Mycobacteria are widespread both in the environment and in animals. The major human pathogens are M. tuberculosis and M. leprae, but awareness of the importance of other species is increasing with their recognition as pathogens in AIDS patients.


Aerobic rods with a Gram-positive cell wall structure, but stain with difficulty because of the long-chain fatty acids (mycolic acids) in the cell wall. Acid-fastness can be demonstrated by resistance to decolorization by mineral acid and alcohol (Ziehl-Neelsen stain). Mycobacteria grow more slowly than many other bacteria of medical importance, but the genus can be divided into: rapid growers (form visible colonies within seven days); slow growers (form visible colonies only after 14 days’ incubation).


Droplet spread aided by ability of organism to survive in the environment (M. tuberculosis, M. leprae). Milkborne spread of M. tuberculosis to humans from infected cattle has been important in the past. Social and environmental factors and genetic predisposition all have a role. Leprosy requires close and prolonged contact for spread.


M. tuberculosis causes tuberculosis in humans and animals. M. leprae is restricted to man and causes leprosy. Mycobacteria other than tuberculosis (MOTT) are associated with a range of conditions, usually in immunocompromised hosts. The avium-intracellulare complex has important association with AIDS patients.



Sample Collection:

  1. Sputum: Spot and the morning sample should be collected in clean, dry, wide necked & leak proof container before any mouthwash is used.
    • Send it to laboratory within 2 hours or kept at 4ºC until delivery is possible.
  2. Gastric wash: children
  3. Body fluids (Synovial, Pleural, Pericardial, Ascitic fluid)
  4. CSF
  5. Urine


Conventional Methods: 

  • Direct Smear Examination:
    • Z N staining: Acid – fast bacilli are seen
    • Fluorescent Staining


  • Culture:
    • Early morning 2 sputum samples
    • Decontamination & concentration


  • Media:
    • Egg-based
      • J. medium
    • Agar-based
      • Middlebrook
    • Incubation
      • 4-6 weeks


  • Biochemical Reactions:

Niacin test and Nitrate reduction test are two important tests for identification of M. tuberculosis


  • Tuberculin Test:

It is valuable diagnostic test in paediatric age group.


Newer Methods:

MGIT (Mycobacteria Growth Indicator Tube):

Culture tube contains Middle brook 7H9 broth and a fluorescent compound embedded in a silicon sensor. Growth is detected visually using an ultraviolet light. Oxygen diminishes the fluorescent output of the sensor; therefore, Oxygen consumption by organisms present in the medium is detected as an increase in fluorescence.

MODS (Microscopic Observation Drug Sensitivity):

It is liquid culture method based on microscopic detection of characteristic mycobacterium tuberculosis morphology. It utilizes the tuberculosis cording phenomenon for identification, which is easily viewed with an inverted microscope. It detects susceptible, mono resistant and multi drug resistant tuberculosis usually within a 5 to 10 days incubation period. It can be used to monitor patient on therapy, detects only living organisms.


LPA (Line Probe Assay):

It is based on nucleic acid amplification technology which allows for rapid detection of Mycobacterium tuberculosis complex along with rifampicin resistant alone or in combination with isoniazid resistant in direct sputum sample or in culture isolates.

Gene Expert:

It is a cartridge based automated new molecular diagnostic test that detect the presence of Mycobacterium tuberculosis as well as detect resistant to drug rifampicin. It can provide reliable results in about 2 hrs. It detects DNA sequence specific for Mycobacterium tuberculosis and drug resistant by PCR.


It is radiometric method which detects the growth of mycobacteria in about a week by using 14C labeled substrates. Culture media contains 14C-labelled palmitic acid. Mycobacteria metabolize the 14C-labelled substrates and release radioactively labeled 14CO2. The instrument measures 14CO2 and reports in terms of ‘growth index’. A growth index of ≥10 is considered as positive. This method can also differentiate between M. tuberculosis and M. bovis.


Sensitivity Testing: 

BACTEC can also be used for sensitivity testing of antituberculus drugs to identify drug resistance.

Luciferase reporter mycobacteriophage (chemiluminescence) has been used for susceptibility testing of M. tuberculosis.

Epsilometer test (E-Test) has also been applied for susceptibility testing of M. tuberculosis.



  1. Specimen:
    • Nasal mucosa, Skin lesion and ear lobules. Skin specimen are obtained by slit and scrape (edge of the lesion) method
  2. Acid Fast Staining:
    • Smear is stain by Ziehl Neelson Method using 5% H2SO4 as decolourising agent. The Smears are graded, based on the number of the Bacilli as follows:


1-10 Bacilli in 100 fields


1-10 Bacilli in 10 fields 2+
1-10 Bacilli per fields 3+
10-100 Bacilli per fields 4+
100-1000 Bacilli per fields 5+
More than 1000 Bacilli 6+


  1. Skin and Nerve Biopsy:
    • Useful in diagnosis and classification of leprosy lesion.
  2. Animal inoculation:
    • Foot pad of mouse and 9 Banded Armadillo are used.
  3. Lepromin Test:
    • This test used to assess the prognosis and response to treatment.
  4. Serological Test:
    • By detection of anti-phenolic glycolipid-1 antibodies.various serological test like latex agglutination, ELISA and M. leprae particle agglutination test.