Additional Mycobacterium Collection Information

Specimen Collection and Transportation for Mycobacteriology/Mycobacterium Specimens 

1. Principle

The quality of specimens collected and the proper transport of those specimens to the laboratory are critical to the successful isolation of etiological agents.

2. Collection of Specimens 

  • Use a sterile, leak proof, disposable plastic container. Do not use waxed containers.  Swabs are not recommended for the isolation of mycobacteria.
  • Label the container with the patient’s name, specimen type, and date and time of collection.
  • Collect initial specimens before antimicrobial therapy is started.
  • Collect specimens aseptically, minimizing contamination with indigenous microbiota.
  • Collect sufficient material (see table below).
  • Screw cap on container securely. Place in the PHOL biohazard bag and seal.
  • Place requisition form in side pouch of bag.
  • Submit one specimen and one form per bag.
  • Do not freeze or use any fixatives or preservatives.

3. Specimen requirements for mycobacterial isolation and acid-fast stain.

 Specimen type  Specimen requirements  Special instructions  Unacceptable specimens
Abscess contents, aspirated fluid As much as possible in sterile plastic container. Cleanse skin with alcohol before aspirating sample. If volume is insufficient for aspiration by needle and syringe, collect specimen on swab and place in multi organism (Amico or Stuarts) aerobic transport medium. Dry swab. Swabs in anerobic transport medium.
Blood
  • 7 mL SPS (yellow top) or
  • 7 mL heparin (green top) blood collection tube or 
  • 10 mL Isolator tube or 
  • 5 mL inoculated directly into Myco/F Lytic Medium.
Disinfect site as for routine blood culture. Mix tube contents immediately after collection. Blood collected in EDTA, which greatly inhibits mycobacterial growth even in trace amounts; coagulated blood; serum or plasma.
Body fluids (pleural, pericardial, peritoneal, etc.) As much as possible (10-15mL minimum) in sterile container. Disinfect site with alcohol if collecting by needle and syringe.   
Bone Bone in sterile container without fixative or preservative.    Specimen submitted in formalin.
Bone marrow As much as possible in sterile collection tube or SPS or heparin tube. Disinfect site with alcohol if collecting by needle and syringe.   
Bronchoalveolar lavage or bronchial washing ≥ 5 mL in sterile container Avoid contaminating bronchoscope with tap water. Saprophytic mycobacteria may produce false-positive culture or slide results.  
Bronchial brushing Sterile container. If small amount of specimen then add sterile saline.    
Corneal Scrapings Physician to inoculate cultures during procedure. Contact the laboratory before the procedure. Laboratory to send one MGIT tube, two LJ slants and a microscope slide to the physican's office. Specimen submitted in formalin.
CSF ≥ 2 mL in sterile container. Send maximum volume attainable. < 0.5 mL
Gastric lavage fluid ≥ 5 – 10 mL in gastric lavage container. Collect in the morning soon after patient awakens in order to obtain sputum swallowed during sleep. Collect fasting early-morning specimen on three consecutive days. Use sterile water. Adjust to neutral pH with 100 mg of sodium carbonate immediately following collection. The PHOL provides collection jars for gastric lavage (N-0043). Specimen that has not been neutralized.
Lymph node Node or portion in sterile container without fixative or preservative. A small amount of sterile saline may be added.  Collect aseptically. Select caseous portion if available. Do not wrap in gauze. Do not freeze. Specimen submitted in formalin.
Skin lesion material Submit biopsy specimens in sterile containers without fixative or preservative. Submit aspirate in syringe with needle removed and Luer lock cap in place.
  • Swabs in transport medium (Amies or Stuarts) are acceptable only if biopsy sample or aspirate is not obtainable. For cutaneous ulcer, collect biopsy sample from periphery of lesion, or aspirate material from under margin of lesion.
  • If infection was acquired in Africe, Australia, Mexico, South America, Indonesia, New Guinea or Malaysia, note on requisition, because Mycobacterium ulcerans may require prolonged incubation for primary isolation.
  • Dry swab.
  • Swabs in anerobic transport medium.
Sputum  
5 – 10 mL in sterile, wax-free, disposable container. Collect an early morning specimen from deep, productive cough on three consecutive days. Do not pool specimens. To obtain a sufficient volume of specimen (5mL) the patient may expectorate several times per collection. For follow-up of patients on therapy, submit theree specimens after two months and again on completion of therapy. 
  • Saprophytic mycobacteria in tap water may produce false-positive culture or slide results.
  • For expectorated sputum, instruct patient on how to produce specimen as distinct from saliva or nasopharyngeal discharge. Do not have patient rinse mouth with tap water which may contain environmental mycobacteria.
  • For induced sputum, use sterile hypertonic saline.  Avoid sputum contamination with nebulizer researvoir water. Indicate on request if specimen is induced sputum, as these watery specimens resemble saliva and risk rejection as inadequate.
 
Faeces  
≥ 1g in sterile, wax-free, disposable container. 
Collect specimen directly into container, or transfer from bedpan or from plastic wrap stretched over toilet bowl. Frozen specimen. Specimen that has been in contact with water in toilet.
Tissue biopsy sample  1g of tissue, if possible, in sterile container without fixative or preservative. Collect aseptically, and avoid indigenous microbiota. Select caeous portion if available. Do not wrap in gauze. Do not freeze. Specimen submitted in formalin. 
Transtracheal aspirate As much as possible in syringe with needle removed and Luer Lock cap in place.  Aspirate can be sent in sterile container. Do not submit specimens in endotracheal tubes; these are unsuitable for processing.  
Urine Cathetor or midstream urine as much as possible (minimum, 40mL) of first morning specimen. For suprapubic tap, as much specimen as possible with needle removed and Luer Lock cap in place. Asirate can be sent in sterile container. Collect first morning specimen on three consecutive days. PHOL will accept only one specimen/day. Organisms accumulate in bladder overnight, so first morning void provides best yield. Specimens collected at other times are dilute and are not optimal. 24 hour pooled specimens; urine from catheter bag; specimens of <40mL unless larger volume is not obtainable. Urine specimens should only be tested if renal TB is suspected, not used for as routine screen.
Wound material (See biopsy or aspirate) (See biopsy or aspirate)  Dry swabs in anaerobic transport medium.

4. Transport of Specimens

  • Transport specimen in accordance with the Transportation of Dangerous Goods Regulations.
  • Transport specimens to the laboratory in as short a time as is practical to avoid overgrowth by contaminating indigenous microbiota.
  • Do not refrigerate blood for mycobacterial culture, hold at room temperature.  Refrigerate all other specimens whenever possible if transport to the laboratory or specimen processing will be delayed more than one hour.
  • Submit specimens, sealed individually in biohazard bags, to the local Public Health Laboratory in blue transport packs provided.

5. Unacceptable Specimens

  • Dry swabs or swabs in anaerobic transport medium are not processed.
  • Swabs are not recommended for the isolation of mycobacteria, since they provide limited material.  They are acceptable only if a specimen cannot be collected by other means. Place swabs in aerobic Amies or Stuarts transport medium.  Negative results obtained from specimens submitted on swabs are not reliable.
  • Waxed containers may produce false-positive slide results.
  • 24 hours collections; they are likely to be diluted and contaminated.
  • Frozen specimens as it will decrease the yield.

Note: Specimens obtained for initial diagnosis after the initiation of antimicrobial therapy may produce false-negative results. Even a few days of antimicrobial therapy may kill or inhibit sufficient numbers of mycobacteria so as to leave laboratory confirmation of disease in doubt.

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Updated 8 April 2019