Phenotypic identification of mycobacteria is based on a set of characteristics, such as resistance to certain growth inhibitors, biochemical reactions, colony morphology, the ammonium ferric citrate test, and the urease, catalase and Tween hydrolysis tests. Final identification of a species is determined by the combination of results from various tests.
The following phenotypic assays are standard available at BCCM/ITM
- Temperature inhibition: This test examines whether bacteria can grow in 37°C as compared to 30°C. With the exception of Mycobacterium ulcerans, for instance, most non-pathogenic mycobacteria that are isolated from the environment are not resistant to these higher temperatures.
- Pigment formation: This test examines whether bacteria are capable of forming pigment with or without prior exposure to sunlight. For this test, it is important to use well-grown, fresh and well-ventilated cultures. The kind of solid medium itself is of little importance.
- Colony morphology: Through the growth on an enriched agar plate, both the colony morphology and the purity of the culture are examined. This also allows for a simple colony subculture to be created in the event of partial contamination.
- MPT64 antigen test: This rapid, lateral flow assay differentiates species from the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria.
The below presented phenotypic assays can be performed upon specific request BCCM/ITM
- Inhibitor series: Here, the extent to which the growth of mycobacteria is inhibited by the presence of certain products (inhibitors) is investigated. This growth is compared to growth in a tube with 1/100 bacterial dilution, i.e. 1% of maximum potential growth.
- Urease test: This test examines whether bacteria have an active urease-enzyme; in other words, whether they can break down urea in this medium. Through the breakdown of urea, ammonium forms, which, in turn, raises the pH and causes the neutral red indicator to colour the medium pink.
- Tween hydrolysis test: This test examines whether bacteria can breakdown the Tween present in the medium. Through the breakdown of Tween, pH rises, which in turn makes the neutral red indicator change the medium from orange to red.
- Catalase test: This, actually, refers to the semi-quantitative catalase test. This test examines whether the bacteria possess the catalase enzyme that can convert hydrogen peroxide into water and oxygen (2H202 → 2H2O + O2). The released oxygen generates the foam in the mixture containing detergent (Tween). The amount of foam produced is measured.
- Niacin production test: This test examines whether bacteria produce niacin in the medium. Nicotinic acid (niacin) plays a vital role in oxide-reduction reactions that occur during the metabolic processes of mycobacteria. Although all mycobacteria produce niacin, it has been shown that, through a blocked metabolic reaction, M. tuberculosis exhibits by far the largest accumulation of niacin, making it a commonly used identification test. The production of media containing egg generates the best niacin production and is recommended when performing this test. The culture must also be at least three weeks old and contain at least 50 colonies. To optimally free the produced niacin from the medium, it is advisable to remove the pre-grown colonies beforehand, especially in cases of fully-grown cultures. Ventilating the culture beforehand is also essential for the success of the test.
- Nitratase Test: This test examines whether bacteria can reduce nitrate. The culture to be tested should be fresh and contain at least 50 colonies. This is achieved by placing the bacterial suspension in contact with a nitrate solution for a minimum of 2 hours, after which the nitrate content is measured by adding sulphanilamide and α-napthylamine.
- Phosphatase test: This test examines whether bacteria can reduce phosphate. The culture to be tested should be fresh and contain at least 50 colonies. This is achieved by placing the bacterial suspension in contact with a di-phosphate solution for at least 2 hours, after which the phosphate content is measured by adding sodium carbonate.
- Ammonium ferric citrate test: This test examines both the growth rate and the capacity to absorb iron. A very small amount of bacteria is inoculated onto a peptone agar medium. Only fast-growing mycobacteria are able to grow on the poor peptone agar medium, and only a portion of them is able to absorb the iron into their cells. Direct contact of the inoculum with the ammonium ferric citrate solution must be avoided.
- Arylsulfatase: This test looks at the ability of the bacteria to produce sufficient arylsulfatase. The enzyme arylsulfate will convert phenolphthalein disulfate to free phenolphthalein, which will give the medium a pink to red color. The test can be read after 3 days (for rapid and slow growers) and after 14 days (slow growers).
Requirements for the analyte: Viable pure mycobacterial culture isolates on solid or in liquid medium.
Expected turn around time: Phenotypic identification may take up to 3 months after arrival, depending on the species (rapid or slow growing) and tests requested.
References: Rigouts & Cogneau, 2021 eBook ISBN9781003099277; Bhalla et al., 2018 doi: 10.1016/j.plabm.2018.e00107;