs: 2 h or less, RTa

24 h or less, RTa

None

If possible, sample both conjunctivae, even if only one is infected, to determine the indigenous microflora. The uninfected eye can serve as a control with which to compare the agents isolated from the infected eye.

    Corneal
    Scrapings

1. Obtain conjunctival swab specimens as described above.
2. Instill 2 drops of local anesthetic.
3. Using a sterile spatula, scrape ulcers or lesions and inoculate scraping directly onto media.
4. Apply remaining material to 2 clean glass slides for staining.

Direct culture inoculation, blood, CHOCa, and SAB agar

15 minutes or less, RTa

24 h or less, RTa

None

It is recommended that swabs for culture be taken prior to anesthetic application, whereas corneal scrapings can be obtained afterward.

Fluid or aspirates

Prepare eye for needle aspiration of fluid.

Sterile screw-cap tube or direct inoculate of small amount of fluid onto media.

15 minutes or less, RTa

24 h or less, RTa

1/day

Include fungal media. Anesthetics may be inhibitory to some etiologic agents.

Feces:
    Routine culture

1. Pass directly into a clean, dry container.
2. Transport the specimen to lab within 1 h of collection or transfer a portion to a Cary-Blair transport system.
3. Cary-Blair: carefully remove the cap and attached spoon to pick several spoonfuls of the stool, especially from areas that are slimy, bloody, or watery. Place the stool into the vial to the fill line. Mix well.

Clean, leak-proof, wide-mouth container or a Cary-Blair transport system; 2 g or greater

Unpreserved: 1 h or less, RT.


Cary-Blair transport system: 24 h or less, RTa

24 h or less, 4oC

 

48 h or less, RTa or 4oC

1/day

Do not perform routine stool cultures on patients whose length of stay was greatter than 3 days and the admitting diagnosis was not gastroenteritis. Culture and toxin tests for Clostridium difficile should be considered in these cases.
Swabs for routine pathogens are not recommended except in infants and in patients with active diarrhea (see Rectal swabs).

     C. difficile

Pass liquid or soft stool directly into a clean, dry container. Soft stool is defined as stool assuming the shape of its container.
A swab specimen is not recommended for toxin testing.

Sterile, leak-proof, wide-mouth container; 5 ml or greater

1 h or less, RT;
1-24 h, 4oC;
Greater than 24 h,      -20oC

2 days, 4oC, for culture;
3 days, 4oC, or longer at –70oC for toxin test

1 /2 days

Patients should be passing 5 or more liquid or soft stools per 24 h. Testing of formed or hard stool is often unproductive and may indicate only commensal carriage.

Escherichia coli
For Shiga-like  (Vero) toxin
(0157 and
other serotypes)

Pass liquid or bloody stool into a clean, dry container.

Sterile, leak-proof wide-mouth container or Cary-Blair transport system

Unpreserved:
1 h or less, RT;
Cary-Blair transport system: less than 24 h, RTa or 4oC

24 h or less, 4oC

48 h or less, RTa

1/day

Bloody or liquid stools collected within 6 days of onset from patients with abdominal cramps have the highest yield.

    Leukocytes

Pass feces directly into a clean, dry container. Transport specimen to lab within 1 h of collection or transfer to ova and parasite transport system (SAF).

Sterile, leak-proof, wide-mouth container 2 ml or greater

Unpreserved:
1 h or less, RTa

24 h or less, 4oC

Indefinite, RTa

1/day

 

    Rectal swab

1. Carefully insert a swab ≈ 1 in. beyond the anal sphincter.
2. Gently rotate the swab to sample the anal crypts.
3. Feces should be visible on the swab for detection of diarrheal pathogens.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

Reserved for detecting Neisseria gonorrhoeae, Shigella spp., Campylobacter spp., HSV, and anal carriage of group B Streptococcus spp. or for patients unable to pass a specimen.

    Fistulas

See Abscess

 

 

 

 

 

Fluids: abdominal, amniotic, ascites, bile, joint, paracentesis, pericardial, peritoneal, pleural, synovial, thoracentesis

1. Disinfect overlying skin with 2% iodine tincture.
2. Obtain specimen via percutaneous needle aspiration or surgery.
3. Transport specimen to laboratory immediately.
4. Always submit as much fluid as possible; never submit a swab dipped in fluid.

Blood culture bottle for bacteria and yeast or sterile screw-cap tube or Transport Tube.

Bacteria, 1 ml or greater;
fungi, 10 ml or greater;
mycobacteria, 10 ml or greater

15 minutes or less, RTa

24 h or less, RTa. Pericardial fluid and fluids for fungal cultures:
24 h or less, 4oC

None

Amniotic and culdocentesis fluids should be transported in anaerobic system and need not be centrifuged prior to Gram staining. Other fluids are best examined by Gram staining of a cytocentrifuged preparation. See Table 4.

Gangrenous tissue

See Abscess

 

 

 

 

Discourage sampling of surface or superficial tissue; tissue biopsy or aspirates are preferred.

Gastric: wash or lavage

Collect early in the morning before patients eat and while they are still in bed.
1. Introduce a nasogastric tube orally or nasally into the stomach.
2. Perform lavage with 25-50 ml. of chilled, sterile, distilled water.
3. Recover sample and place it in a leakproof, sterile container.
4. Before removing the tube, release suction and clamp it.

Sterile, leak-proof container.

15 minutes or less, RTa

24 h or less, 4oC

1/day

The specimen must be processed promptly since mycobacteria die rapidly in gastric washings.

Genital (female):
   Amniotic

1. Aspirate via amniocentesis, cesarean section, or intrauterine catheter.

Transport Tube or sterile tube, 1 ml or greater

15 min or greater, RTa

24 h or greater, RTa

None

Swabbing or aspiration of vaginal membrane is not acceptable because of the potential for culture contamination by commensal vaginal flora.

    Bartholin

1. Disinfect skin with a iodine preparation.
2. Aspirate fluid from ducts.

Transport Tube
1 ml or greater

2 h or less, RT

24 h or less, RTa

1/day

 

    Cervical

1. Visualize the cervix using a speculum without lubricant.
2. Remove mucus and secretions from the cervix with a swab and discard the swab.
3. Firmly, yet gently, sample the endo-cervical canal with a newly obtained sterile swab.

ESwab transport system.
GC Selective plate.

2 h or less, RTa

24 h or less, RTa

1/day

See information on virus and chlamydia collection and transport needs.
Neisseria gonorrhoeae is found in exudates, whereas chlamydiae infect specific cells.

    Cul-de-sac

Submit aspirate or fluid

Transport Tube greater than 1 ml

2 h or less, RTa

24 h or less, RTa

1/day

 

    Endometrial

1. Collect transcervical aspirate via a telescoping catheter.
2. Transfer the entire amount to an anaerobic transport system.

Transport Tube 1 ml or greater

2 h or less, RTa

24 h or less, RTa

1/day

 

    Products of
    conception

1. Submit a portion of tissue in a sterile container.
2. If obtained by cesarean section, immediately transfer it to an anaerobic transport system

Sterile tube or Transport Tube

2 h or less, RTa

24 h or less, RTa

1/day

Do not process lochia. Culture of this specimen may or may not provide clinically relevant results, and such results can be misleading.

    Urethral

Collect 1 h after patient has urinated.
1. Remove exudate from the urethral orifice.
2. Collect discharge material on a swab by massaging the urethra against the pubic symphysis through the vagina.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

If no discharge can be obtained, wash the external urethra with betadine soap with rinse with water. Insert a urethrogenital swab 2-4 cm into the urethra; rotate swab for 2 seconds.

    Vaginal

1. Wipe away an excessive amount of secretion or discharge.
2. Obtain secretions from the mucosal membrane of the vaginal vault with a sterile swab or pipette.
3. If a smear is also requested, use a second swab.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

For intrauterine devices, place entire device in a sterile container and submit at RTa.
Gram staining is recommended for confirmation of bacterial vaginosis. Results from cultures are often inaccurate and misleading.

Genital (female or male):
  Lesion

1. Clean the lesion with sterile saline and remove the surface of the lesion with a sterile scalpel blade.
2. Allow transudate to accumulate.
3. Pressing the base of the lesion, firmly sample exudate with a sterile swab.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

Specimens for syphilis should not be submitted for culture.

Genital (male):
     Prostate

1. Cleanse the glans with soap and water.
2. Massage the prostate through the rectum.
3. Collect fluid on a sterile swab or in a sterile tube.

ESwab transport system or sterile tube

2 h or less, RTa

24 h or less, RTa

1/day

More relevant results may be obtained by adding a urine specimen immediately before and after massage to indicate urethral and bladder organisms.
Ejaculate can also be cultured.

     Urethra

Insert a urethrogenital swab 2-4 cm into the urethral lumen, rotate the swab, and leave it in place for at least 2 seconds to facilitate absorption.

ESwab transport system. GC selective place

2 h or less, RTa

24 h or less, RTa

1/day

See information on virus and chlamydia collection and transport needs.

Hair:
  Dermatophytosis

1. With forceps, collect at least 10-12 affected hairs with the base of the shaft intact.
2. Place in a clean tube or container.

Clean container, 10 hairs

24 h or less, RTa

 

1/day/ site

Collect scalp scales, if present, along with scrapings of active borders of lesions. Note any antifungal therapy taken recently.

Nail:
  Dermatophytosis

1. Wipe the nail with 70% alcohol using gauze (not cotton).
2. Clip away a generous portion of the affected area and collect material or debris from under the nail.
3. Place material in a container.

Clean container.
Enough scrapings to cover the head of a thumb tack.

24 h or less, RTa

 

1 day

 

Pilonidal cyst

See Abscess

 

 

 

 

 

Respiratory, lower:
Bronchoalveolar
lavage,
bronchial brush or
wash, tracheal
aspirate

1. Place aspirate or washing in a sputum trap.
2. Place brush in a sterile container with saline.

Sterile container,
Greater than 1 ml

2 h or less, RTa

24 h or less, 4oC

1/day

 

  Sputum, 
     expectorate

1. Collect the specimen under the direct supervision of a nurse or physician.
2. Have the patient rinse or gargle with water to remove superficial flora.
3. Instruct the patient to cough deeply to produce a lower respiratory specimen (not postnasal fluid). Collect in a sterile container.

Sterile container, greater than 1 ml.
Minimum amounts:
Bacteria, greater than 1 ml;
fungi, 3-5 ml;
mycobacteria, 5-
10 ml; parasites, 3-5 ml

2 h or less, RTa

24 h or less, 4oC

1/day

For pediatric patients unable to produce a specimen, a respiratory therapist should collect a specimen via suction. The best specimen should have 10 squamous cells or less per 100x field.

  Sputum, induced

1. Have the patient rinse mouth with water after brushing the gums and tongue.
2. With the aid of a nebulizer, have the patient inhale ≈25 ml of 3-10% sterile saline.
3. Collect the induced sputum in a sterile container.

Sterile container

2 h or less, RTa

24 h or less, RTa

1/day

Histoplasma capsulatum and Blastomyces dermatitidis survive for only short periods of time once a specimen is obtained. Fungal recovery is primarily for Cryptococcus spp. and some filamentous fungi; other yeasts rarely cause lower respiratory tract infection.

Respiratory, upper:
   Oral

1. Remove oral secretions and debris from the surface of the lesion with swab and discard swab.
2. Using a second swab, vigorously sample the lesion, avoiding any areas of normal tissue.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

Discourage sampling of superficial tissue for bacterial evaluation. Tissue biopsy or needle aspirates are the specimens of choice.

    Nasal

1. Insert a swab, premoistened with sterile saline ≈2 cm into the nares.
2. Rotate the swab against the nasal mucosa.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

Anterior nose cultures should be reserved for detecting staphylococcal and streptococcal carriers or for nasal lesions.

    Throat

1. Depress the tongue with a tongue depressor.
2. Sample the posterior pharynx, tonsils, and inflamed areas with a sterile swab.

ESwab transport system.

2 h or less, RTa

24 h or less, RTa

1/day

Throat cultures are contraindicated for patients with an inflamed epiglottis. Swabs for N. gonorrhoeae should be inoculated or GC selective plates and transport at RTa.

Skin:
  Dermatophytosis
 

1. Cleanse the affected area with 70% alcohol.
2. Gently scrape the surface of the skin at the active margin of the lesion. Do not draw blood.
3. Sample the posterior pharynx, tonsils, and inflamed areas with a sterile swab.

ESwab transport system.

24 hr or less, RTa

 

1/day/ site

If the specimen is submitted between glass slides, tape the slides together and submit in an envelope.

Tissue

1. Submit in a sterile container.
2. For small samples, add several drops of sterile saline to keep moist.
3. Do not allow tissue to dry out.
4. Place in a sterile, moist jar.

Sterile, screw-cap jar. Saline may need to be added.

15 minutes or less, RTa

24 h or less, RTa

None

Always submit as much tissue as possible. If possible, save an amount of surgical tissue at –70oC in case further studies are needed.

Urine:
    Female,
        midstream

1. Thoroughly cleanse the urethral area with soap and water.
2. Rinse the area with wet gauze pads.
3. While holding the labia apart, begin voiding.
4. After several milliliters have passed, collect a midstream portion without stopping the flow of urine.

Sterile wide-mouth container, 1 ml or greater, or urine transport kit

Unpreserved:
2 h or less, RTa
Preserved:
24 h or less, RTa

24 h or less, 4oC

1/day

 

   Male,
       midstream

1. Cleanse the glans with soap and water.
2. Rinse with wet gauze pads.
3. Holding the foreskin retracted, begin voiding.
4. After several milliliters have passed, collect a midstream portion without stopping the flow of urine.

Sterile wide-mouth container, 1 ml or greater, or urine transport kit

Unpreserved:
2 h or less, RTa
Preserved:
24 h or less, RTa

24 h or less, 4oC

1/day

 

   Straight catheter

1. Thoroughly cleanse the urethral area with soap and water.
2. Rinse the area with wet gauze pads.
3. Aseptically, insert a catheter into the bladder.
4. After allowing ≈15 ml to pass, collect urine to be submitted in a sterile container.

Sterile, leakproof container

Unpreserved:
2 h or less, RTa
Preserved:
24 h or less, RTa

24 h or less, 4oC

1/day

If preparation is inadequate, the procedure may introduce urethral flora into the bladder and increase the risk of iatrogenic infection.

   Indwelling
   catheter

1. Disinfect the catheter collection port with 70% alcohol.
2. Use a needle and syringe to aseptically collect 5-10 ml of urine.
3. Transfer to a sterile tube or container or urine transport system.

Sterile, leakproof container or urine transport kit

Unpreserved:
2 h or less, RTa
Preserved:
24 h or less, RTa

24 h or less, 4oC

1/day

 

Wound

See Abscess

 

 

 

 

 

 

a EtoH, ethanol; RT, room temperature; I.V., intravenous; AFB, acid-fast bacilli; CSF, cerebrospinal fluid; BAP, blood agar plate; CHOC, chocolate agar; BHI, brain heart infusion; PVA, polyvinyl alcohol fixative.
b All specimens should be transported in leakproof plastic bags having a separate compartment for the requisition.

Reference: Miller, J Michael, A Guide to Specimen Management in Clinical Microbiology, 2nd edition, ASM Press, Washington, D.C., 1999.

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