Microbiology

Quest Diagnostics Nichols Institute offers a complete spectrum of diagnostic microbiology services. Please use tight sealing sterile containers or tubes of transport medium that will maintain viability, prevent drying out of the specimen/swab, and prevent overgrowth of nonpathogenic microorganisms. It is important to label the container with the patient’s name and source. The inoculated containers should show no leakage.

GENERAL CONSIDERATIONS:

  1. Whenever possible, specimens should be obtained before antibiotics or other antimicrobial agents have been administered.
  2. Clinical material should be collected in leak-proof specimen containers that are tightly sealed.
  3. Material should be collected where the suspected organism is most likely to be found and with as little external contamination as possible (this is particularly important for draining lesions).
  4. The stage of the disease is sometimes an important consideration in the successful isolation of the causative agent.
  5. Specimens should be of sufficient quantity to permit completion of all tests ordered.
  6. Provisions should be made for the prompt delivery (within one hour after collection) of the specimen to the laboratory.
  7. Most clinical material can be held for several hours in a refrigerator before culturing if it cannot be processed immediately. This is particularly true with the following specimen types: urine, sputum, and material on swabs taken from a variety of sources. DO NOT refrigerate body fluids such as CSF or blood.
  8. Specimens for Neisseria gonorrhoeae isolation MUST be submitted on appropriate isolation plates (Martin-Lewis or Neigon agar plates). Do not refrigerate inoculated plates.
  9. All stools for Ova and Parasite exam require preservation in a formalin fixative and PVA or equivalent immediately after collection.
  10. Soft and liquid stools require PVA fixative to maintain the integrity of the trophozoites for the performance of the trichrome stain. Please order O&P with Trichrome Stain.
  11. Collect one stool for culture/O&P per day. From hospitalized patients, stool cultures/O&P exams should not be performed if the length of stay is greater than 3 days and the admitting diagnosis is gastroenteritis.
  12. In hospitalized patients, collect watery, loose, or unformed stools and submit promptly to the laboratory for Clostridium difficile PCR testing. Repeat testing appears rarely useful, except for patients with evidence of a new infection.
  13. For mycobacterial (TB) culture, it is recommended to collect three (3) sputum specimens for acid-fast smears and culture in patients with clinical and chest x-ray findings compatible with tuberculosis. These three (3) samples should be collected at 8-24 hour intervals (24 hours when possible) and should include at least one first morning specimen.

SELECT AGENTS

If a CDC Select Agent (i.e. anthrax, plague, etc.) is suspected of producing an infection, please notify the Clinical Microbiology/Virology Laboratory at Quest Diagnostics Nichols Institute for instructions. Most clinical samples (i.e. blood, wound, etc.) from patients potentially infected with CDC Select Agents can be collected and processed by routine Clinical Microbiology and Virology Laboratories. If environmental contamination with a CDC Select Agent is suspected, please contact your local Public Health Department for more details on how to handle this sample type. Do not submit environmental samples potentially contaminated with CDC Select Agents to Quest Diagnostics Nichols Institute. A complete list of CDC Select Agents can be found at http://www.selectagents.gov/

RECOMMENDATIONS FOR COLLECTING BLOOD CULTURES

  1. For each septic episode, draw 2 to 3 separate sets within a 24-hour period, spaced as far apart as possible (a minimum of 30 minutes between sets).
  2. Specific recommendations for initial cultures:
    • Suspected sepsis: 2 separate sets before antimicrobial therapy is started; spaced a minimum of 30 minutes apart.
    • Bacterial endocarditis: 3 separate sets before therapy.
    • Fever of unknown origin (FUO) (3 weeks of documented fever without an obvious cause): 2 separate sets initially, then 2 sets the next day just before the expected fever spike.
  3. Except as noted above for FUO, wait at least 72 hours from the time of first set for identification and sensitivity results before obtaining additional cultures.
  4. If cultures are still negative after 72 hours and the clinical condition warrants, draw a maximum of 3 more blood cultures over the next 24 hours. Wait another 72 hours for results.

SPECIMEN COLLECTION AND HANDLING

BLOOD CULTURE COLLECTION PROCEDURE

Materials Required:

  • Gloves
  • Syringe
  • Alcohol preps
  • 1-2% tincture of iodine or 2% chlorhexidine
  1. Locate vein for venipuncture site and put on gloves.
  2. Using an alcohol prep, vigorously cleanse the intended venipuncture site for 30 seconds back and forth followed by tincture of iodine which is allowed to dry for at least 30 seconds or 2% chlorhexidine, allowed to dry for at least 30 seconds. The drying action kills the microorganisms on the skin surface.
  3. Remove the caps from one aerobic blood culture bottle and one anaerobic blood culture bottle. Clean each rubber septum with an alcohol prep.
  4. Prepare the 20 mL syringe and perform the venipuncture,drawing 20 mL in adult patients. A smaller draw volume,1-3 mL, is sufficient for pediatric patients. Pediatric blood culture bottles are available. 

Note: A separate fee will be charged for each blood culture bottle.

VIRAL AND CHLAMYDIAL TESTS

Viral and Chlamydial Cultures:

  • Viral Chlamydial Transport Medium (VCM). IMPORTANT: After sample collection, refrigerate culture specimens until pick up.
  • DNA Probe:
  • GEN-PROBE® PACE® 2 Collection Kit (male or female); stable at ambient temperatures until printed expiration date.

COLLECTION FROM NORMALLY STERILE SITES AEROBIC CULTURE

Specimen collection from normally sterile sites requires a needle puncture or a surgical procedure. Decontamination of the skin must be performed prior to the collection of specimens such as blood, cerebrospinal fluid and other normally sterile body fluids.

To decontaminate the site effectively, first clean the puncture site with a povidone-iodine preparation or alternate disinfectant to remain on the skin for at least one minute. Then clean the site with 70% alcohol and wait for the alcohol to air dry. After the puncture site has been disinfected, avoid any finger probing unless fingers have also been disinfected.

BLOOD COLLECTION (USED FOR BRUCELLA CULTURE)

Specimens for blood cultures must be submitted in blood bottles. After removing the protective cardboard covering, decontaminate the diaphragm tops of two bottles by swabbing with iodine followed by alcohol to remove iodine. Allow alcohol to air dry. After collection of the blood specimen, inject approximately 10 mL into each of the two bottles. Swirl bottles gently to mix, but do not vent. Keep at room temperature (15-30°C) until sent to the laboratory.

CEREBROSPINAL FLUID (USED FOR THE CULTURE OF MYCOBACTERIA, FUNGI, BACTERIA, OR BACTERIAL ANTIGEN TESTING)

Submit a separate sterile screw-capped tube containing at least 0.75 mL of cerebrospinal fluid for each test ordered. For microbiological analysis, it is best to submit the second or third tube drawn. Do not send the collection tube.

OTHER STERILE BODY FLUID

Follow standard procedures and obtain the specimen by aspiration. Transport the specimen in aerobic or anaerobic transport kits or blood culture bottles depending on clinical condition. Specimens may be submitted in sterile containers for aerobic culture only.

COLLECTION FROM NONSTERILE SITE

SPUTUM SPECIMENS

For routine sputum specimens, collection in the early morning is recommended. Patient should gargle with water before collection. The most suitable specimen is the expectoration obtained after a deep cough. Collect specimen in a leakproof sterile screw-capped container, such as the sterile urine container available from Quest Diagnostics Nichols Institute.

URINE SPECIMENS

All patients should void the first part of the specimen into the toilet, then collect the remainder of the specimen in a sterile container. Urine samples for routine culture must be transported in the urine transport tubes provided by Quest Diagnostics Nichols Institute.

Urine samples for mycobacteria or fungus culture may be submitted in a sterile screw cap container. Keep urine refrigerated.

To obtain a clean catch sample of urine from a female patient, a thorough cleansing of the periurethral area is essential before specimen collection. Wash the area with a disinfectant, thoroughly rinse with clean water, and make all efforts to avoid any contact until urination is complete.

For a male patient, a thorough cleansing of the glans penis with disinfectant followed by a complete rinse with clean water is required.

For in-dwelling catheters, obtain the specimen with a needle and syringe. Select a puncture site 1-2 inches distal to the meatus and clamp below the puncture site. Cleanse the area to be punctured with 70% alcohol. Aspirate exactly 5 mL of urine with a sterile needle and syringe. Deliver this volume directly into a sterile urine transport tube available from Quest Diagnostics Nichols Institute. Specimens obtained from the collection bag are not suitable for analysis. Foley tips will not be accepted.

For cytoscopic or suprapubic aspiration, follow standard procedure and obtain the specimen by aspiration. Transport the specimen in anaerobic transport tubes available from Quest Diagnostics Nichols Institute. Keep urine refrigerated.

STOOL CULTURE

Collect stool without contaminating with urine. Select portions of stool containing pus, blood or mucus and immediately mix into modified Cary-Blair transport media. Stool samples will not be accepted unless they are submitted in transport media. Frozen specimens are not acceptable. N ote: Stools for C. difficile are not acceptable in Cary Blair transport and must be refrigerated or frozen at -70° C or below.

ANAEROBIC CULTURE

Specimens from the following sites are acceptable when submitted in appropriate transport media including the anaerobic transport tubes available from Quest Diagnostics Nichols Institute.

  • Transtracheal aspirations
  • Suprapubic urines from percutaneous suprapubic bladder, nephrostomy tube or suprapubic catheter
  • Genital specimens from cul-de-sac aspiration, culdocentesis, percutaneous aspiration, placenta, fallopian tube, or prostatic or seminal fluid
  • Surgical specimens
  • Exudates, aspirated pus from deep wounds or abscesses
  • Body fluids - normally sterile

Specimens from the following sites are not acceptable:

  • Throat and nasopharyngeal swabs
  • Sputum and bronchoscopy specimens
  • Feces and rectal swabs except for C. difficile Cultures
  • Voided or catheterized urines
  • Specimens from sites contaminated with intestinal contents such as colostomy sites, draining pilonidal sinus and traumatic perforation of the bowel
  • Superficial wounds
  • Vaginal or cervical swabs

MYCOBACTERIA CULTURE

Blood: Refer to the database for current specimen requirements.

BRONCHIAL WASH

Collect 3 mL of bronchial washings into a sterile container that seals tightly, or submit a bronchial brush in a sterile container with sterile saline. Do not send collection containers.

CSF

Collect 1 mL CSF (minimum) in a sterile plastic conical tube, tightly sealed. Tubes supplied in spinal fluid collection kits are not designed for specimen transport. To avoid leakage, transfer fluid aseptically into a sterile plastic conical tube.

MYCOBACTERIA CULTURE

Blood: Refer to the database for current specimen requirements.

GASTRIC

Collect a fasting early-morning specimen. Use sterile saline. Adjust to neutral pH (7.0-7.5) with 100 mg sodium carbonate immediately following collection. 4% NaOH is an acceptable substitute for sodium carbonate. Gastric samples cannot be accepted unless they have been adjusted to neutral pH.

SPUTUM

Collect sputum using a sputum collection kit. Remove and cap the conical tube. Seal tightly to leakage and send the conical tube to the laboratory. A first morning specimen consisting prevent of 5-10 mL of sputum is preferred. Do not send saliva. Specimens sent in 70% alcohol are not acceptable.

URINE

Collect the first morning urine specimen, up to 50 mL. Send in a sterile plastic container that seals tightly.

BONE MARROW

Collect as much as possible into an SPS or heparin blood collection tube.

BODY FLUIDS

Submit at least 10-15 mL in a leakproof sterile container. Collect bloody specimens into a SPS blood collection tube (yellow-top). Do not use ACD as an anticoagulant for culture submission.

TISSUE BIOPSY SAMPLE

Submit I gram of tissue, if possible, in a sterile container without fixative or preservative. Keep moist with a small quantity of sterile saline or nutritive broth. Collect aseptically and avoid indigenous microbiota. Select caseous portion if available. Refrigerate. Do not freeze.

LYMPH NODE

Submit node or portion in a sterile container without fixative or preservative. Collect aseptically and avoid indigenous microbiota. Refrigerate. Do not freeze

SKIN LESION MATERIAL

Submit biopsy specimen in sterile container without fixative or preservative. Swabs in transport medium are acceptable only if biopsy sample material or aspirate is not obtainable. For a cutaneous ulcer, collect the biopsy sample from the periphery of the lesion, or aspirate material from under the margin of the lesion. If the infection was acquired in Africa, Australia, Mexico, South America, Indonesia, New Guinea, or Malaysia, note this on the test request form; as Mycobacterium ulcerans may require prolonged incubation.

CULTURE FOR FUNGI

Collect specimens from the following sites using a culture swab transport medium system: mouth, nose, nasopharynx, ear, eye, wound, vagina, cervix, or urethra. Use a sterile plastic container for respiratory secretions, body fluids, tissue, bone marrow, CSF, urine, hair, skin, nail, contact lens fluid and/or contact lenses. Refer to the database for the current collection method for blood. Store and transport specimens at 4°C. Dermatological specimens may be shipped at 15-30°C.

MYCOPLASMA/UREAPLASMA CULTURES

General considerations: All specimens must be transported in the multi-microbe transport media (VCM supplied at your request). Samples submitted without suitable transport media will not be accepted. Refrigerated stability for all mycoplasma/ureaplasma cultures is 48 hours. Specimens transported and stored at -70°C are stable indefinitely. Freezing is preferred if transport time is expected to exceed 24 hours. It is preferable to vigorously agitate the swab in the transport media for 30 seconds, and express the material from the swab into the transport media. Discard the swab. Inhibitory agents may be present in the material of the swab tip or shaft. If viral cultures are requested, submit a separate sample according to the standards outlined in the virology section.

MYCOPLASMA PNEUMONIAE

Collect tracheal aspirate, sputum, throat or nasopharyngeal swabs and submit in the multi-microbe transport medium (VCM) supplied upon request. Refrigerate (48-hr stability) or freeze at -70°C (indefinite stability; transport on dry ice: do not thaw). Freezing is preferred if transport time is expected to exceed 24 hours.

UREAPLASMA UREALYTICUM AND M. HOMINIS

Collect tracheal aspirates from newborns to detect pneumonia caused by ureaplasma urealyticum Submit in multi-microbe transport medium (VCM) supplied upon request. Refrigerate (48-hr stability) or freeze at -70°C (indefinite stability; transport on dry ice; do not thaw). Freezing is preferred if transport time is expected to exceed 24 hours. Adult genital specimens include: vaginal, cervical or urethral swabs, amniotic fluid, CSF, urine or semen submitted in multi-microbe transport refrigerated or frozen as detailed above.

NUCLEIC ACID TESTS (BDNA, PCR, TMA, GENOTYPING, ETC.)

Standard quantitative assay: 1 mL frozen PPT-potassium EDTA plasma (white-top tube); (bDNA, PCR, TMA, Genotyping, etc.) 0.5 mL minimum

Ultrasensitive assay: 2.0 mL frozen PPT-potassium EDTA plasma (white-top tube); 0.6 mL minimum

Expanded range assay: 2.5 mL frozen PPT-potassium EDTA plasma (white-top tube); 1.0 mL minimum

Centrifuge blood within 2 hours of collection and freeze without removing the plasma. Do not thaw. Alternatively, submit EDTA (lavender-top tube) or ACD (yellow-top tube) frozen plasma that has been removed from cells and frozen within 2 hours of collection. Avoid repeated freezing and thawing. Note that specimens collected in ACD anticoagulant will have results that are 15% lower than those collected in EDTA, owing to the dilution effect of the liquid anticoagulant.

PARASITOLOGY

OVA AND PARASITE STUDIES

A series of three specimens submitted on separate days within a 10 day period is usually recommended. Collect stool without contamination by urine. Immediately, within one hour maximum, mix amount of stool indicated by fill line into both a modified PVA and 10% formalin containers. These transport tube sets are available upon request. Transport and store at room temperature. Do not freeze.

Note: specimens should be obtained prior to or at least 7 days after radiologic studies involving barium sulfate. Unpreserved specimens will not be accepted.

MALARIA/BLOOD PARASITE STUDIES

Blood smears should be prepared within 24 hours after collection. Both thin (prepared as for hematology examination with a feathered edge) and thick (3 small drops pooled together- the size of a dime) smears should be submitted in a slide carrier. Submit several of each and include a backup EDTA tube of whole blood for each exam requested. These slides must be air dried without applying heat or fixing by any method.

MISCELLANEOUS

CLOSTRIDIUM DIFFICILE TOXIN, EIA OR TISSUE CULTURE, STOOL

Collect fresh stool in sterile, leakproof container without media, serum, preservative, or metal ion. For patients requiring the use of diapers, first line the diaper with clean plastic to prevent absorption. Then transfer 2 g or 2 mL of the stool specimen from the plastic-lined diaper to the sterile container. Do not submit the diaper itself. Do not use VCM or equivalent. Do not use any media, preservative, or additive. Freeze at -70° C or refrigerate. Freezing is preferred if transport time is to expected to exceed 24 hours. Stable for 3 days refrigerated, longer if frozen.

NEISSERIA GONORRHEAE PROBE (GENPROBE)

Swab only

Use GenProbe Specimen Collection Kit for urethral specimens or GenProbe Specimen Collection Kit for cervical specimens. Only female endocervical or male urethral specimens are acceptable. Store at room temperature (15-30°C) or refrigerated. (2-10°). Do not freeze. Stable up to a week.