B4.3 BIOCHEMICAL AND INHIBITION TEST FOR MYCOBACTERIUM Flashcards
BIOCHEMICAL TESTS used for Mycobacterium
a. Niacin Accumulation
b. Nitrate Reduction
c. Catalase Test
d. Tween 80 Hydrolysis
e. Iron Uptake
f. Arylsulfatase
g. Pyrazinamidase
h. Tellurite Reduction
i. Urease
What is the result of Niacin Accumulation for M. tuberculosis?
Positive result: Yellow color
Reagents of Niacin Accumulation for M. tuberculosis?
Cyanogen Bromide and Aniline
Niacin Accumulation Performed on
L-J medium, cultures at least 3 weeks old and with at least 50 colonies
Most commonly used for M. tuberculosis
Niacin Accumulation
Nitrate Reduction test principles
Nitroreductase catalyzes the reduction of nitrate to nitrite
Nitrate Reduction test Reagents
Hydrochloric acid,
Sulfanilamide,
N-(1-naphthyl)enediamine dihydrochloride
Nitrate Reduction test Indicator
Zinc
Nitrate Reduction test Positive result
Red color
Nitrate Reduction test Positive organisms
M. kansasii,
M. szulgai,
M. fortuitum, and
M. tuberculosis
Measurement of the height of the oxygen bubble column formed by untreated enzyme
Semiquantitative Catalase Test
What are the results for the Semiquantitative Catalase Test
<45 mm of bubbles = Negative
45 mm of bubbles = Positive
What are the conditions for the Semiquantitative Catalase Test?
Addition of Tween 80 and hydrogen peroxide
Tests the ability of catalase enzyme to remain active after heating at 68°C for 20 minutes
Heat-stable Catalase Test
Heat-stable Catalase Test Positive result spp present
M. tuberculosis,
M. bovis,
M. gastri,
M. haemophilum,
M. marinum remain active
Tween 80 Hydrolysis principle
Lipase hydrolyzes Tween 80 into oleic acid and polyoxyethylated sorbitol
pH indicator used in Tween 80 Hydrolysis
Neutral Red bound to Tween 80
Tween 80 Hydrolysis Positive result
Pink color
Tween 80 Hydrolysis Positive organisms
M. asiaticum,
M. gastri,
M. marinum,
M. malmoense,
M. scrofulaceum,
M. flavescens,
M. terrae;
M. kansasii
Iron Uptake test principle
Converts ferric ammonium citrate to an iron oxide
Iron Uptake test Positive result
Rusty brown colonies
What does the Iron Uptake test distinguish
M. chelonae (-) from other rapid growers (+)
What is the biochemical reaction for Arylsulfatase?
Breaks down phenolphthalein disulfate into phenolphthalein
Arylsulfatase Positive result
Pink color
Arylsulfatase Reagents
Tripotassium phenolphthalein sulfate and sodium bicarbonate
Arylsulfatase Positive organisms
M. fortuitum complex,
M. chelonae,
M. xenopi,
M. triviale (3 days);
M. marinum and
M. szulgai (14 days)
Pyrazinamidase activity principle
Hydrolyzes pyrazinamide to pyrazinoic acid and ammonia
Pyrazinamidase activity Positive result
Red pigment formed with ferrous ammonium sulfate
Pyrazinamidase activity Distinguishes
M. marinum from M. kansasii and
M. bovis from M. tuberculosis
What is the biochemical result in the Tellurite Reduction test?
> reduction of colorless POTASSIUM TELLURITE to BLACK METALLIC TELLURIUM in 3 to 4 days
Tellurite Reduction test Positive result
Black color within 3-4 days
Tellurite Reduction test Distinguishes
MAC from other nonchromogenic species
What species are able to reduce tellurite within 3 days
All rapid growers
What is the Urease test used to differentiate?
Distinguishes M. scrofulaceum (+) from M. gordonae (-)
Urease test Positive result
Pink to red color
->differentiates MTB from the scotochromogens and MAC
Nitrate Reduction
Nitrate Reduction operating principle
> incubated in 2 ml of sodium nitrate at 37? C for 2 hours
> differentiates species of photochromogens,
nonchromogens, and scotochromogens
Tween 80 Hydrolysis
What are the inhibitory tests used for the identification of mycobacteria?
NAP (p-nitroacetylamino-Bhydroxypropiophenone)
Thiophene-2-Carboxylic Acid Hydrazide (TCH)
Sodium Chloride Tolerance
Growth on MacConkey Agar
is a precursor in the synthesis of chloramphenicol
NAP (p-nitroacetylamino-Bhydroxypropiophenone)
NAP (p-nitroacetylamino-Bhydroxypropiophenone) test Selectively inhibits
M. tuberculosis complex
NAP reagent
5 mg of NAP in BACTEC-NAP vial
NAP Specimen
Colonies from agar or broth in BACTEC medium
NAP Positive result
20% increase in growth index (indicates M. tuberculosis complex)
What does Thiophene-2-Carboxylic Acid Hydrazide (TCH) test differentiate?
Distinguishes M. bovis (Sensitive) from M. tuberculosis (Resistant)
What does the Sodium Chloride Tolerance test inhibit
Inhibits M. flavescens, M. triviale, and most rapidly growing Mycobacterium species
Sodium Chloride Tolerance test component
5% NaCl in egg-based media
What is the result of Growth on MacConkey Agar for M. fortuitum-chelonae complex?
Positive result: Growth in 5 days
Negative result: Group IV rapid growers do not grow on MacConkey agar
