Biochemical Test of Mycobacterium tuberculosis

Mycobacterium tuberculosis is pathogenic bacterium causes human tuberculosis (TB) .It is a distinct biochemical bacillus that reacts quickly to acid. A battery of tests is used to distinguish Mycobacterium tuberculosis from other mycobacteria and germs in order to identify it. The following is a summary of the essential biochemical tests and traits used to diagnose Mycobacterium tuberculosis:

Mycobacterium tuberculosis

Acid-Fast Stain (Ziehl-Neelsen Stain)

• Acid-fast positive as a result.
• Even after decolorization with acid-alcohol, Mycobacterium tuberculosis preserves the primary stain (carbol fuchsin), showing as red rods against a blue background. Its cell wall’s high mycolic acid concentration is the cause of this. This test is a quick and affordable way to screen for tuberculosis. However, it is not exclusive to M. tuberculosis and may also test positive for other bacteria that ferment quickly.

Culture Characteristics

• Middlebrook 7H10/7H11 agar or Löwenstein-Jensen (LJ) medium is the medium.
• Colony Morphology: Rough, buff-colored colonies with a possible appearance time of two to six weeks.

Niacin Accumulation Test

• Concept: Niacin, or nicotinic acid, is produced by M. tuberculosis and builds up in the culture medium.
• Method: Use a colorimetric test to determine whether niacin is present in the medium after the bacteria have grown on LJ medium.
• Result: Positive (niacin is present as indicated by the yellow hue).

Nitrate Reduction Test

• Concept: Nitrate can be reduced to nitrite by M. tuberculosis.
• Method: Introduce the bacteria into the nitrate broth, let it sit for a while, then add the nitrate reagents and watch for a color shift.
• Positive outcome (red hue after reagent addition denotes nitrate reduction).

Catalase Test

• Concept: Hydrogen peroxide is broken down by catalase into oxygen and water.
• Procedure: Treat a bacterial culture using hydrogen peroxide.
• Positive outcome (catalase enzyme is present as bubbles). Observe that at 68°C, M. tuberculosis exhibits catalase positivity.

Heat-Stable Catalase Test

• Principle: After 20 minutes at 68°C, some strains of Mycobacterium tuberculosis stop producing catalase.
• Method: Preheat the culture and measure the amount of catalase present.
• Result: Heating frequently yields unfavorable results for M. tuberculosis.

Arylsulfatase Test

• Concept: Phenolphthalein disulfate is hydrolyzed by the enzyme arylsulfatase to produce free phenolphthalein.
• Process: Check for the presence of phenolphthalein after incubating the bacteria in arylsulfatase broth.
• The outcome for M. tuberculosis is usually negative (no color change).

Pyrazinamide Test

• Concept: Pyrazamidase, produced by Mycobacterium TB, hydrolyzes pyrazinamide to create pyrazinoic acid.
• Method: Test for pyrazinoic acid after incubating bacteria with pyrazinamide substrate.
• Result: Positive; pyrazinoic acid is present as shown by the reddish-brown color.

Urease Test

• Concept: Urea is hydrolyzed by the urease enzyme to produce carbon dioxide and ammonia.
• Method: Inoculate the bacteria into agar or urea broth, then watch to see if the color changes.
• Outcome: Negative (no color shift suggests no urease action)

Molecular Methods

• Through the use of PCR (Polymerase Chain Reaction), certain M. tuberculosis DNA sequences can be amplified and identified.
• Gene probes: Hybridization methods to pinpoint particular M. tuberculosis genetic markers.

Summary

• Positive tests include Pyrazamidase, catalase, nitrate reduction, acid-fast staining, and niacin buildup.
• Heat-stable catalase (post-heating), urease, and arylsulfatase are the negative tests.

To accurately identify and diagnose M. tuberculosis infections, biochemical testing, genetic techniques, and clinical correlation are essential.

Important Notes

• The particular set of tests chosen for a diagnosis varies according to the patient’s characteristics, the clinical setting, and the resources that are available.
• The clinical setting and other pertinent variables must be carefully taken into account when interpreting the results.
• It is significant to remember that these are only a few of the most often used biochemical tests for Mycobacterium tuberculosis identification. Depending on the particular requirements of the case, other tests might be employed.

Frequently Asked Questions(FAQ)

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• pyrazinamide substrate.
• niacin