Phenotypic testing of Mycobacteria
Encyclopedia
A variety of phenotypic
tests can be used to identify and distinguish mycobacteria strains and species from each other.
The most commonly used tests are described below:
Acetamide, use as solce C and N source
Media: KH2PO4 (0.5 g), MgSO4*7H20 (0.5 g), purified agar (20 g), distilled water (1000ml).
The medium is supplemented with acetamide
to a final concentration f 0.02M, the medium is adjusted to pH
7.0 and sterilized by autoclaving at 115 ºC for 30 minutes. After sloping the medium is inoculated with one loopful of the cultures and incubated. Growth is read after incubation for 2 weeks (rapid growers) or after incubation for 4 weeks (slow growers). (2)
Acid phosphatase
The acid phosphatase of some mycobacteria splits at pH 5.4 free phenolphthalein
from phenolphthalein sulphate. Prepare a suspension of the organism to be tested and inoculate 0.5 ml into a vial of 0.5 ml substrate. Incubate at 35C to 37C for 4 hours. Add 0.5 ml sodium carbonate. The development of a red colour is a positive test for acid phosphatase. (4)
Catalase, semiquantitative activity
Most mycobacteria produce the enzyme
catalase
, but they vary in the quantity produced. Also, some forms of catalase are inactivated by heating at 68C for 20 minutes, and others are stable. Organisms producing the enzyme catalase have the ability to decompose hydrogen peroxide
into water and free oxygen. The test differs from that used to detect catalase in other types of bacteria by using 30% hydrogen peroxide in a strong detergent solution (10% Tween 80). (2)
Citrate
Utilization as a sole carbon
source. (2)
Egg medium
Growth on Löwenstein-Jensen medium
L-Glutamate
Utilization as a sole carbon and nitrogen
source. (2)
Growth rate
The growth rate refers to the length of time required to form mature colonies that are visible without magnification on solid media. Mycobacteria that form colonies clearly visible to the naked eye within 7 days on subculture are termed rapid growers, while those requiring longer periods are termed slow growers. (3)
Iron uptake
The ability to take up iron
from an inorganic iron containing reagent
helps to differentiate some species of mycobacteria. (2)
Lebek medium
Growth characteristics on. Lebek is a semisolid medium which can be used to test the oxygen preferences of mycobacterial isoloates. Aerophilic growth is indicated by growth on the surface and above the surface on glass wall of the tube, whereas microaerophilic growth is indicated by growth below the surface. (1)
MacConkey agar without crystal violet
(2)
Niacin accumulation
Paper strip method. Niacin
is formed as a metabolic byproduct by all mycobacteria, but some species possess an enzyme that converts free niacin to niacin ribonucleotide
. M. tuberculosis as well as some other species lack this enzyme and accumulate niacin as a water soluble byproduct in the culture medium. (2)
Nitrate reduction
Mycobacteria containing nitroreductase
catalyze the reduction from nitrate
to nitrite
. The presence of nitrite in the test medium is detected by addition of sulfanilamide and n-naphthylethylendiamine. If nitrate is present, red diazonium dye is formed. (2)
Photoreactivity of mycobacteria
Some mycobacteria produce carotenoid
pigment
s without light. Others require photoactivation for pigment production. Photochromogens produce nonpigmented colonies when grown in the dark and pigmented colonies only after exposure to light and reincubation.
Scotochromogens produce deep yellow to orange colonies when grown in either the light or dark.
Nonphotochromogens are nonpigmented in the light and dark or have only a pale yellow, buff or tan pigment that does not intensify after light exposure. (3)
Picrate tolerance
Ability to grow on Sauton agar containing picric acid
(0.2% w/v) after 3 weeks. (2)
Pigmentation
Some mycobacteria produce carotenoid pigments without light. Others require photoactivation for pigment production. (see photoreactivity) (3)
Pyrazinamide sensitivity (PZA)
The deamidation
of pyrazinamide
to pyrazinoic acid
(which is assumed to be the active component of the drug PZA) in 4 days is a useful physiologic characteristic by which M. tuberculosis complex members can be distinguished. (2)
Sodium chloride tolerance
Growth on LJ containing 5% NaCl. (2)
Thiophene-2carboxylic acid hydrazide (TCH) sensitivity
The growth of M. bovis
and M. africanum subtype II is inhibited by thiophene-2carboxylic acid hydrazide, whereas the growth of M. tuberculosis and M. africanum subtype I is not inhibited. (2)
Tween 80 hydrolysis
This is a test for lipase. Tween 80 is the trade name for the detergent polyoxyethylene sorbitan monooleate. Certain mycobacteria possess a lipase that splits it into oleic acid
and polyoxyethylated sorbitol
. The test solution also contains phenol red
which is stabilised by the Tween 80; when Tween 80 is hydrolysed, the phenol red turns from yellow to pink. (2)
Urease (adaptation to mycobacteria)
With an inoculating loop, transfer several loopfuls of test colonies of mycobacteria to 0.5ml of urease
substrate. Mix to emulsify. Incubate at 35C for 3 days. Observe for colour change from amber-yellow to pink-red. (2)
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
tests can be used to identify and distinguish mycobacteria strains and species from each other.
The most commonly used tests are described below:
Acetamide, use as solce C and N source
Media: KH2PO4 (0.5 g), MgSO4*7H20 (0.5 g), purified agar (20 g), distilled water (1000ml).
The medium is supplemented with acetamide
Acetamide
Acetamide is an organic compound with the formula CH3CONH2. It is the simplest amide derived from acetic acid. It finds some use as a plasticizer and as an industrial solvent...
to a final concentration f 0.02M, the medium is adjusted to pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
7.0 and sterilized by autoclaving at 115 ºC for 30 minutes. After sloping the medium is inoculated with one loopful of the cultures and incubated. Growth is read after incubation for 2 weeks (rapid growers) or after incubation for 4 weeks (slow growers). (2)
Acid phosphatase
The acid phosphatase of some mycobacteria splits at pH 5.4 free phenolphthalein
Phenolphthalein
Phenolphthalein is a chemical compound with the formula C20H14O4 and is often written as "HIn" or "phph" in shorthand notation. Often used in titrations, it turns colorless in acidic solutions and pink in basic solutions...
from phenolphthalein sulphate. Prepare a suspension of the organism to be tested and inoculate 0.5 ml into a vial of 0.5 ml substrate. Incubate at 35C to 37C for 4 hours. Add 0.5 ml sodium carbonate. The development of a red colour is a positive test for acid phosphatase. (4)
Catalase, semiquantitative activity
Most mycobacteria produce the enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
catalase
Catalase
Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it catalyzes the decomposition of hydrogen peroxide to water and oxygen...
, but they vary in the quantity produced. Also, some forms of catalase are inactivated by heating at 68C for 20 minutes, and others are stable. Organisms producing the enzyme catalase have the ability to decompose hydrogen peroxide
Hydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...
into water and free oxygen. The test differs from that used to detect catalase in other types of bacteria by using 30% hydrogen peroxide in a strong detergent solution (10% Tween 80). (2)
Citrate
Utilization as a sole carbon
Carbon
Carbon is the chemical element with symbol C and atomic number 6. As a member of group 14 on the periodic table, it is nonmetallic and tetravalent—making four electrons available to form covalent chemical bonds...
source. (2)
Egg medium
Growth on Löwenstein-Jensen medium
Lowenstein-Jensen medium
The Lowenstein–Jensen medium, more commonly known as LJ medium, is a growth medium specially used for culture of Mycobacterium, notably Mycobacterium tuberculosis....
L-Glutamate
Utilization as a sole carbon and nitrogen
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
source. (2)
Growth rate
The growth rate refers to the length of time required to form mature colonies that are visible without magnification on solid media. Mycobacteria that form colonies clearly visible to the naked eye within 7 days on subculture are termed rapid growers, while those requiring longer periods are termed slow growers. (3)
Iron uptake
The ability to take up iron
Iron
Iron is a chemical element with the symbol Fe and atomic number 26. It is a metal in the first transition series. It is the most common element forming the planet Earth as a whole, forming much of Earth's outer and inner core. It is the fourth most common element in the Earth's crust...
from an inorganic iron containing reagent
Reagent
A reagent is a "substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs." Although the terms reactant and reagent are often used interchangeably, a reactant is less specifically a "substance that is consumed in the course of...
helps to differentiate some species of mycobacteria. (2)
Lebek medium
Growth characteristics on. Lebek is a semisolid medium which can be used to test the oxygen preferences of mycobacterial isoloates. Aerophilic growth is indicated by growth on the surface and above the surface on glass wall of the tube, whereas microaerophilic growth is indicated by growth below the surface. (1)
MacConkey agar without crystal violet
(2)
Niacin accumulation
Paper strip method. Niacin
Niacin
"Niacin" redirects here. For the neo-fusion band, see Niacin .Niacin is an organic compound with the formula and, depending on the definition used, one of the forty to eighty essential human nutrients.Niacin is one of five vitamins associated with a pandemic deficiency disease: niacin deficiency...
is formed as a metabolic byproduct by all mycobacteria, but some species possess an enzyme that converts free niacin to niacin ribonucleotide
Ribonucleotide
A ribonucleotide or ribotide is a nucleotide in which a purine or pyrimidine base is linked to a ribose molecule and exactly one phosphate group. In living organisms the most common bases for ribonucleotides are adenine , guanine , cytosine , or uracil ....
. M. tuberculosis as well as some other species lack this enzyme and accumulate niacin as a water soluble byproduct in the culture medium. (2)
Nitrate reduction
Mycobacteria containing nitroreductase
Nitroreductase
Nitroreductases are a family of evolutionarily related proteins involved in the reduction nitrogen containing compounds including those containing the nitro functional group...
catalyze the reduction from nitrate
Nitrate
The nitrate ion is a polyatomic ion with the molecular formula NO and a molecular mass of 62.0049 g/mol. It is the conjugate base of nitric acid, consisting of one central nitrogen atom surrounded by three identically-bonded oxygen atoms in a trigonal planar arrangement. The nitrate ion carries a...
to nitrite
Nitrite
The nitrite ion has the chemical formula NO2−. The anion is symmetric with equal N-O bond lengths and a O-N-O bond angle of ca. 120°. On protonation the unstable weak acid nitrous acid is produced. Nitrite can be oxidised or reduced, with product somewhat dependent on the oxidizing/reducing agent...
. The presence of nitrite in the test medium is detected by addition of sulfanilamide and n-naphthylethylendiamine. If nitrate is present, red diazonium dye is formed. (2)
Photoreactivity of mycobacteria
Some mycobacteria produce carotenoid
Carotenoid
Carotenoids are tetraterpenoid organic pigments that are naturally occurring in the chloroplasts and chromoplasts of plants and some other photosynthetic organisms like algae, some bacteria, and some types of fungus. Carotenoids can be synthesized fats and other basic organic metabolic building...
pigment
Pigment
A pigment is a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption. This physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which a material emits light.Many materials selectively absorb...
s without light. Others require photoactivation for pigment production. Photochromogens produce nonpigmented colonies when grown in the dark and pigmented colonies only after exposure to light and reincubation.
Scotochromogens produce deep yellow to orange colonies when grown in either the light or dark.
Nonphotochromogens are nonpigmented in the light and dark or have only a pale yellow, buff or tan pigment that does not intensify after light exposure. (3)
Picrate tolerance
Ability to grow on Sauton agar containing picric acid
Picric acid
Picric acid is the chemical compound formally called 2,4,6-trinitrophenol . This yellow crystalline solid is one of the most acidic phenols. Like other highly nitrated compounds such as TNT, picric acid is an explosive...
(0.2% w/v) after 3 weeks. (2)
Pigmentation
Some mycobacteria produce carotenoid pigments without light. Others require photoactivation for pigment production. (see photoreactivity) (3)
Pyrazinamide sensitivity (PZA)
The deamidation
Deamidation
Deamidation is a chemical reaction in which an amide functional group is removed from an organic compound. In biochemistry, the reaction is important in the degradation of proteins because it damages the amide-containing side chains of the amino acids asparagine and glutamine.In the biochemical...
of pyrazinamide
Pyrazinamide
Pyrazinamide is a drug used to treat tuberculosis. The drug is largely bacteriostatic, but can be bacteriocidal on actively replicating tuberculosis bacteria.-Abbreviations:...
to pyrazinoic acid
Pyrazinoic acid
Pyrazinoic acid is a pyrazinamide metabolite....
(which is assumed to be the active component of the drug PZA) in 4 days is a useful physiologic characteristic by which M. tuberculosis complex members can be distinguished. (2)
Sodium chloride tolerance
Growth on LJ containing 5% NaCl. (2)
Thiophene-2carboxylic acid hydrazide (TCH) sensitivity
The growth of M. bovis
Mycobacterium bovis
Mycobacterium bovis is a slow-growing , aerobic bacterium and the causative agent of tuberculosis in cattle...
and M. africanum subtype II is inhibited by thiophene-2carboxylic acid hydrazide, whereas the growth of M. tuberculosis and M. africanum subtype I is not inhibited. (2)
Tween 80 hydrolysis
This is a test for lipase. Tween 80 is the trade name for the detergent polyoxyethylene sorbitan monooleate. Certain mycobacteria possess a lipase that splits it into oleic acid
Oleic acid
Oleic acid is a monounsaturated omega-9 fatty acid found in various animal and vegetable fats. It has the formula CH37CH=CH7COOH. It is an odorless, colourless oil, although commercial samples may be yellowish. The trans isomer of oleic acid is called elaidic acid...
and polyoxyethylated sorbitol
Sorbitol
Sorbitol, also known as glucitol, Sorbogem® and Sorbo®, is a sugar alcohol that the human body metabolizes slowly. It can be obtained by reduction of glucose, changing the aldehyde group to a hydroxyl group. Sorbitol is found in apples, pears, peaches, and prunes...
. The test solution also contains phenol red
Phenol red
Phenol red is a pH indicator that is frequently used in cell biology laboratories.-Chemical structure and properties:...
which is stabilised by the Tween 80; when Tween 80 is hydrolysed, the phenol red turns from yellow to pink. (2)
Urease (adaptation to mycobacteria)
With an inoculating loop, transfer several loopfuls of test colonies of mycobacteria to 0.5ml of urease
Urease
Urease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The reaction occurs as follows:In 1926, James Sumner showed that urease is a protein. Urease is found in bacteria, yeast, and several higher plants. The structure of urease was first solved by P.A...
substrate. Mix to emulsify. Incubate at 35C for 3 days. Observe for colour change from amber-yellow to pink-red. (2)