Biochemical Tests to identify Mycobacteria, NTM

Various biochemical tests can be performed for the identification of Mycobacteria and Non-tuberculous Mycobacteria . An overview of some of these biochemical tests is mentioned in this blog post.

Niacin accumulation test

All species of Mycobacterium produce niacin (nicotinic acid) and Mycobacterium tuberculosis accumulates the most. A positive niacin test provides preliminary evidence that an organism that exhibits a buff-colored, slow-growing rough colony may be M. tuberculosis.

Biochemical test for Mycobacteria and NTM

Nitrate Reduction test

Mycobacterium tuberculosis is a strongly nitrate-positive organism. This test is valuable for the identification of M. tuberculosis, M. kansasii, M. szulgai and M. fortuitum.

Rapid growers such as M. fortuitum can be tested within 2 weeks, but slow growers should be tested after 3-4 weeks of luxuriant growth.  Both chemical procedures and commercially available nitrate strips are available. Control strains must be used while performing and interpreting the result.

Catalase test

A semiquantitative catalase test is used for the identification of Mycobacteria. Catalase is an enzyme that splits hydrogen peroxide into water and oxygen and a positive catalase test is indicated by the formation of gas bubbles.  Most species of Mycobacteria, except for certain strains of M. tuberculosis complex (some isoniazid-resistant strains) and M. gastri, produce catalase enzyme.

Tween 80 hydrolysis test

Tween 80 hydrolysis test is used to separate the species of Photochromogens, scotochromogens, and nonchromogens. Nonpathogenic slow-growing scotochromogens and nonchromogens produce a lipase that is able to hydrolyze Tween 80 (the detergent polyoxyethylene sorbitan monooleate) into oleic acid and polyoxyethylated sorbitol, whereas pathogenic species do not.

Tellurite reduction test

The ability of mycobacterial species to reduce tellurite in 3 to 4 days is used to distinguish members of M. avium complex from most other non-chromogenic species. All rapid growers reduce tellurite in 3 days.

Arylsulfatase test

Arylsulfatase enzyme is present in most mycobacteria. The rate by which arylsulfatase enzyme breaks down phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts is used to differentiate certain strains of Mycobacteria. Three-day arylsulfatase test is used to identify potentially pathogenic rapid growers such as M. fortuitum and M. chelonae. Slow-growing M. marinum and M. szulgai are positive in the 14-day arylsulfatase test.

Urea Hydrolysis test

Many Mycobacterium species possess a urease enzyme that hydrolyzes urea to form carbon dioxide and ammonia. The ammonia released increases the pH of the medium turning the indicator pink.

References and further readings

  • Gurpreet S. Bhalla, Manbeer S. Sarao, Dinesh Kalra, Kuntal Bandyopadhyay, Arun Ravi John, Methods of phenotypic identification of non-tuberculous mycobacteria, Practical Laboratory Medicine, Volume 12,2018, e00107, ISSN 2352-5517, (

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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