Mycobacterium tuberculosis

Useful For

Diagnosis of respiratory and non-respiratory tuberculosis.


Testing Algorithm

Reporting Name Available separately Always performed
AFB microscopic NO YES
AFB culture NO YES
Mycobacterium multiplexed molecular assay NO YES
Drug susceptibility testing NO YES


*Mycobacterium multiplexed molecular assay performed on all non-fixed patient clinical specimens submitted to PHML for MTBC/Mycobacteria spp. testing. Refer to Mycobacterium multiplexed molecular assay for details



Method Name

Auramine fluorescent microscopy


Liquid and solid media culture (gold standard for diagnosis)


Drug susceptibility testing


Mycobacterium multiplexed molecular assay (Real-time polymerase chain reaction with fluorescent dye-labeled oligonucleotide probe detection).



Reporting Name

AFB Microscopy

Mycobacterium multiplexed molecular assay

TB Culture







General Specimen Requirements

  • All specimens should be collected in sterile, leak-proof, laboratory-approved containers.
  • Specimens should accompany a carefully completed requisition form providing the patient’s demographic data, the physician’s name, the date and time of collection, and the specimen type and site.
  • If possible, specimens collected for initial diagnosis should be obtained before the initiation of anti-TB therapy.
  • Once collected, specimens should be transported to the laboratory promptly.
  • Clinical specimens should be handled, processed and transported in an environment in which biosafety procedures are in place.


Note: Specimens obtained in >7 days will still be processed. However, specimen quality might be compromised, interpret results with caution.


Respiratory Tuberculosis


  • For specimens below, if processing within 1 hour is not possible, samples should be refrigerated at 4 °C (not frozen) and protected from light.



At least three sputum specimens of 5-10 mL each should be collected. The three sputum specimens (either spontaneous or induced) can be collected on the same day, at least 1 hour apart. (Same day procedure may help reduce patient drop-out and make faster decisions about TB infection control and discharge from respiratory isolation).



Bronchoscopy may be used when spontaneous sputum and induced sputum are unavailable, or all samples are smear-negative. Postbronchscopy sputum specimens are recommended to be collected from all adults with suspected pulmonary TB who undergo bronchoscopy.


Gastric aspirate

Primary indications are for children who cannot expectorate sputum or, for the same reason, elderly demented patients. Maximum volume 15 ml; neutralize with 100 mg of sodium carbonate within 1 hour of collection.


Refer to Canadian Tuberculosis Standard for detailed guidance.


Non-respiratory Tuberculosis

Specimen depends on the anatomic site of involvement. Tissue biopsy yields higher positive findings as compared to fluid aspiration. Both are superior to swabs (swabs will be rejected).



Tissue: 1 cm -10 cm. Submit fresh or in a small amount of sterile saline. Histopathologic examination requires the specimen to be placed in formalin (renders Mycobacteria unviable).


CSF (TB meningitis)

Serial sampling of CSF for AFB smear and culture may increase yield. The sensitivity may be increased by using the last CSF tube collected and obtaining a large sample volume (5 – 10 ml). CSF should not be refrigerated.



Transport Temperature

Specimen Room temperature Refrigerated Frozen
Respiratory NO YES NO
Non-respiratory (excl. CSF) NO YES NO


Reject Due To

Sputum/Bronch washes/Gastric aspirates < 3ml REJECT
Pooled specimens REJECT
Gastric aspirate without neutralization received at PHL >4 hours post collection REJECT

Tuberculosis (TB) is one of the top 10 causes of death worldwide and a leading killer of HIV-positive persons. In 2018, Mycobacterium tuberculosis caused approximately 1.5 million deaths and accounted for 10 million newly diagnosed cases globally. In Canada, in 2017 there were 1,796 cases of active tuberculosis reported. Two populations — foreign-born individuals and Indigenous Peoples — accounted for the majority of cases. 1,290 cases of active tuberculosis disease were reported among foreign-born individuals and 313 cases reported among Canadian-born Indigenous Peoples. Canadian-born, non-Indigenous people accounted for 125 cases.


M. tuberculosisis spread from person-to-person via respiratory transmission, and has the potential to become resistant if proper antimycobacterial treatment is not administered. Rapid and accurate detection of M tuberculosisin patient specimens is of clinical and public health importance.



AFB Microscopic


The threshold of detection of AFB in concentrated specimens using flourochrome stain is 5,000 – 10,000 bacteria/ml of sputum. The specificity of the AFB smear is high for mycobacteria (ALL nontuberculous mycobacteria will be AFB positive). Rarely organisms, such as Nocardia and Actinomycetes, can be weakly acid-fast. A positive AFB smear almost always indicates the presence of mycobacteria, but not necessarily M. tuberculosis.


Number of bacteria seen on microscopy and laboratory interpretation:


Flourochrome (250X magnification) Laboratory Report
0 in 30 fields Negative
1-2 per 30 fields Report exact number
1-9 per 10 field 1+
1-9 per field 2+
10 – 90 per field 3+




Refer to Mycobacterium multiplexed molecular assay for details



AFB culture


As few as 10 – 100 viable bacteria can be detected by culture.


Solid and liquid cultures are incubated for 8 weeks and 6 weeks respectively before being reported as NEGATIVE.

Procedure Days Turnaround time Specimen Retention
AFB Microscopic Mon – Sun 24 hr 8 weeks
TB PCR Mon – Sun 48 hr 8 weeks


Culture Mon – Sun 8 weeks 8 weeks



Methods Description


AFB Microscopic: Auramine fluorescent microscopy

Mycobacterium multiplexed molecular assay


1) BACTTEC MGIT Liquid Media

2) LJ Solid Media



Performing Laboratory Location


Public Health Microbiology Laboratory

100 Forest Road

St. John’s

NL A1A 3Z9





  1. Forbes et al. Practice Guidelines for Clinical Microbiology Laboratories: Mycobacteria, Clinical Microbiology Reviews, 2018.
  1. Canadian Tuberculosis Standards 7th Edition: 2014.

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