Mycobacterium – Culture
Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.
Background
This page provides culture testing information for acid-fast bacilli of the Mycobacterium species including the Mycobacterium tuberculosis complex (MTBC) and non-tuberculosis mycobacteria at Public Health Ontario (PHO) for the following primary specimen types: blood or bone marrow, corneal scrapings, cerebrospinal fluid (CSF), urine, body fluids, bone or tissue, faeces, gastric lavage, respiratory, and superficial abscess, lesion, wound or aspirate. Included also in this page is information about referred Mycobacterium cultures for identification.
For information regarding other testing options, refer to the following PHO webpages:
- MTBC:
- Non-tuberculosis mycobacteria:
Updates
- The addition of bone marrow under specimen type, along with related information under submission and collection notes #3.
- The addition of information about neutralization of gastric lavage based on the collection kit and updating volume requirement under submission collection notes #8
Testing Indications
Acceptance/Rejection Criteria
- Any specimens received frozen, in formalin, dry swabs or swabs in anaerobic transport medium, and non-sterile containers will not be tested.
- Pooled respiratory specimens, saliva, 24-hour pooled collections, and specimens submitted in endotracheal tubes will not be tested.
- Blood collected in EDTA, coagulated blood, blood in blood culture medium other than a Myco/F Lytic bottle, serum, or plasma will not be tested.
- Faeces specimens submitted with fixative, frozen, or that have been in contact with toilet water will not be tested.
- Gastric lavage specimens that have not been neutralized and arrive at the PHO’s laboratory sites greater than 6 hours after collection will not be tested.
- Urine specimens in fixative, 24-hour pooled collections, urine from a catheter bag, specimens <40.0 ml (unless a larger volume is not obtainable) will not be tested.
- Contaminated or non-viable referred cultures will not be tested.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
Mycobacterium – Culture |
Blood or bone marrow |
See note 3 |
Blood culture tube (see note 3) |
|
Mycobacterium – Culture |
Corneal scrapings (see note 4) |
N/A |
MGIT tube, 2 LJ slants and a slide – provided as needed prior to collection by PHO |
|
Mycobacterium – Culture |
CSF (see note 5) |
0.5 ml |
||
Mycobacterium – Culture |
Fluids (see note 6) |
5.0 ml |
||
Mycobacterium – Culture |
Bone or tissue (see note 7) |
1.0-2.0 grams |
||
Mycobacterium – Culture |
Faeces |
1.0 grams |
||
Mycobacterium – Culture |
Gastric lavage (see note 8) |
≥5.0 ml |
||
Mycobacterium – Culture |
Respiratory including sputum, tracheal aspirate, bronchoalveolar lavage, bronchial washing, and esophageal & bronchial brushing (see notes 9 to 12) |
Sputum: 5.0-10.0 ml |
||
Mycobacterium – Culture |
Superficial abscess, wound, or aspirate fluids & biopsy or skin lesions (see note 13 to 15) |
Abscess, wound, aspirate fluid: ≥5.0 ml |
||
Mycobacterium – Culture |
Urine (see note 16 and 17) |
40.0 ml |
||
Mycobacterium – Culture |
Referred cultures |
N/A |
Appropriate solid or liquid media to support growth of acid-fast bacilli |
Submission and Collection Notes
Complete all fields of the requisition form, including:
- Test(s) requests and testing indications/criteria for testing
- Patient Setting & Signs/Symptoms
- Specimen Information
- Patient Information
- Submitter/Health Care Provider (HCP) Information
Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens and Additional Mycobacterium Collection Information. Failure to provide this information may result in rejection or testing delay.
Collect bone marrow or whole blood using aseptic technique in one of the following blood culture tubes as appropriate:
- As much as possible (bone marrow) or 10.0 ml (whole blood) in SPS (yellow top tube) or
- As much as possible (bone marrow) or 10.0 ml (whole blood) in heparin (green top tube) or
- BACTEC™ Myco/F Lytic medium – (minimum volume 5.0 ml whole blood) or
- Sterile container (bone marrow)
Corneal scrapings should be obtained by the physician and directly inoculated into the MGIT tube and 2 Lowenstein Jensen slants. A smear should be made and placed in a slide holder for safe shipping to PHO’s laboratory site. Contact PHO’s laboratory Customer Service before procedure to obtain proper media and slides for specimen collection.
CSF volume equal to 0.5 ml will have culture performed only (no smear). Submit maximum volume attainable for culture and smear.
Small volumes of fluid may be submitted in a syringe with needle removed and Luer Lock cap in place.
Collect tissue using aseptic technique, select the caseous portion if available. Do not wrap in gauze or freeze.
- Multiple tissues submitted from the same surgical site should be limited to 3 specimens. If more than 3 specimens are required for testing, contact PHO’s laboratory Customer Service.
- If submitting tissue from lymph nodes, place a small amount of sterile saline in the specimen container(s).
Collect ≥5mL of fasting early-morning gastric lavage specimen soon after patient awakens in order to obtain sputum swallowed during sleep on 3 consecutive days. Use sterile water. If using PHO’s Gastric Lavage Kit Order #: 390043 for collection, follow kit instructions for neutralization. If a different sterile container is used, adjust the gastric lavage specimen to neutral pH with 100 mg of sodium carbonate immediately following collection.
Collect 3 sputum specimens (either spontaneous or induced) on the same day, a minimum of 1 hour apart or optimally, from an early morning collection from a deep productive cough on 3 consecutive days.
- For follow-up patients on therapy, submit 3 specimens after 2 months and again after completion of therapy.
- Do not pool the 3 specimens or have the patient rinse mouth with tap water before producing sputum.
If collecting tracheal aspirates, collect as much material as possible in a syringe with Luer lock cap, needle removed, or in a sterile container.
If collecting bronchial washings, avoid contaminating the bronchoscope with tap water as saprophytic mycobacteria may produce false-positive culture or smear results.
Esophageal and bronchial brushings may be submitted in a small volume of sterile saline.
If collecting superficial specimens, indicate source/anatomical site on the General Test Requisition Form so that appropriate culture media is used to support the growth of mycobacteria species that prefer to grow at lower temperatures.
- Indicate if infection was acquired in Africa, Australia, Mexico, South America, Indonesia, New Guinea, or Malaysia on the General Test Requisition Form as Mycobacterium ulcerans requires prolonged incubation for primary isolation.
- Swabs in transport medium (Amies or Stuarts) are acceptable only if specimen cannot be collected by other means.
If collecting abscess contents/aspirates/skin lesions specimens, do not use gauze. Small volumes may be submitted in a syringe with needle removed and Luer Lock cap in place. Swabs in transport medium (Amies or Stuarts) are acceptable only if specimen cannot be collected by other means.
If collecting cutaneous ulcer specimens, collect the biopsy specimen from periphery of lesion, or aspirate material from under margin of lesion. Swabs in transport medium (Amies or Stuarts) are acceptable only if specimen cannot be collected by other means.
Urine specimens may be collected from the following:
- Catheter or midstream urine of the first morning specimen. Organisms accumulate in the bladder overnight, therefore first morning void provides the best yield. Only one urine sample per day will be processed for Mycobacterium – Culture.
- Suprapubic tap can be collected in the Tuberculosis Kit or in a syringe with needle removed and Luer Lock cap in place.
Indicate on the General Test Requisition Form if the patient has been treated with bladder instillation of M. tuberculosis variantbovis BCG.
Limitations
Specimens obtained for initial diagnosis after the initiation of antimicrobial therapy may produce false-negative results.
Saprophytic mycobacteria in tap water may produce false-positive smear and/or culture results.
Blood may contain antimicrobials which can delay or inhibit the growth of mycobacteria.
Swabs are not recommended for the isolation of mycobacteria since they provide limited material and the bacteria adsorb onto the swab material which may contain toxic substances. Negative results obtained from specimens submitted on swabs are not reliable.
Storage and Transport
Blood and CSF specimens should be stored and transported at room temperature following collection.
Other specimens should be stored at 2-8°C if shipment to PHO’s laboratory site is delayed more than one hour.
Before packaging, place specimen container(s) in a biohazard bag and insert the completed requisition in the pocket on the outside of the sealed biohazard bag (requisitions should not be placed inside the biohazard bag with the specimen). Do not place containers of specimens collected from different patients into one biohazard bag.
Continue to incubate referred cultures if shipment to PHO’s laboratory site is delayed more than one hour.
All clinical specimens should be shipped to PHO’s laboratory site as soon as possible and in accordance to the Transportation of Dangerous Good Act. Delayed shipment of specimens may promote overgrowth by contaminating indigenous microbiota.
Test Frequency and Turnaround Time (TAT)
Mycobacterium cultures of primary specimens are performed daily Monday to Saturday at Public Health Ontario’s laboratory - Toronto, London, and Ottawa sites.
Speciation of referred cultures is performed daily Monday to Friday at Public Health Ontario’s laboratory - Toronto.
Turnaround time (TAT) for smear results from the concentrated specimen is up to 2 calendar days from receipt at PHO’s laboratory sites.
TAT for negative cultures is up to 49 calendar days from receipt at PHO’s laboratory sites.
TAT for negative blood cultures is up to 42 calendar days from receipt at PHO’s laboratory-Toronto.
TAT for a positive culture is reported within 24 hours of growth. Depending on the species and treatment of the patient, a culture may grow within 1-2 weeks or take as long as 6-7 weeks. TAT for speciation of mycobacteria cultures is up to 7 days from a positive culture or receipt at PHO’s laboratory sites.
All new isolates of M. tuberculosis complex are phoned to the submitter within 24 hours of identification.
STAT and Critical Specimens Testing
STAT testing is not available. For URGENT AFB smear requests, contact PHO’s Laboratory Customer Service prior to shipment of specimen to PHO’s laboratory sites to request approval.
If URGENT testing is approved, mark URGENT on the outside of the package as well as on the requisition. A phone number and contact person must be provided for phoning the result.
URGENT smear specimens that are received in the laboratory by 1:30 p.m. will have a smear performed and result phoned the same day. Specimens that arrive after 1:30 p.m. will have the smear result phoned the following morning.
URGENT testing is only available Monday to Saturday.
A smear, prepared from the concentrate, is stained using the Auramine-Rhodamine (AR) fluorochrome stain and examined through fluorescence microscopy. A negative AFB smear does not rule out TB, the culture may still be positive upon incubation. Smears are not performed for blood specimens.
Specimens are set up for mycobacterial culture on solid and liquid media and incubated for up to 7 weeks. Whole blood specimens are inoculated into BACTEC™ Myco/F Lytic culture medium and tested using the BACTEC™ FX Blood Culture System (Becton Dickinson).
Organism identification of positive cultures and phenotypic susceptibility testing for MTBC are performed at PHO’s laboratory- Toronto. Mycobacteria are identified by MALDI-TOF MS, GenoType Mycobacterium line probe tests (HAIN Lifesciences), Bioline™ TB Ag MPT64 linear flow test (Abbott), or by genetic sequence analysis.
Drug susceptibility testing on non-tuberculous mycobacteria (NTM) must be requested after culture identification by contacting PHO’s laboratory Customer Service for approval. Requests must be made within 3 months of organism identification. (See Mycobacterium – NTM-Susceptibility).
Interpretation
|
Test |
Result |
Comments |
|---|---|---|
|
AFB Smear (Fluor. stain of concentrate) |
No Acid Fast Bacilli seen Few, 1+, 2+, 3+ or 4+ |
AFB Quantitation (x200) Few 3-9/smear, 1+ 1-9/10 fields, 2+ 1-9/field, 3+ 10-90/field, 4+ >90/field |
|
Mycobacterial Culture |
[Mycobacterial genus and species name] and method of identification |
Mycobacterial culture results are only final when all culture media has been completed |
Reporting
Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.
Specimens positive for acid-fast bacilli by smear and for the M. tuberculosis complex from cultures are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.
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