Phenotypic testing of Mycobacteria

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, utilization as solce C and N sourceMedia: KH2PO4 (0.5g), MgSO4*7H20 (0.5g), purified agar (20g), 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 115C 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 phosphataseThe acid phosphatase of some mycobacteria splits at pH 5.4 free phenophlalein 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 phosphotase. (4)

Catalase, semiquantitative activityMost 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)

CitrateUtilization as a sole carbon source. (2)

Egg mediumGrowth on Löwenstein-Jensen medium

L-GlutamateUtilization as a sole carbon and nitrogen source. (2)

Growth rateThe 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 uptakeThe ability to take up iron from an inorganic iron containing reagent helps to differentiate some species of mycobacteria. (2)

Lebek mediumGrowth 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 reductionMycobacteria 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 mycobacteriaSome mycobacteria produce caotenoid pigments 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 toleranceAbility to grow on Sauton agar containing picric acid (0.2% w/v) after 3 weeks. (2) PigmentationSome 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 toleranceGrowth on LJ containing 5% NaCl. (2)

Thiophene-2carboxylic acid hydrazide (TCH) sensitivityThe 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 hydrolysisTween 80 is the trade name for the detergent polyoxyethylene sorbitan monooleate. Certain mycobacteria possess a lipase that splits Tween 80 into oleic acid and polyoxyethylated sorbitol, which modifies the optical characteristics of the test solution from a straw 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) Koneman, E. 1988. Diagnostic microbiology. J.B. Lippincott Company, Philadelphia, USA.]

References

(1.) Deutsches Institut für Normung. 1993. Medical microbiology: diagnosis of tuberculosis. Part 9: minimum requirements for the identification of tubercle bacilli (DIN 58943-9). Beuth Verlag, Berlin.

(2.) Koneman, E. 1988. Diagnostic microbiology. J.B. Lippincott Company, Philadelphia, USA.

(3.) Metchock, B.G, F.S. Nolte and R.J. Wallace. 1999. Mycobacterium. P. 399-427. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C Tenover and R.H. Yolken (ed.), Manual of Clinical Microbiology. ASM Press, Washington, D.C.

(4.) Tsukamura, M. 1966a. Adansonian classification of mycobacteria. J. Gen. Microbiol. 45:252-273.