Microbiology Sample Collection Guidelines and Rejection Criteria

Proper collection and transport of the biological specimens to the laboratory for culture is the most crucial step in the recovery of pathogenic organisms responsible for infectious diseases. A poorly collected specimen may lead to failure to recover the causative organism(s) and/or result in the recovery of contaminating organisms. This will lead to incorrect or harmful therapy if treatment is directed toward commensal or contaminant organisms. This post discusses the specimen rejection criteria in brief.

Microbiology Sample Collection Criteria

  1. Collect the specimen from the actual site of infection, avoiding contamination from adjacent tissues, organs, or secretions
  2. Collect the specimen at optimal times (for example, early morning sputum for the culture of acid-fast bacillus) to provide the best chance of recovering causative microorganisms.
  3. Whenever possible, collect specimens prior to administration of antimicrobial agents.
  4. A sufficient quantity of material must be obtained to perform the test.
  5. Properly label the specimen and complete the test request form. Mention the specific source of the specimen.  Each culture container must have a legible label with the following minimum information:
    1. Patient name
    2. Patient identification number
    3. Source of specimen
    4. Name of clinician
    5. Date/hour of collection
  6. To ensure optimal recovery of microorganisms:
    1. Use appropriate collection devices: sterile, leak-proof specimen containers.
    2. Use appropriate transport media (anaerobe transport vials, Cary-Blair media, M4RT for viral and Chlamydia cultures, and urine boric acid transport).
    3. Use appropriate culture media
    4. Minimize transport time. Maintain an appropriate environment between a collection of specimens and delivery to the laboratory.
  7. Ensure that the proper transport system is utilized for orders with more than one test. (For example, anaerobic culture requests need to be submitted in anaerobic transport media; bacteriology requests should not be in viral transport media; AFB requests should not be in anaerobic transport media.)

Microbiology Specimen Rejection Criteria

The microbiology laboratory should have a policy that defines conditions in which a specimen is unacceptable for processing. Specimen rejection criteria should be distributed to all clinical practitioners. Requesting physicians or nursing in charge should always be notified upon receipt of these specimens before discarding them. Such communications should be documented in the laboratory information systems (LIS) with the date and time.

It may be necessary to process some specimens even though they are not clearly identified if they are difficult to repeat. Such  irretrievable specimens include CSF, surgical fluid/tissue/swabs, post mortem specimens, blood cultures/ tips. A notation regarding improper collection should be added to the final report so that physician is able to determine the validity of the results.

This post outlines some of the essential criteria for the rejection of clinical specimen for microbiology tests. 

Clerical Errors

  1. Unlabeled or incorrectly labeled specimens: Unlabeled (no identification on the container) or incorrectly marked specimens (when compared with its accompanying request form) should not be accepted for analysis. When a discrepancy is seen between patient identification on the requisition form and specimen container label, request a new specimen or have the requesting physician or nurse correct the error in person in the laboratory (identification of a mislabeled specimen or requisition should not be made over the telephone).
  2. Specimens received without a request form: If a specimen is received without a request form, ask for a correctly completed request. However, if the request form is not received on the same day, do not process the specimen.
  3. Request forms received without specimen: If a matching specimen is not received by the end of the working day, issue a report stating that no specimen was received with the request form.
  4. Specimens received with a request form devoid of any patient demographic details: Request for a correctly completed request form. However, if the correctly completed request form is not received on the same day, do not process the specimen.
  5. Missing vital information in the requisition form: If the specimen source or type or requested test name is not noted in the requisition form, call the physician or charge nurse to ascertain the missing information.

Inappropriate Containers/Conditions

  • Specimens are received in improper or non-sterile containers, leaking containers, or transport media: Leaking or damaged specimens should not be accepted for analysis. Return requisition with appropriate comment. Notify the physician and request a properly handled specimen.
  • Specimen for anaerobes not received in an appropriate container: If specimens for anaerobic bacteria are submitted in aerobic transport, notify the physician or charge nurse. If the physician insists specimen be processed, refer to supervisory personnel or comment in the laboratory record and report form that inappropriate transport may have influenced the recovery of significant anaerobic bacteria.
  • Specimen received in fixative (formalin); exception, stool for parasites and ova: If the specimen is received in a fixative (formalin), it kills any microorganism present in the sample. In such cases, the laboratory should notify the physician and request to send a new proper specimen; record on requisition “specimen unsatisfactory; received in preservative.”

Delay in Transit or Improper transport

  1. Specimens received that have been delayed in transit (specimen transport time exceeds 2 hours post collection and the specimen is not preserved): Reject the samples that have been delayed in transit as unsuitable for processing.
  2. The specimen has been transported at the improper temperature: Reject the sample and request a new specimen.

Inappropriate specimens

  1. Dry swab: Notify physician or charge nurse and request to submit proper specimen in an appropriate transport device. If a physician insists dry swabs be cultured, note on the laboratory record and report with a caveat: “microorganisms recovered may not reflect actual microbiota.”
  2. Foley catheter tip: Processing specimens like Foley catheter tip may produce information of questionable medical value, so the physician should be notified that the specimen is not suitable for microbiological analysis. Note rejection on requisition and return.
  3. Unpreserved urine held in the refrigerator for >24 hours: Notify the physician or nurse in charge and request a new specimen. Return requisition with appropriate comment.
  4. Sputum specimen with 25 < WBC, >10 epithelial cells/lpf: Inform physician or charge nurse that specimen is mostly saliva and is not appropriate for culture. Request a repeat specimen.
  5. 24-h collection of urine or sputum for AFB or fungus culture: Inform physician or nurse that according to laboratory manual, three separate first morning specimens of sputum or of urine are the best samples for analysis; reject 24-h specimens.
  6. Gram stain for Neisseria gonorrhoeae on specimens from cervix, vagina, and anal crypts: As these anatomic locations may harbor nongonococcal Neisseria giving false-positive results, the smear should not be examined. Notify the physician or charge nurse why these smears are unsuitable for processing.
  7. Specimen received for anaerobic culture from a site known to have anaerobes as part of the normal flora such as mouth, vagina, fistula or intestinal contents, samples from ileostomy or colostomy, etc. should not be processed and the physician should be informed accordingly.

Inadequate specimens

Many times laboratory receives an insufficient quantity of specimens for testing; in those cases, the laboratory should seek additional material or test prioritization. For example;

  1. If only one swab is submitted with multiple requests for various organisms (bacteria, AFB, fungi, virus, ureaplasma, etc.), ask the physician to send the additional samples or to prioritize test requests.
  2. If sputum swabs for AFB or fungal culture are received, notify the physician or charge nurse that the specimen is inadequate in quantity for the isolation of these microorganisms. Request properly collected samples.

Excess specimens

  1. If more than one specimen of urine, stool, sputum, wound or routine throat specimens were submitted on the same day from the same source. Notify the physician or charge nurse that, as stated in the laboratory manual, only one specimen will be processed per day.

Summary of Specimen Collection and Transport Guidelines for Bacteriological Examination

SpecimenCollection EquipmentTransportInstruction
BloodNeedle and springer; Two Brain-heart infusion (BHI) broth bottles for a setIf delayed, incubate at 35℃ OR Keep at room temperature (DO NOT refrigerate)Use antiseptics (mentioned below) at puncture site: 
a. 70% isopropyl alcohol-providone iodine or
b. 70% isopropyl alcohol-2% chlorhexidine
c. <2 months child: omit the iodine step.Clean two more times with separate 70% isopropyl alcohol or ethyl alcohol pads.
d. >2 months child: Chlorhexidine gluconate (DO NOT touch the site after cleaning!
e. Blood to broth ratio 1:10 for adults in adult clood culture bottle;1:5 for children in paediatric blood culture bottle OR as recommended by manufacturer. 
Bone marrowSurgical preparation; BHI brothIf delayed, incubate at 35 OR Keep at room temperature.DO NOT refrigerate.Surgical preparation of site
Cerebospinal Fluid (CSF)Surgical preparation; Sterile screw capped tubesIf delayed, incubate at 35℃. DO NOT refrigerate.Handle CSF as EMERGENCY specimen
Sterile body fluidsSkin asepsis for percutaneous aspiration. BHI broth (Blood culture bottle), sterile screw capped tube.If delayed, incubate at 35℃. DO NOT refrigerate.a. Place recommended volume (5:10 ml) in blood culture bottle. 
b. Place 1ml in sterile tube for gram staining and direct plating. 
Conjunctiva (bacterial conjunctivitis)Lid Margin (blepharoconjunctivitis)Separate cotton swabs for each eye; Sterile Kimura spatula for conjunctival scrapingImmediately inoculate the material at bedside, on blood agar and chocolate agar.a. Obtain material using premoistened swabs before topical medications are applied. 
b. Take swabs from both eyes.
c. Prepare smears for Giemsa and/or Gram staining. 
d. Fix with 95% methanol for 5 minutes. 
Cornea (bacterial keratitis)Sterile Kimura spatulaImmediately inoculate the material at bedside, on blood agar and chocolate agar using a ‘C’ formation for each scarping (3-5 scrapings per cornea).a. Obtain corneal scrapings from advancing edge of ulcer.
b. Scrape multiple areas of ulceration and suppuration. 
c. Keep the eyelid open. DO NOT touch the eyelashes. 
d. Prepare smears by compressing material between two clean glass slides and pulling the slides apart. 
Fecal specimenSterile disposable collection bottle (around 40ml)Refrigerate if not plated within 1 hour.
Transport media if prolonged delay/shipment: Stuart’s or Cary-Blair transport media.
Collect in the container directly. DO NOT collect from bedpan. DO NOT allow contact with urine.
GenitalCollect two swabs (Dracon or rayon swabs for gonococci)For gonococci, bedside inoculation and immediate incubation at 35℃ with 5% CO2, Amies transport medium. DO NOT refrigerate.a. Collect endocervical swab through a speculum. 
b. Avoid touching swab to uninfected mucosal surfaces. 
c. Clean external urethra before taking urethral specimen. 
d. Prepare slide for staining using second swab. 
NasopharynxCotton-tipped flexible swabDO NOT refrigerate. Transport medium (Annie’s/Stuart’s) if delay expected.a. Tilt head backward at 70 degrees. 
b. Pass through nose into nasopharynx. 
c. Allow to remain for a few seconds. Carefully withdraw. 
NoseSwab pre moistened with sterile normal saline; sterile tubeTransport medium (Annie’s Stuart’s) if delay expected. Dry swabs are not acceptable.Swab anterior nares only. Culture immediately.
SputumSterile containerTransport immediately. Refrigerate if delayed for >2 hours.Avoid refrigeration if fastidious organisms are suspected.a. Instruct patient to rinse mouth and cough deeply. DO NOT mix with saliva. 
b. First morning specimen is preferred. 
Endotracheal aspirateSterile technique using a 22 inch, 12F suction catheter; mucus collection tubeImmediately send to laboratory and culture within 1 hour of collection.a. Introduce catheter for at least 30 cm through endotracheal tube. 
b. Discard the first aspirate done without instilling saline. 
c. Collect aspirate in a mucous collection tube after tracheal instillation of 5 ml saline. [Perform chest vibration or percussion for 10 minutes to increase the retrieved volume (>1ml)]
ThroatTwo cotton swabs; Tongue depressor; Sterile tubeTransport medium (Annie’s/Stuart’s) if delayed for >2 hoursa. Use tongue depressor.
b. Sample ONLY back of throat between and around the tonsillar area thoroughly.
c. Avoid checks, teeth, and tongue. 
Urine (freshly voided midstream clean catch)Sterile screw-capped wide mouth containerTransport in a collection container. Refrigerator within 30 minutes after collection. (It may be refrigerated up to a maximum of 24 hours before plating).a. Give patient clear and detailed instructions for collection. 
b. Clean external genitalia with soap , and not disinfectant. 
c. Discard the first part and collect the middle part of the urine stream. 
Urine(catheter)-Sterile screw-capped tube
-Needle ad syringe
Sterile tube.
Refrigerate quickly if delay expected.
a. Clean from catheter line. 
b. Decontaminate line as with vein puncture or use port. DO NOT puncture silicone catheter. DO NOT collect from the drainage bag. DO NOT culture Foley tips. 
Pus or tissue (from closed wound or abscess)Syringe and needle; sterile container or blood collection tube without anticoagulantDeliver aspirates and tissues to the laboratory within 30 minutes. 
Keep tissues moist in normal saline.DO NOT refrigerate or incubate before or during transport.
If delay, keep the sample at room temperature.
a. Clean overlying skin with: 2% chlorhexidine or 70% alcohol followed by povidone-iodine. 
b. Remove iodine with alcohol prior to specimen collection.
c. Place some in anaerobic medium if an anaerobic culture is required. 
Wounds (open wounds)Cotton swabs/curette/
Transport medium (Stuart’s) if delay expected. Refrigerate, if delayed.a. Clean surrounding skin with antiseptics. Debride, thoroughly clean wound with sterile saline. 
b. Sample viable infected tissue, NOT superficial debris. 
c. Specimen advancing margin of lesion or base of wound. 
d. Collect swabs only when tissue or aspirate cannot be obtained. 
e. Gently roil swab over the inflamed surface of the wound approximately face times. 
f. Simple different areas of the burn wound since organisms may not be distributed evenly. 
TissueSterile container with normal salineRefrigerate, if delayed.Never place tissue for bacterial culture in formalin.
Collection of Different Specimen for Aerobic Bacteriological Examination

References and further readings

  1. Andrea J. Linscott, 2016. 2.1. Collection, Transport, and Manipulation of Clinical Specimens and Initial Laboratory Concerns, Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch2.1
  2. Procop, G. W., & Koneman, E. W. (2016). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology (Seventh, International edition). Lippincott Williams and Wilkins.
  3. Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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