Specimen Collection Guidelines

Specimen Collection, Transport, and Processing

• General Guidelines for Specimen Collection:
  • Collect during the acute or early phase of illness.
  • Select the correct anatomical site.
  • Collect using proper technique, minimizing normal biota contamination.
  • Collect the appropriate specimen quantity.
  • Collect before administering antimicrobials.
  • Package in containers suitable for specimen viability and to prevent hazards.
  • Label specimens accurately.
  • Transport promptly or store specimens to avoid degradation of suspected organisms.
• Swabs are generally less desirable than tissue or needle aspirates when collecting specimens.
• Lesions, wounds, and abscesses should be collected from the advancing margin, preferably via aspiration.
• Rejection of Unacceptable Specimens:
  • Unidentified or improperly labeled.
  • Label information does not match requisition.
  • Improper transport temperature.
  • Improper transport medium/container.
  • Insufficient specimen quantity.
  • Leaking specimen.
  • Grossly contaminated specimen.
  • Transport time exceeds 2 hours post-collection.
  • Received in a fixative (formalin).
  • Received for anaerobic culture from a site with normal flora.
  • Specimen is dried.
  • Test would produce information of questionable medical value.
  • Duplicate specimen received.
  • More than one specimen from one source on the same day.
  • One swab submitted with multiple organism requests.
  • Gram stain of expectorated sputum reveals fewer than 25 WBCs and more than 10 epithelial cells per LPF with mixed bacterial flora.

Collection Procedures

• Sterile containers: used for stool specimens.
• Swabs: used for upper respiratory tract, external ear, eye, and genital tract specimens. Cotton, Dacron, or Calcium Alginate are appropriate swab materials.

Labeling and Requisitions

• Patient Identification: include the patient's name, identification number, room number, physician, culture site, date of collection, and time of collection.
• Requisition Form: include the patient's name, age, date of birth, sex, room number, location, physician's name and address, specific anatomical site, date and time of collection, clinical diagnosis, relevant patient history, antimicrobial agents, and the name of the individual who transcribed the orders.

Specimen Transport

• Ideally transport specimens within 30 minutes of collection, preferably within 2 hours.
• Anaerobic bacteria should be transported in under 10 minutes.
• CSF should arrive within 15 minutes.
• All specimens should be transported in leak-proof, sealable containers, ideally placed inside a leak-proof plastic bag.

Specimen Preservation

• Urine: use boric acid
• Stool: refrigerate; if delay is longer than two hours, add to Cary-Blair Transport Media for Clostridium difficile toxin assay.

Transport or Holding Medium

• Stuart's Medium
• Amies Medium
• Cary and Blair Medium
• Transgrow Medium
• John E Martin Biological Environmental Chamber (JEMBEC)

Anticoagulants

• 0.025% sodium polyanethol sulfonate (SPS): inhibits phagocytosis, complement activation, neutralizes aminoglycoside antibiotics, neutralizes bacterial effect of plasma, and precipitates fibrinogen, β-lipoproteins, and β1-C globulin (10-30 ml for adults, 5-10 ml for children).
• SPS may inhibit Neisseria gonorrhoea, N. meningitidis, Gardnerella vaginalis, Streptobacillus moniliformis, and Peptostreptococcus anaerobius
• Heparin: used for viral cultures and isolating Mycobacterium spp. from blood.
• Citrate and EDTA (ethylenediaminetetraacetic acid): should not be used for microbiology.

Specimen Storage

• Refrigerator temperature (4°C): Urine, stool, viral specimens, sputum, swabs, and foreign devices (catheters) and viral specimens
• Incubator temperature (35°C): CSF samples intended for Gram staining, culture, and sensitivity tests.
• Ambient temperature (22°C–25°C): specimens for anaerobic culture, sterile body fluids, genital specimens, and swabs
• Freezer temperature (-20°C or -70°C): serum for serologic studies(-20°C), tissues or specimens for long-term storage (-70°C), fecal specimens for studying C. difficile toxin (-70°C)

Specimen Priority

• Specimens are categorized by priority level based on criticality or invasive nature, preservation needs, and quantitation requirements.

Specimen Processing

• Preparation of Smears: Specimens are processed by rolling the swab back and forth over glass slide areas and spread evenly for a thin layer of sample material.
• Smears from Thick Liquids or Semisolids: These smears require preparation from thick liquids or semisolids.
• Methods of Direct Examination
  • Gram staining
  • Acid-fast staining
  • Fluorescent Methods
• Processing of Specimens
  • Homogenization
  • Concentration
  • Decontamination

Specimens Used for Bacteriological Studies (Blood)

• Cleanse the venipuncture site with 70% alcohol, then 2% iodine or iodophore.
• Allow the site to air dry or dry the site with the iodophore.
• Collect specimens are best drawn just before a temperature increase, collecting 2-4 sets.
• For suspected fever of unknown origin (FUO), 4 sets of blood should be collected from separate arms during a two-day period.
• Use aerobic and anaerobic culture bottles to preserve the microorganisms.

Specimen Processing and Culture

• 0.025%-0.05% SPS added to media
• Media used: TSB, BHIA, Supplemental Peptone, THIO, Columbia Broth, Brucella Broth, and Castaneda Medium
• Incubate specimen at 35°C for 7 days
• Endocarditis, brucellosis, and fungemia have longer incubation times (2 to 4 weeks)
• Growth is usually indicated by the following: hemolysis of red cells, gas bubbles in the medium, turbidity, or colony formation.

Newer Methods of Blood Culture

• Lysis-Centrifugation (Isolator)
• Self-contained Subculture (Septi-Check and Vacutainer Agar Slant)
• Detection of microorganisms through changes in electrical impedance (BACTOMETER)
• Detection of CO2 end product metabolism (BACTEC System)

Cerebrospinal Fluid (CSF)

• Collection and Handling: Insert a needle into the subarachnoid space at the lumbar spine level. Collect at least three or four test tubes.
• Processing: Refrigerate test tubes up to 24 hours or freeze at -70 °C for viral studies. Centrifuge and decant, and use the sediment for smear and culture.

Examples of Serologic Tests

• Counter Current Immune Electrophoresis
• Coagglutination Test PHADEBACT
• Latex Agglutination Test

Culture Media

• Enrichment Broth (TSB of THIO): for gram-negative cocci.
• CAP: for gram-negative cocci.
• BAP: for gram-positive cocci.
• EMB, & MAC: for gram-negative bacilli.
• Fungal media: for fungal organisms.
• Tissue culture: for tissue specimens.

Major Laboratory Results for the Differential Diagnosis of Meningitis

• Bacterial: Elevated WBC count, presence of neutrophils, marked protein elevation, decreased glucose levels.
• Tubercular: Elevated WBC count, lymphocytes and monocytes present, moderate protein elevation, decreased glucose levels, pellicle formation.
• Viral: Elevated WBC count, lymphocytes present, moderate protein elevation, normal glucose levels, normal lactate levels.
• Fungal: Elevated WBC count, presence of lymphocytes and monocytes, moderate protein elevation, decreased glucose levels, positive India Ink and/or immunologic test for C. neoformans.

Throat and Nasopharyngeal Specimens

• Throat Swab: Collect from the uvula, to both sides of the throat. Moistened with Stuart's or Amies medium.
• Nasopharyngeal Swab: Used for isolation and detection of carriers, especially for B. Pertussis, Respiratory syncytial Virus, Parainfluenza Virus, and viruses causing rhinitis.

Sputum

• Methods: First morning specimen, 2-3 consecutive early-morning specimens, deep cough or collection via aerosol induction.

Other Means of Obtaining Sputum

• Gastric aspiration
• Transtracheal aspiration
• Transthoracic needle biopsy
• Bronchoscopy
• Thoracentesis
• Open lung biopsy
• Bronchoalveolar lavage (BAL)
• Protected catheter bronchial brushing.

Culture

• Culture on MAC, BAP, and CAP. Anaerobic culture by percutaneous aspiration and protected bronchial brush. Perform Gram and Acid fast staining on smear.

Gastrointestinal Tract (Stool)

• Collection and Handling: Collected as walnut-sized specimens, delivered within 1 hour.

Staining of Smears

• Gram Staining: used to observe clumps of gram-positive cocci for staphylococcal infection, thin gram-negative bacilli for Campylobacter and Vibrio, and many gram-positive bacilli for C. difficile.
• Acid-Fast Staining: detects Cryptosporidium spp. and Mycobacteria.

Culture

• General supportive media for yeast spp., staphylococci, streptococci, and Gram-negative bacilli
• Moderately selective media (MAC, EMB), HEA, & XLD for Salmonella and Shigella
• Highly selective agar (Brilliant Green, Bismuth Sulfite for Salmonella, Campy-blood for Campylobacter, TCBS for Vibrio cholera, CIN for Yersinia enterocolitica, CCFA for C. difficile.

Colony Count

• Interpretation: Colony count > 105CFU/ml (100, 000CFU/ml) for 1 or 2 bacterial species, >105 CFU/ml for 3 or more bacteria, >10³/ml of a bacterial species from a male's midstream urine, 10² - 10³ /ml of 1 or 2 bacterial species in female lower urinary tract symptoms, or <5,000CFU/ml, Organism presence in any quantity obtained by suprapubic aspiration.

Abscess (Lesion, Wound, Pustule, Ulcer)

• Superficial Abscesses; swab along the leading edge of the wound
• Deep Abscesses; aspirate from the wall or advancing margin of lesion.

Body Fluids

• Include amniotic, abdominal, ascites/peritoneal, bile, synovial, pericardial, and pleural fluid. These may require homogenization.

Ear

• Clean the ear canal with mild soap, and aspirate fluid with a needle for inner ear specimens, or use a swab if the eardrum is ruptured.
• Outer ear collection involves removing debris and crusts using a saline-moistened swab.

Foreign Bodies (IUD)

• Intrauterine device (IUD) specimens are cultured for Actinomyces spp.
• Use Maki Roll Technique for catheter and prosthetic valve specimens. More than 15 colonies are needed to perform identification and susceptibility tests. A whole Foley catheter specimen should not be cultured.

Genital Tract

• Specimens should be collected from the cervix, vagina, endocervical/vaginal mucosa, urethra, or prostate gland. Using a flexible swab, the urethra should be swabbed for 2 to 3 seconds, and the swab inserted 2-4 cm into the urethra. The secretions should then be collected from swab or tube.

Tissue

• Tissue specimens require disinfection, and tissue should not dry out.

Critical (Panic) Values

• Critical values are those needing prompt action:
  • Positive blood cultures.
  • Positive CSF Gram stain or culture.
  • Streptococcus pyogenes (group A Streptococcus) in a surgical wound;
  • Gram stain suggestive of gas gangrene;
  • positive acid-fast stain;
  • positive antibiotic-resistant bacteria (e.g., Vancomycin-resistant S. aureus);
  • positive for Legionella, Francisella, and Brucella.

This quiz covers essential guidelines for specimen collection, transport, and processing in clinical settings. Learn about the correct techniques, labeling practices, and factors that lead to the rejection of specimens. Test your knowledge on what constitutes an acceptable specimen for effective diagnosis.