Mycobacterium tuberculosis : Culture and Biochemical Reactions

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LJ medium for Mycobacterium tuberculosis

Mycobacterium tuberculosis is an obligate aerobe that grows extremely slowly in media. The optimal temperature is 37°C, and bacteria do not grow at temperatures below 25°C or above 40°C. The ideal pH range is 6.4–7.0. Mycobacterium bovis is microaerophilic during primary isolation but becomes aerobic during subculture.

Mycobacterium tuberculosis is a slow-growing bacillus with a generation time of 14–15 hours on average. Prolonged incubation is thus required to demonstrate bacterial growth. The colonies usually appear in less than two weeks, but they can take up to eight weeks to appear. Mycobacterium tuberculosis can thrive in a variety of enriched solid and liquid media.

Culture of Mycobacterium tuberculosis on solid media

Solid media include:

  • Egg-containing media (Lowenstein–Jensen [LJ] medium, Petragnani and Dorset egg medium)
  • Blood-containing media (Tarshis medium); serum-combining media (Loeffler’s serum slope)
  • Potato-based media (Pawlowsky medium). Mycobacterium tuberculosis grows dry, rough, raised, and irregular colonies with a wrinkled surface on these media.

LJ medium: LJ medium without starch is the most widely used of these media and is also recommended by the International Union Against Tuberculosis (IUAT). LJ medium is made up of coagulated whole eggs, asparagines, malachite green, mineral salt, and glycerol or sodium pyruvate. Other bacteria than mycobacterium are inhibited by malachite green. The addition of 0.75 percent glycerol promotes the growth of M. tuberculosis but inhibits the growth of M. bovis. M. tuberculosis produces yellowish or buff-colored colonies on LJ medium after 6–8 weeks of incubation. They are tenacious and difficult to emulsify.

LJ medium for Mycobacterium tuberculosis
LJ medium for Mycobacterium tuberculosis

Culture in liquid media

Soloac’s solutions, Dubos medium, and Middlebrook’s and Beck’s medium are examples of commonly used liquid media. M. tuberculosis grows in liquid media by first appearing at the bottom, then gripping up to the sides and producing a surface pellicle that may extend along the sides above the medium. Bacteria grow diffusely in Dubos medium containing Tween 80. They form a whitish wrinkled pellicle on the surface and a granular deposit at the bottom of glycerol broth. In liquid media, virulent strains typically form long serpentine cords. Avirulent strains, on the other hand, grow in a more dispersed manner in the medium. The liquid media are not used for routine bacilli culture.

They are typically used for

  • Preparing mycobacterial antigens for vaccines
  • Testing M. tuberculosis antibiotic sensitivity.

Biochemical reactions of Mycobacterium tuberculosis

M. tuberculosis exhibits the following biochemical characteristics:

  • The niacin test is used to distinguish niacin-positive human strains of M. tuberculosis from niacin-negative M. bovis strains. Niacin tests show that M. tuberculosis human strains are niacin positive. Niacin is also present in M. simiae and a few strains of M. chelonae. When grown on an egg-containing solid medium, they produce niacin as a metabolic byproduct.
  • M. tuberculosis is usually catalase positive. When they become resistant to isoniazid, they lose their catalase activity (INH). Catalase-negative strains of M. tuberculosis are not virulent to guinea pigs.
  • M. tuberculosis produces amidase. It generates amidase enzymes like nicotinamidase and pyrazinamidase, which split amide substrates.
  • M. tuberculosis is positive for nitrate reduction test. The nitrate reduction test for M. tuberculosis is positive. It produces the enzyme nitrate reductase, which converts nitrate to nitrite. M. bovis and M. avium lack the enzyme nitroreductase and thus fail the nitrate reduction test.
  • Both M. tuberculosis and M. bovis test positive for neutral red. They can bind neutral red in an alkaline buffer solution.
  • M. tuberculosis is arylsulfate test negative

Other properties of M. tuberculosis

Susceptibility to physical and chemical agents:

  • Mycobacteria are killed by heating to 60°C for 15–20 minutes. The nature of the clinical specimen in which the bacteria are present determines whether or not the bacteria are killed. Mycobacteria in sputum can live for 20–30 hours, but in dried sputum protected from sunlight, they can live for up to 6 months. They can survive in droplet nuclei for 8–10 days. Bacteria are killed when exposed to direct sunlight for 2 hours, but they can survive at room temperature for 6–8 months.
  • Tubercle bacilli are sensitive to formaldehyde and glutaraldehyde. They are destroyed in 5 minutes by iodine tincture and in 2–10 minutes by 80% ethanol. They are generally more resistant to chemical disinfectants than other nonsporeforming bacilli. They can withstand 5% phenol, 15% sulfuric acid, 3% nitric acid, 5% oxalic acid, and 4% sodium hydroxide.
  • M. tuberculosis is sensitive to pyrazinamide, whereas M. bovis and other mycobacteria are not. M. tuberculosis is resistant to the antibiotic thiophene-2-carboxylic acid hydrazide (TCH), whereas M. bovis is susceptible.

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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