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Department of Pathology and Laboratory Medicine

Department of Pathology and Laboratory Medicine

Microbiology

Christopher R. Polage, M.D. (Director)

Lynda Braun, CLS (Supervisor)

Lynne Lenhart, CLS (Supervisor)

Janet Kashiwada, CLS (Technical Specialist and Training)

 

General Information

The Microbiology Laboratory is located at the Specialty Testing Center.  Services are provided from 6 am to 12:30 a.m., seven days a week.  Gram stains are prepared 24 hours per day with service limited to STATs during the period from 12 a.m. to 6 a.m..  The laboratory has the following sections:

  • Bacteriology, which includes isolation of anaerobic bacteria and antimicrobial susceptibility testing
  • Mycology
  • Virology, which includes testing by cell culture, antigen assays and serology.  Organisms identified in addition to viruses are and Chlamydia trachomatis
  • Parasitology

The quality of the laboratory results depends on the quality of the specimen.  Special requests for culture of unusual isolates (e.g. C. diphtheriae, Leptospira, Brucella, Haemophilus ducreyi, etc.) require prior notification of the laboratory in addition to noting the request on the requisition order. 

I. Specimen Collection Policies

A. All clinical specimens for microbiological examination must be collected in clean, sterile and properly sealed containers. The outer portion of the container must not be contaminated.

B. All specimens must be enclosed in a zip-lock plastic bag for transport to the lab.

C. Optimal specimens are aseptically obtained fresh pus, fluid or tissue that is rapidly and safely transported to the lab. Direct aspiration of pus or body fluids into a syringe is recommended. The needle must be removed and the syringe capped during transport to provide anaerobic conditions. Submit tissues in a sterile cup. Do not use swabs if fluid or tissue can be obtained. Collect enough material for the desired studies.

D. Swabs without transport medium (e.g., "culturettes" with the transport medium ampule not crushed) are not satisfactory since they allow drying of the specimen and loss of viability.

E. Specimens and laboratory requisition forms must be labeled with the patient's family name and individual name (up to 15 characters each) and a distinct patient identification number (such as a medical record number or account number); the requisition form must have the following additional information:

1. Date of birth and gender.
2. Hospital unit or clinic originating the request.
3. Requesting physician's name and physician index (PI) number.
4. Test or procedure requested.
5. Source of specimen.
6. Disease suspected.
7. Date and time of collection.
8. Surname of person collecting specimen.
9. Outpatients : ICD9 code(s).

(Refer to UCDHS Policies and Procedures Manual, Section 3090, Specimen Identification/Documentation Errors [Unlabeled/Mislabeled].)

F. A specimen may be rejected for any of the following reasons:

1. Specimen mislabeled or unlabeled.
2. Specimen inappropriate for test requested.
3. Specimen container leaking.
4. Specimen improperly handled (e.g., "culturette" ampule not crushed on genital specimen).
5. Duplicate specimens.
6. Quantity insufficient for analysis.
7. Specimen container grossly contaminated on outside.
8. Specimen, other than feces, submitted in nonsterile container.
9. Specimen too old for requested test.

If the container or specimen is inappropriate in any way, a new specimen will be requested by telephone.

II. Collection of Specimens for Microbiological Examination

(These instructions can be found in the UC Davis Health System Policies and Procedures Manual, Section 2025, Collection of Clinical Specimens for Microbiological Examination, accessible at http://intranet.ucdmc.ucdavis.edu/policies/hosp/2025.HTM.)

A. Using Transport Swabs

1. Remove culturette from package.
2. Remove cap/swab from tube.
3. Take sample, and return cap/swab to tube.
4. Label culturette.
5. Hold cap-end down, crush ampule at midpoint by squeezing the tube through the plastic shield.
6. Push cap to bring swab into contact with moistened pledget.

B. Blood Cultures

1. Preparation of patient and collection

a. When the optimal venipuncture site has been selected, meticulous attention to skin disinfection is essential to reduce the incidence of contamination.

b. Remove the flip-off caps from the vials, swab the rubber diaphragm of the two culture bottles (one aerobic/one anaerobic) with 70% alcohol, and allow the alcohol to act for approximately one minute.

c. After palpation, scrub the venipuncture site with 70% alcohol for a minimum of 30 seconds.

d. Apply iodine solution (1-2% tincture of iodine or 10% povidone-iodine) in concentric circles away from the puncture site covering a circular area 1-2" in diameter. Use of individually packaged pledgets or swabsticks of iodine or iodophor for cleansing are preferable and convenient. Allow the iodine solution to dry/act for 1-3 minutes.

e. For patients with iodine hypersensitivity, cleanse with 70% alcohol only. Scrub for 60 seconds and let the area dry prior to the venipuncture.

f. Apply a tourniquet, and locate the vein to be punctured being careful not to touch the skin at the proposed site of needle entry. If further palpation of the vein is necessary before or during venipuncture, disinfect the finger of the glove or use a sterile glove.

g. Perform the venipuncture, and withdraw approximately 20ml of blood into a sterile syringe (about 10ml per bottle). For children and infants, withdraw 1-6 ml. Do not change the needle prior to inoculating the blood culture bottles.

h. Inoculate blood culture vials, one aerobic vial (grey label) and one anaerobic vial (purple label). Aseptically inject half of the specimen into each vial with the anaerobic vial being used first to preclude entry of air from the syringe. Inject a maximum of 10 ml into each vial. 10ml per bottle is the optimum amount to culture in adults; bottles with less than 10 ml will be cultured nevertheless. Smaller volumes of blood can be cultured in pediatric patients as the concentration of organisms is higher. Gently mix bottles by inversion to prevent clotting.

i. Remove iodine from the skin with 70% alcohol. Avoid use of adhesive or tight dressings on skin cleansed with tincture of iodine, as iodine burns may result.

j. Label each vial, taking care not to cover the peel-off barcode strips, and submit to the lab using one requisition per set.

2. Number and time of collection (general guide)

a. In cases of suspected sepsis or acute septic shock, collect 2-3 separate venipunctures (two bottles each) immediately before starting treatment. There are no published data to indicate that the practice of separating venipunctures by any arbitrary period of time improves detection of septic episodes.

b. In suspected infective endocarditis or chronically ill patients, obtain three blood culture sets during the first 1-2 hours of evaluation; if all are negative 24 hours later, obtain three more sets. From patients who have received antimicrobial agents within two weeks prior to admission, obtain two separate blood cultures on each of three successive days.

c. For suspected bacteremia in patients already on antimicrobial therapy, if therapy cannot be suspended for a few days, draw 4-6 cultures within the first 48 hours. Cultures should be taken immediately before the next dose of antimicrobial agent if the patient is receiving intermittent parenteral therapy.

d. For fever of unknown origin (e.g., occult abscess, typhoid fever, or brucellosis), obtain two or three blood cultures initially. Then 24-36 hours later, obtain two more cultures immediately before the expected (usually afternoon) temperature elevation.

e. One to two blood cultures usually suffice for diagnosing sepsis of the newborn. The physician should determine the volume of blood. Inject 0.5-3ml into each bottle.

f. Routine blood cultures are incubated for five days, unless the lab is notified to hold for a longer period (e.g., when brucellosis or systemic fungal infections such as histoplasmosis are suspected). The routine 5-day incubation period is adequate for recovery of most yeasts (e.g., Candida species).

g. For blood culture for Mycobacterium avium complex, submit 4 ml heparinized blood (green top Vacutainer) for AFB culture and smear.

h. Blood for culture should not be drawn through an indwelling intravenous or intra-arterial catheter unless it cannot be obtained by venipuncture or unless it is being drawn to specifically evaluate a potential catheter-related infection, in which case peripheral blood should be simultaneously drawn by venipuncture from another site.

i. Lysis-centrifugation blood culture tubes are available from the Microbiology laboratory for additional studies in culture-negative endocarditis or other difficult cases.

3. Requisition

Order blood culture. Use a fungal culture (mycology) or AFB culture/smear requisition when appropriate. Record the site of the blood culture draw on the requisition as well as the blood culture bottles. Place padding around the bottles if using the pneumatic tube system.

C. Urine Cultures

Collect urine specimens by the clean-voided midstream technique, by diagnostic catheterization, by suprapubic aspiration or from an indwelling catheter. The first morning specimen is preferred. Urinary catheter tips are not cultured because the tip is contaminated as it is removed from the urethra. A pooled, 24-hour collection is unacceptable for culture, as is more than one specimen within 24 hours.



1. Preparation and collection

  1. Indwelling catheter
    1) If necessary, clamp the catheter tubing to collect urine in the tube but do not allow the clamp to remain for more than 30 minutes. Urine obtained from the catheter bag is unacceptable for culture.
    2) Clean the sampling port (or tubing site if a port is unavailable) with an alcohol sponge.
    3) If a needleless urine sampling port is not available, use a 21-gauge needle and syringe to enter the sampling port or tubing. Withdraw approximately 10ml of urine.
  2. Clean-voided urine

  1. Clear verbal and/or written instructions must be given to the patient to ensure understanding of the procedure.
  2. Gather the following supplies for each patient:



i) Gauze sponges.
ii) Antibacterial soap (ordinary soap is acceptable) or commercially prepared wipes or soap towelette packages.
iii) Rinse water.
iv) Wide-mouthed sterile covered container, e.g., screw-cap specimen container.

3) Instruct the female patient to:

i) Remove undergarments.
ii) Wash hands thoroughly with soap and water, rinse and dry.
iii) Sit comfortably on the toilet and swing one knee to the side as far as possible. With one hand, spread the labia and keep continuously apart during cleaning and collection of the specimen.
iv) Take a single sponge dripping with soap or a prepared wipe; wash the vulva well, passing only from front to back. Discard the used sponge or wipe.
v) Repeat the front to back wash, discarding the sponge or prepared wipe after use.
vi) Rinse twice with water-soaked sponges from front to back, discarding the sponge after each rinse.
vii) Grasp the collection cup on the outer surface only. Fingers should be kept away from the rim and inner surface of the container. Pass a small amount of urine, 20-25ml, into the toilet, then move the cup into place without stopping the stream. Collect at least 15-20ml or fill the cup half full.
viii) Void remaining urine into the toilet. Securely affix the cap onto the cup.

  1. Instruct the male patient to:
    i) Wash hands thoroughly with soap and water, rinse and dry.
    ii) Completely retract the foreskin (if uncircumcised), and cleanse the glans penis using liquid soap-soaked sponges or two packaged wipes. Rinse well with two water-soaked sponges. Discard each item after use.
    iii) Grasp the collection cup on the outer surface only. Fingers should be kept away from the rim and inner surface of the container. Allow the first portion of urine to be passed into the toilet, then, without stopping the flow of urine, collect a portion (at least 15-20ml) into the specimen container or fill the cup half full.
    iv) Void remaining urine into the toilet. Securely affix the cap onto the cup.
  2. For infants or other patients unable to cooperate: Attach a plastic bag device after careful preparation, as outlined above. Check the bag frequently so that the urine specimen can be collected immediately after it is voided. If the patient has not voided within 30 minutes, remove the bag, rescrub the patient and attach a new collection bag. Do not submit urine collected from diapers.
  3. Immediately after collection, aspirate the urine into the urine culture transport tube up to the fill line (4 ml). If the available specimen volume is less than 4 ml, it should be submitted in a sterile container on ice. Under no circumstances should a urine sample be taken from urinal or bedpan, nor should the specimen be brought from home.


2. Requisition

Order urine culture if indicated by urinalysis (submit a separate sample for urinalysis). Order Gram stain if desired.

3. Transport

Deliver specimens in urine culture transport tubes to the lab at room temperature. Specimens with volume less than 4 must be immediately placed and delivered on ice. Urine samples are stable for approximately 24 hours using the urine culture transport tube.

D. Throat Cultures

Routine throat cultures are used to ascertain whether Group A streptococci are present in pharyngitis. Arcanobacterium haemolyticum, associated with pharyngitis in older children, will also be detected in a routine throat culture. Neisseria gonorrhoeae can cause pharyngitis but is not included in a routine culture and must be specifically requested.

1. Preparation and collection

a. Gather the transport medium for the culture (e.g., use "culturette" swab).

b. Using a tongue depressor to hold the tongue down, carefully examine the back of the throat and the tonsillar area for localized areas of inflammation and exudate. Carefully but firmly rub the swab over several areas of exudate or over the tonsils and posterior pharynx. Care should be taken to avoid the tongue and uvula, as well as the cheeks or teeth when withdrawing the swab.

c. Place the swab back into the transport device, crush the ampule of transport medium and press the swab firmly down onto the moistened pledget. Label the specimen.

2. Requisition

Order beta strep culture only, and when suspected, GC culture (gonococcus). Order miscellaneous culture and notify the lab if Corynebacterium diphtheriae is suspected, as a special culture medium is prepared for this request. Posterior nares culture is also recommended when diphtheria is suspected. A direct Gram stain is not useful in the diagnosis of routine bacterial pharyngitis, but may be helpful in the diagnosis of diphtheria, oral candidiasis or ulcerative gingivitis.

3. Transport

Transport swabs at room temperature, and deliver to the lab promptly.

E. Nasopharyngeal Cultures

Swabs from the nasopharynx or from secretions aspirated through the nose from the nasopharynx may be helpful in making the diagnosis of pertussis or diphtheria and in identifying meningococcus carriers. The disease or organism suspected must be noted, as special culture media are required in each case.

1. Preparation and collection

a. If pertussis is suspected, obtain a pertussis culture kit (contains Regan Lowe plate, appropriate swab and instructions) from the laboratory on North 2 Room 2225. Check the expiration date.

b. Remove excess secretions or exudate from the anterior nares.

c. Collect nasopharyngeal specimens by the pernasal route using a special calcium alginate/dacron swab firmly supported on the end of a flexible wire.

d. Insert the swab gently through the nose and into the nasopharynx. Rotate the swab on the nasopharyngeal membrane and allow it to remain in place for 10-15 seconds to absorb organisms.

e. Remove the swab carefully, place it in the transport device and crush the ampule of transport medium. (In suspected pertussis, use the swab to inoculate the culture plate as described in the instructions.)

f. Alternatively, bend the wire at an angle and insert it into the throat. Then move the swab upward into the nasopharyngeal space.

g. For a nasopharyngeal aspirate, attach a sterile suction catheter to a Lukens trap and introduce the end of the catheter into the nasopharynx until resistance is encountered. Withdraw the catheter 1-2cm and apply suction to aspirate a sample.

2. Requisition

Order respiratory culture, and specify disease suspected, e.g., pertussis, diphtheria, meningococcus.

3. Transport

Label the specimen and send to the lab at room temperature without delay. Send inoculated pertussis culture plate (tape the lid onto the plate) back to the lab immediately.

F. Nasal (Nares) Cultures

Anterior nares cultures, without indicating the presence of a lesion, are routinely examined only for Staphylococcus aureus and beta-hemolytic streptococci. Nasal cultures do not predict the etiologic agent of sinus, middle ear or lower respiratory tract infections and should not be submitted in lieu of specimens from these sites.

1. Preparation and collection:

a. Obtain a culturette swab. A nasal speculum may be needed for some patients.

b. Carefully insert the swab at least 1cm into the nares.

c. Firmly sample the membrane by rotating the swab and leaving it in place for 10-15 seconds.

d. Withdraw the swab and place it in the transport device. Crush the ampule and label the specimen.

2. Requisition:

a. Order miscellaneous culture, specify nares.

b. Order MRSA only culture if MRSA (Methicillin-resistant Staphylococcus aureus) surveillance screen is ordered.

3. Transport to the laboratory promptly at room temperature.

G. Expectorated Sputum

Lower respiratory tract specimens are screened for quality microscopically. A properly collected specimen containing a minimum of squamous epithelial cells and significant numbers of polymorphonuclear leukocytes usually provides clinically relevant results. Specimens of lesser quality provide misleading results. Careful attention to the instructions given the patient greatly reduces the number of unsatisfactory specimens. The first early morning specimen is preferred. Pooled samples are not recommended for culture. Blood culture is often an accurate specimen for diagnosis of bacterial pneumonia.

1. Preparation and collection

a. Have the patient rinse his/her mouth with water (preferably brush teeth). For patients with dentures, remove the dentures first.

b. Instruct the patient in the difference between sputum and spit. The patient should cough up material from deep in the lungs and expectorate a single bolus into a wide-mouthed sterile container. This should be done under direct supervision, preferably when the patient first awakens in the morning.

c. To facilitate acquiring an adequate specimen from patients with nonproductive coughs, use ultrasonic nebulization with 10% saline, hydration, chest physiotherapy or postural drainage.

2. Requisition

Order respiratory culture/Gram stain.

3. Transport

Carefully and tightly affix the specimen cap. Leaking samples will not be processed. Send the specimen to the lab without delay, as there is no effective transport medium.

H. Bronchoscopy, Endotracheal Aspirates and Transtracheal Aspirates

1. Preparation and collection

Collect specimens according to protocol. Specimens include brushings (Bartlett protected brush), transbronchial biopsies or bronchial secretions that are aspirated through the inner channel of the bronchoscope with or without an irrigating solution.

2. Requisition

Order respiratory culture/Gram stain. Order anaerobic culture/Gram stain for Bartlett protected brushes. Note: Legionella culture is routinely done on Bartlett brush, bronchoalveolar lavage (BAL) and lung tissue.

3. Transport

Transport specimen without delay in a sterile container. Add precisely 1ml sterile saline to the tube containing the Bartlett brush in order to result in accurate quantitation.

I. Body Fluids (abdominal, amniotic, ascites, bile, joint, paracentesis, pericardial, peritoneal, pleural, synovial, thoracentesis)

1. Preparation of patient and collection

a. Disinfect overlying skin with 2% tincture of iodine.

b. Obtain specimen via percutaneous needle aspiration or surgery.

c. Aspirate fluids that collect in pericardial, pleural, peritoneal and synovial spaces with the utmost precaution to avoid introducing microorganisms and to avoid contamination of the specimen.

d. If directly aspirating into a syringe (recommended), remove the needle and cap the syringe for transport. If the material cannot be aspirated into a syringe, place it into a sterile tube or container. Label the specimen.

e. Inoculate ascitic fluid specimens (in spontaneous bacterial peritonitis) at the bedside into blood culture bottles. Put 10ml of fluid into aerobic and anaerobic blood culture bottles (20ml total). Submit additional fluid in a sterile container, as much as possible for Gram stain and additional lab processing (e.g., acid-fast bacilli [AFB], fungal cultures). Do not inject body fluids other than ascitic fluid into blood culture bottles.

2. Requisition

Order anaerobic culture/Gram stain (anaerobic culture includes aerobic culture). An anaerobic request should have a STAT sticker placed to flag it for prompt delivery and setup. Anaerobic cultures will not be done on swab specimens, nor are swabs needed as additional specimen for aerobic culture.

3. Transport

Transport properly labeled specimens immediately in a capped syringe with the needle removed, or in a sterile container.

J. Skin and Contiguous Tissue Specimens (Wound, Abscess, Burn, Exudate)

A closed abscess is the specimen site of choice. Collect exudate and a sample of the abscess wall. For most open lesions, remove the superficial flora before collecting a specimen from the advancing margin or base. For dry, encrusted lesions, culture is not recommended unless an exudate is present. Culture burn wounds only after extensive cleaning and debridement. Biopsy specimens are recommended.

1. Preparation and collection

a. Closed abscess: Decontaminate the skin overlying the abscess with 70% alcohol and aspirate the abscess contents with a syringe. After excision and draining, a portion of the abscess wall may be submitted for culture. Remove the needle and cap the syringe. Label the specimen.

b. Open lesions and abscesses: Remove as much of the superficial flora as possible by decontaminating the skin using 70% alcohol. Remove exudate and firmly sample the base or margin of the lesion with a culturette swab. Return the swab to the culturette base and crush the ampule. Label the specimen.

c. Burn wounds: Debride the area and disinfect the wound. As exudate appears, sample it firmly with a culturette swab. Biopsy tissue is the specimen of choice; surface specimens usually represent colonization. Return the swab to the culturette base and crush the ampule. Label the specimen.

d. Pustules or vesicles: Select an intact pustule. Apply alcohol and allow it to dry. Unroof the pustule with a needle. Collect fluid and basal cells by rotating the swab vigorously in the pustule. If the lesion is older, the crust should be removed and the moist base of the lesion sampled by swabbing with a premoistened (sterile saline) culturette swab. For bacterial or fungal culture, return the swab to the culturette base and crush the ampule. For viral culture, place the swab in M4 viral transport medium (refer to detailed viral culture collection instructions in section S below).

2. Requisition

Order anaerobic culture/Gram stain (includes aerobic), miscellaneous culture/Gram stain or wound culture/Gram stain. Specify the body site of wounds, drainages, exudates, etc.

3. Transport

Transport swabs at room temperature, and deliver to the lab without delay. Deliver tissue and syringe specimens to the lab immediately for optimum culture of anaerobic organisms.

K. Cervical, Urethral and Vaginal Cultures

DNA probe or other molecular testing is in most cases preferable to culture for detecting GC and chlamydia in genital sources because of improved sensitivity, faster turnaround time, and greater stability of samples during transit. However, culture of these agents is recommended when testing pediatric patients, with forensic specimens or with specimens that are very bloody. When testing for GC and chlamydia in non-genital sources (e.g., rectal, throat), collect culture specimens.

1. Preparation and collection for culture

a. Cervix (endocervix) : Wipe the cervix clean of vaginal secretion and mucus. Use a speculum without lubricant - it may be toxic to certain bacteria. Under direct vision, gently compress the cervix with the blades of speculum, and use a rotary motion with a culturette swab. Obtain exudate from endocervical glands. Alternatively, insert the swab into the cervical os, allow it to remain in place for a few seconds, and remove it. Return the culturette swab to the base.

b. Urethra: Collect specimen one hour or more after urinating. Wipe urethral meatus clean with sterile gauze or swab. If discharge cannot be obtained by "milking" the urethra, use a swab to collect material from about 2cm inside the urethra. Return swab to culturette device.

c. Vagina (this is a less productive source than the cervix): Use a speculum without lubricant. Swab mucosa high in the vaginal canal under direct visualization. For detection of Group B streptococci in pregnant women, public health guidelines suggest obtaining one or two swabs of the vaginal introitus and the anorectum for optimal recovery. Cervical swabs are not acceptable.

2. Requisition

Order genital culture, GC culture or Beta strep culture only (for recto-vaginal source). Order Gram stain if desired. Gram stain is helpful in diagnosing GC in males but for females it is not recommended. Other organisms in the vaginal or cervical flora may have morphology similar to gonococci.

3. Transport

Return the swab to the culturette device, crush ampule of transport medium, label and send to the lab immediately at room temperature (do not refrigerate). NOTE: Failure to crush the ampule of the culturette will result in rejection of the specimen. Specimen must be received within 12 hours of collection to allow recovery of Neisseria gonorrhoeae. Viability of this organism rapidly diminishes over time. DNA probe specimens are stable even after prolonged transit times (see below).

L. Cervical and Urethral Specimens for DNA Probe (Chlamydia trachomatis and Neisseria gonorrhoeae)

NOTE: Standard culture techniques are recommended for pediatric patients and forensic purposes. Culture is also required for excessively bloody cervical specimens as a large amount of blood interferes with the DNA probe testing method.
Molecular methodology for detection of these agents is a dynamic field; therefore careful attention to the current product's collection instructions is essential.

1. Preparation and collection


  1. For female cervical specimens:

    1) Remove excess mucus from cervical os and surrounding mucosa using one of the swabs provided in the collection kit. Discard this swab.
    2) Insert second swab from collection kit 1-1?cm into endocervical canal.
    3) Rotate swab for 30 seconds in endocervical canal to ensure adequate sampling.
    4) Withdraw swab carefully, avoiding any contact with vaginal mucosa.
  2. For male urethra specimens:


1) Ensure that the patient has not urinated for at least one hour prior to sample collection.
2) Insert swab from collection kit 2-4cm into urethra.
3) Once inserted, rotate swab gently, using sufficient pressure to ensure swab comes into contact with all urethral surfaces. Allow swab to remain inserted for 2-3 seconds.
4) Withdraw swab.

c. Insert swab into DNA Probe transport tube.

d. Snap off shaft at score line, or cut shaft to fit tube.

e. Tightly cap tube. NOTE: Specimens collected for DNA probe cannot be used for culture. Only the swabs supplied with the collection kit should be used.

2. Requisition

Order DNA probe-Chlamydia and GC.

3. Transport

Label the tube and transport to the lab at room temperature. The specimen is stable for several days. Specimens containing no swab, two swabs, swabs not provided with the collection kit, or those that are excessively bloody will be rejected.

M. Stool, Gastrointestinal (GI) Specimens and Rectal Swabs for Bacteriology

1. Specimen collection method and volume

a. Collect early in the course of illness and prior to the administration of antimicrobics.

b. Collect feces in a clean, wide-mouth container. Commode collection systems in which a plastic collection device ("hat") fits over the rim of a toilet seat are available. Feces may also be collected from a sterile bedpan. However, if there is any contamination with urine, residual soap, detergent, or disinfectant in the pan, the sample is unsatisfactory. Do not take a specimen from the water in a toilet. For young children, lining a diaper with plastic wrap facilitates retrieval of feces for testing.

c. Submit approximately 50gm (walnut size) of stool, 20ml if fluid. Select those portions of stool that contain pus, blood or mucus for examination. Submit the sample in a specimen cup with a tight, leak-proof lid.

d. If fresh stool cannot be delivered to the lab within 1-2 hours of passage, Cary Blair enteric transport medium is recommended. Add stool (approximately 10gm) to the red fill line on the transport vial label. The medium should be red prior to addition of the sample; if the indicator is yellow, the product should not be used. Check the expiration date. Mix the stool thoroughly into the medium.

e. If feces are not readily obtainable, submit a rectal swab. Generally, swabs are recommended only for infants. Insert the culturette swab beyond the anal sphincter, carefully rotate then withdraw swab. The swab must show feces. Return the swab to the culturette base and crush the ampule of transport medium.

f. Submit sigmoidoscopic, duodenal, colostomy, ileostomy or colorectal aspirates in sterile containers with tight, leak-proof lids and deliver to the lab immediately.

g. Submit rectal biopsy material in a sterile container with tight, leak-proof lid. Add a small amount of sterile water to prevent desiccation. Specimens allowed to dry out are unacceptable. Deliver immediately.

h. Label all specimens.

i. For Clostridium difficile toxin assay collection instructions, refer to section S.3 below.

2. Recommended number

a. One stool specimen per day. Duplicate specimens within a 24-hour period will be rejected.

b. No more than 2 specimens/GI episode.

c. Feces collected from in-house patients more than 3 days after admission will not be cultured. This utilization restriction has been widely implemented in hospitals because of the low yield of bacterial pathogens from patients whose diarrhea began after admission. EXCEPTION: Request for screening for overgrowth of Staphylococcus aureus, Candida spp. or Pseudomonas aeruginosa.

d. Special circumstances should involve consultation with the Microbiology Lab director or supervisory staff.

3. Requisition

a. Routine culture (R/O Salmonella spp., Shigella spp. and Campylobacter jejuni), order stool culture.

b. If other organisms are suspected (e.g., Escherichia coli 0157:H7, Yersinia enterocolitica, Vibrio spp., Aeromonas spp., S. aureus), note specific organism(s) on requisition. Additional culture techniques must be employed to recover these other agents. If blood has been observed in the stool, please note this on the lab form.

c. If WBC examination is desired, order Gram stain. NOTE: WBCs rapidly disintegrate in stool. Deliver immediately.

4. Transport

a. Transport fresh, unpreserved specimens at room temperature to the lab within 1-2 hours of collection.

b. Submit rectal swabs at room temperature. Swabs over 24 hours old or swabs in which the ampule was not crushed at time of collection are unacceptable.

c. Submit Cary Blair transport specimens at room temperature. Samples in this medium are stable for up to 4 days.

N. Stool, GI and Miscellaneous Specimens for Parasitology

1. Fecal specimen collection

a. Collect fecal specimens prior to the administration of antibiotics or antidiarrheal agents. Avoid the use of mineral oil, bismuth and barium prior to fecal collection, since these substances interfere with the detection or identification of intestinal parasites. Wait at least one week if any of these have been used.

b. Collect the fecal specimen in a clean, wide-mouthed container or on a newspaper, and transfer it to a container with a tight-fitting lid.

c. Avoid contamination with urine or water from the toilet.

d. Transport the specimen to the lab promptly.

e. Fecal specimens that cannot be delivered within 4 hours of collection should be preserved in the ParaPak O&P specimen collection kit. Add feces to the fill line and thoroughly mix the sample into the preservatives. Preserved specimens are stable indefinitely. Unpreserved specimens received more than 12 hours after collection will not be processed.

f. Since shedding of some parasites is intermittent, a minimum of 24 hours should elapse between collection of fecal specimens. Only one specimen per day will be processed. Feces collected from inpatients greater than 3 days after admission will not be examined (except when a series of specimens is in progress). This utilization restriction has been implemented in hospitals due to the low yield of parasites from patients whose diarrhea began after admission.

g. Rectal swabs are not satisfactory for examination.

2. Recommendations for specific parasites and miscellaneous specimen types

a. Acanthamoeba--corneal scrapings, biopsy material; obtain non-nutrient agar plate from microbiology lab prior to specimen collection. Telephone the lab at 4-2544 for instructions.

b. Amoebiasis--loose, watery, mucous or bloody stool for Entamoeba histolytica requires immediate examination. No specimen older than 1 hour will be examined for motile amoebae. A minimum of 6 specimens collected over a 10-14 day period is recommended to rule out amebiasis.

c. Cryptosporidium/Isospora/Microsporidium/Cyclospora--special stains required; must be requested.

d. Duodenal aspirate--appropriate for examination for Giardia, Clonorchis, Strongyloides, Cryptosporidium, and Isospora. Keep specimen warm and submit immediately to lab.

e. Giardia--4-5 specimens may be required to rule out Giardia. Antigen testing for Giardia is available.

f. Helminths--a minimum of 3 specimens collected over a 7-10 day period is recommended.

g. Pinworm--collect the specimen on arising in the morning, before bathing or going to the bathroom. Apply clear cellophane tape, sticky side toward the body, to the uncleansed perianal area. Remove the tape and then apply it, sticky side down, to a clean microscope slide. Label the slide. A minimum of 4-6 consecutive collections are necessary to rule out infection. "Pinworm paddles" are usually available in pediatric outpatient clinics.

h. Sigmoidoscopic material. Gently insert the lubricated sigmoidoscope into the rectum. Pass the instrument to the point where the sigmoid colon can be seen. With a pipette and bulb, aspirate material from visible lesions and from the mucosal surface. A curette can be used to gently scrape suspicious areas of the mucosal wall. Place the sample in a sterile culture tube. Add a drop of saline to prevent drying. Keep warm and submit immediately to the lab. Do not submit the material on a swab.

i. Trichomonas--submit vaginal fluid or exudate in 0.5ml of saline. Keep warm and submit immediately to the lab; specimen must be received within 1 hour of collection. If unable to transport the specimen to the lab immediately, make a thin smear of the fluid or exudate on a microscope slide, air dry, and submit this to the laboratory for staining.

j. Urine--appropriate for examination for Schistosoma ova. Submit all urine passed between noon and 3 p.m. A 24-hour collection without preservative is also acceptable. Urine may be submitted for examination for Trichomonas vaginalis in the male patient. Submit the first AM urine or urine passed after prostatic massage.

k. Worm identification--submit entire specimen (roundworms, tapeworms, proglottids).

3. Requisition

Order Ova and Parasites or Giardia antigen. Specify Cryptosporidium, Isospora or other specific parasites when required.

4. Transport

In general, all unpreserved material (e.g., special GI collections, vaginal secretions) for parasitology exam must reach the microbiology lab and be examined quickly for valid results. Use of Parapak O&P vials for fecal samples extends the allowable transit time and these preserved specimens are stable at room temperature indefinitely.

O. Acid-Fast Bacilli (AFB) Specimens

1. Preparation of patient and collection

a. Sputum--collect only material brought up from the lungs after a productive cough. Do not collect sputum immediately after a mouth wash. A series of three daily early morning specimens, each submitted promptly to the lab after collection, is recommended. 5-10ml volume is adequate. For patients who have difficulty in raising sputum, inhalation of hypertonic saline or nebulized solutions often proves productive. Submit the specimen in a sterile, labeled container. Assure the lid is tightly sealed.

b. Urine--first morning midstream specimens are recommended. Instruct patient to wash and rinse the genital area well as described above for urine culture prior to collection of specimen. Submit at least 50ml in sterile labeled cup (urine transport tube volume is inadequate for AFB culture).

c. Other specimens--submit spinal fluids in sterile lumbar collection tubes, other fluids in sterile containers. Pieces of tissue must be kept moist with a small amount of sterile saline. Submit as much CSF, other body fluid or tissue as possible.

d. Blood--submit 4 ml heparinized blood (green top Vacutainer).

2. Requisition

Order CULTURE—AFB. If Mycobacterium marinum is suspected, i.e., cutaneous infections on extremities, note this on requisition.

3. Transport

Specimens should be delivered promptly to the lab at room temperature (urine should be delivered on ice). Specimens are processed at the Sacramento County Public Health Laboratory. They are picked up from the Microbiology Lab at 0800, Monday through Friday, excluding County holidays. To assure specimens are processed on the day of collection, samples from hospitalized patients should be received at the North 2 laboratory no later than 0600.

P. Mycology (Fungal Culture) Specimens

1. Preparation of patient and collection

a. Skin--clean site with 70% alcohol to help eliminate surface contaminants. Using a scalpel, skin scrapings should be made from the active periphery of the lesion. Submit scrapings in a sterile Petri dish or other sterile container.

b. Nails--clean site with 70% alcohol on gauze, not cotton. Collect nail shavings and material from under the nail plate. Scraping should be deep enough to assure acquiring recently invaded tissue. Submit nail clippings and scrapings in a sterile Petri dish or other sterile container.

c. Hair--use forceps to pluck 10-12 involved hairs from the edges of the patches. Select hairs that fluoresce under a Wood's lamp. Submit hair, including shaft, in a sterile Petri dish or other sterile container.

d. Other specimens--collect and submit specimens as described for specific type. Specimens associated with the systemic and deep-seated mycoses are obtained from a wide variety of sources. They should be obtained, whenever possible, under aseptic conditions and in sufficient quantity for both microscopic and cultural examinations. Indicate specific organism suspected, if possible.

2. Requisition

Order fungal culture (mycology); order KOH mount if direct examination is desired.

3. Transport

Transport properly labeled specimens promptly to the laboratory at room temperature.

Q. Cryptococcal Antigen Testing

1. Cryptococcal antigen testing may be done on CSF and serum.

2. Collect CSF sterile container. Submit blood in a 3ml red top tube. Transport specimens to the laboratory promptly at room temperature.

3. Requisition

Order Cryptococcal antigen.

R. Chlamydia trachomatis Testing

1. DNA probe (refer to collection instructions, paragraph L above). Testing can be performed on genital and conjunctival sources.

2. Specimens for chlamydia culture must be submitted in M4 multimicrobe transport medium. M4 is available on the nursing units, from the North 2 Lab Room 2225 or from the Microbiology Lab in the Clinical Lab Building. It should be refrigerated at 2-6oC. Check expiration date.

  1. Specimen collection NOTE: Use wire- or plastic-shafted dacron swabs for specimen collection. Do not use wooden-shafted swabs.


1) Ocular--scrape epithelial cells from lower conjunctiva with a curette or swab. Care must be taken to collect a sufficient number of involved epithelial cells. Place swab in a tube of M4.
2) Female genital tract--scrape epithelial cells from the transitional zone of the cervix using a dacron swab. Rotate the swab in the endocervical canal for 5-6 seconds; place swab in a tube of M4.
3) Throat, posterior nasopharynx--using a swab, scrape epithelial cells from the posterior nasopharynx and throat. Place swab in a tube of M4.
4) Rectal swabs--scrape rectal mucosa with swab. Place swab in a tube of M4.
5) Bubo pus--aspirate approximately 0.5ml material. Place aspirated material in a tube of M4.
6) Male urethra--insert a dacron swab 2-4cm into urethra, rotate swab, withdraw and place in M4.

b. Requisition: Order Chlamydia culture. Include source of specimen.

c. Transport: Immediately transport specimens in M4 transport medium on wet ice to the lab.

S. Virology Laboratory Procedure Information

The Virology Laboratory is staffed from 0800 to 1630 daily. Isolation techniques are available for the following agents:

Adenovirus

Chlamydia trachomatis

Cytomegalovirus (CMV)

Enterovirus (Poliovirus, ECHO, Coxsackie)

Influenza virus

Herpes simplex virus (HSV)

Measles virus (Rubeola)

Mumps virus

Parainfluenza virus

Respiratory syncytial virus (RSV)

Varicella zoster virus (VZ)

Direct examination of clinical material by immunofluorescence is available for the following agents:

Adenovirus

Herpes simplex

Influenza A and B

Parainfluenza 1,2,3

Pneumocystis carinii

Respiratory syncytial virus

Varicella zoster

Antigen detection in clinical material is available for the following agents:

Respiratory syncytial virus

Rotavirus

1. Virus isolation and immunofluorescence

Specimens for virus isolation should be collected as soon as possible after the onset of symptoms. Media for the transportation of viral specimens should be available on the wards. If M4 multimicrobe transport medium is not available, it may be obtained in the North 2 Lab Room 2225 or in the Microbiology Lab in the Clinical Lab building. Store M4 medium refrigerated at 2-6oC until use. Check the expiration date. Virus isolation specimens should be sent to microbiology as soon as possible after collection. A brief clinical history is required. It is recommended that specimens be collected whenever possible during the early dayshift hours to permit immediate processing.

a. Specimen collection NOTE: Use wire- or plastic-shafted swabs for collecting specimens for viral culture. Do not use wooden-shafted swabs.

  1. Autopsy and biopsy specimens: Collect tissues from probable sites of pathology using a separate sterile instrument for each sample. Samples should be about ?-1cm cubes. Place each specimen in a separate container; add a small amount of sterile saline.

  1. In cases of central nervous system disease, submit samples of brain (temporal lobe cortex, mid-brain, medulla) and spinal cord. A 2-3" segment of descending colon, tied off with contents, should be included for the recovery of enteroviruses.
  2. For influenza and other respiratory diseases, include samples of lung tissue, bronchus and trachea. Heart muscle, liver and kidney are less common sources of virus but may be included if involvement is suspected.

  1. Conjunctival scrapings: Vigorously scrape the conjunctiva with an ophthalmologic spatula or a swab. For virus culture, place the scrapings or break off the swab into a vial of M4. For direct immunofluorescence, prepare a smear of the scrapings in one dime-sized area on a clean microscope slide; air dry. Prepare a separate slide for each agent to be tested; prepare 2 dime-sized smears on one slide for HSV.
  2. Throat/nasopharyngeal swabs and throat washing: Swab the throat and/or nasopharynx using the appropriate swab type (nasopharyngeal swabs are best collected using dacron-tipped wire swabs). Break the swab off into a vial of M4. For throat washing, have the patient gargle 10-15ml of Hank's solution (available from the North 2 Lab Room 2225 or the Microbiology Lab in the Clinical Laboratory Building.
  3. Urine: For cytomegalovirus (CMV) collect 10-20ml (volumes as low as 2ml are acceptable) and deliver immediately to the Microbiology Lab on wet ice. Do not freeze. Do not submit in a urine culture transport tube.
  4. Vesicular lesion material
    a) Obtain cellular scrapings from the base of vesicles with a scalpel, swab or applicator stick. Rupture a young vesicle, absorb vesicular fluid with a swab and scrape the base of lesion. Do not draw blood. If pus is present, clean lesion and discard swab prior to taking the specimen.
    b) For virus culture, place the scrapings or break the swab off into a tube of M4.
    c) For direct immunofluorescence, prepare two smears of the lesions in two dime-sized areas on each of two clean microscope slides; air dry.
  5. Nasal washing (for respiratory syncytial virus [RSV]): Wash the posterior nasopharynx using 5ml sterile phosphate buffered saline (PBS) and a rubber bulb. Place 2ml of nasal washing in a tube of M4 if viral culture is requested, and the remainder of the washing in the tube that contained the PBS if direct detection by immunofluorescence is requested. Submit either or both containers (depending on test request) on ice; deliver immediately to the lab.
  6. Blood (buffy coat): Collect 3-7 ml blood in an EDTA (purple top) tube. Transport specimen at room temperature immediately to the lab.
  7. Stool: Collect 5gm fresh stool in a clean container.
  8. Rectal swab: Scrape rectal mucosa with a swab and break off swab into vial of M4.

  1. Requisition
    1) Virus isolation--order virus isolation. Include source of specimen, date of onset and clinical history.
    2) Immunofluorescence--order immunofluorescence. Specify virus (e.g., RSV) and include source of specimen.
  2. Transport


1) Virus isolation--all specimens except blood should be transported on wet ice immediately to the lab. EDTA blood should be transported at room temperature.
2) Immunofluorescence--transport primary specimen as above or transport slides promptly to the lab in a clean cardboard slide holder or specimen cup.

2. Rotavirus detection

a. Collection: Collect fecal specimens during the acute phase of the illness, preferably within the first 3 to 5 days. Liquid stools can be collected from pediatric patients by several means: to prevent absorption of liquid stool into diapers, a clean diaper can be lined with plastic wrap or a disposable diaper can be put on inside out. A pediatric urine bag can be placed over the anal area to collect watery stool.

b. Requisition: Order Rotavirus.

c. Transport: Submit 5gm (5-10ml liquid) of fresh stool (not a rectal swab) in a clean container promptly to the lab at room temperature. If transit time is expected to exceed 2 hours from the time of collection, submit the sample on ice.

3. Clostridium difficile toxin

a. Requisition: Order C. difficile toxin assay.

b. Transport: Submit at least 5gm of fresh soft stool or 10-20 ml liquid stool (not a rectal swab) in a clean container promptly to the lab at room temperature. If transit time is expected to exceed 2 hours from the time of collection, submit the sample on ice.

c. Duplicate samples within a 24-hour period will not be processed.


III. ROUTINE SUSCEPTIBILITY TESTING:

A quantitative microdilution technique is used to yield MICs (minimum inhibitory concentration) and requires 18 - 24 hours of incubation once the isolate is obtained in pure culture. The procedure is initiated in the Microbiology Laboratory on appropriate organisms and sources. The panels offered are as follows:

Enterobacteriaceae:

amikacin ciprofloxacin

ampicillin gentamicin

cephalothin piperacillin

cefazolin piperacillin/tazobactam

cefepime tobramycin

cefotaxime trimethoprim/sulfamethoxazole

ceftazidime nitrofurantoin (urine)

ceftizoxime

Pseudomonas aeruginosa

amikacin gentamicin

cefepime piperacillin

ceftazidime piperacillin/tazobactam

ciprofloxacin tobramycin

Acinetobacter spp. and other miscellaneous Gram negative bacilli

amikacin gentamicin

cefepime piperacillin

cefotaxime piperacillin/tazobactam

ceftazidime tobramycin

ceftizoxime trimethoprim/sulfamethoxazole

ciprofloxacin

Staphylococcus spp.

cefazolin oxacillin

ciprofloxacin rifampin

clindamycin tetracycline

erythromycin trimethoprim/sulfamethoxazole

gentamicin vancomycin

penicillin (B-lactamase detection) available on request

 Enterococcus spp.

ampicillin gentamicin synergy screen

penicillin streptomycin synergy screen

vancomycin B-lactamase detection on request

Streptococcus pneumoniae


cefotaxime penicillin

ceftriaxone tetracycline

chloramphenicol trimethoprim/sulfamethoxazole

clindamycin vancomycin

erythromycin

Haemophilus influenzae and Moraxella catarrhalis

B-lactamase detection

Coagulase-negative staphylococci

As these organisms are often contaminants, clinical input is needed to guide the Microbiology Laboratory as to which isolates need to be tested. Isolates tested routinely include those from multiple positive blood cultures and CSF from shunts; other isolates are tested on request.

Anaerobic organisms

Anaerobic susceptibility testing must be approved by an Infectious Disease physician or the Microbiology Laboratory director. There is no panel - each antimicrobic is requested and tested individually.

Fungi (yeasts and molds)

Isolates for fungal susceptibility testing are sent to the University of Texas Health Science Center at San Antonio, a prominent fungal research center.

Other antimicrobics available for testing (or reported for resistant organisms) include:

amoxicillin/clavulanic acid, ampicillin/sulbactam, aztreonam, cefuroxime, imipenem, levofloxacin, linezolid, meropenem, moxifloxacin, quinupristin/dalfopristin (Synercid), ticarcillin/clavulanic acid.

There are some organisms for which routine susceptibility testing is either unnecessary or has not been standardized. Consult with the Infectious Diseases service physicians with questions regarding treatment of infections for which no susceptibility testing is reported. They can assist in emperic therapy selection or suggest specific agents for the laboratory to test outside of our routine practices.

Appendix A: Anaerobic Bacteriology

Anaerobic bacteriology is time-consuming and expensive and therefore performed only on appropriate specimens. Collect and submit aspirates in a syringe and tissues in a sterile specimen cup. Swabs are not satisfactory for anaerobic culture. Aerobic culture is done automatically so a separate specimen is not necessary. Specimens should be promptly delivered to the laboratory or transported STAT by a lab courier (place a STAT sticker on the request form). Inappropriate specimens are those which contain indigenous anaerobic flora or which are unavoidably contaminated with it during collection. Indigenous flora is often present in high numbers (109 - 1012 organisms per ml) so even minimal contamination can result in misleading results.

Appropriate Specimen
Inappropriate Specimen

Pulmonary Bartlett (protected) brush sputum, expectorated or induced

transtracheal aspirate aspirated sputum (ET or NT)

percutaneous lung aspirate standard BAL

lung tissue bronchial washing

thoracentesis fluid

"protected" BAL

Urinary suprapubic aspirate voided or catheterized urine

Female genital tract culdocentesis fluid vaginal, cervical endometrial suction or protected collector tubo-ovarian abscess material

Paranasal sinuses maxillary sinus needle aspirate nasal drainage other sinuses during surgery endoscopically obtained secretions

Abdominal peritoneal fluid bowel contents abscess contents obtained at surgery, or under CT or ultrasound guidance

Decubitus/foot ulcers scrapings from base of ulcer surface material after surface debridement, aspirate under skin flap or deep pocket through uninvolved skin, submarginal irrigation aspiration