Phenotypic testing of mycobacteria

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In microbiology, the phenotypic testing of mycobacteria uses a number of methods. The most-commonly used phenotypic tests to identify and distinguish Mycobacterium strains and species from each other are described below.

Tests

Acetamide as sole C and N sources

Media: KH2PO4 (0.5 g), MgSO>4*7H20 (0.5 g), purified agar (20 g), distilled water (1000 ml). The medium is supplemented with acetamide to a final concentration of 0.02M, adjusted to a pH of 7.0 and sterilized by autoclaving at 115°C for 30 minutes. After sloping, the medium is inoculated with one loop of the cultures and incubated. Growth is read after incubation for two weeks (rapid growers) or four weeks (slow growers). [1]

Arylsulfatase test

Arylsulfatase enzyme is present in most mycobacteria. The rate by which arylsulfatase enzyme breaks down phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts is used to differentiate certain strains of Mycobacteria. 3 day arylsulfatase test is used to identify potentially pathogenic rapid growers such as M. fortuitum and M. chelonae. Slow growing M. marinum and M. szulgai are positive in the 14-day arylsulfatase test. [2]

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 68°C for 20 minutes (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% polysorbate 80). [1]

Citrate

Sole carbon source [1]

Egg medium

Growth on Löwenstein–Jensen medium (LJ medium)

L-Glutamate

Sole carbon and nitrogen source [1]

Growth rate

The growth rate is the length of time required to form mature colonies visible without magnification on solid media. Mycobacteria forming colonies visible to the naked eye within seven days on subculture are known as 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 differentiate some species of mycobacteria. [1]

Lebek medium

Lebek is a semisolid medium used to test the oxygen preferences of mycobacterial isolates. Aerophilic growth is indicated by growth on (and above) the surface of the glass wall of the tube; microaerophilic growth is indicated by growth below the surface. [4]

MacConkey agar without crystal violet
[5]
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 (and some other species) lack this enzyme, and accumulate niacin as a water-soluble byproduct in the culture medium. [1]

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. [1]

Photoreactivity of mycobacteria;

Some mycobacteria produce carotenoid pigments without light; others require photoactivation for pigment production. Photochromogens produce non-pigmented colonies when grown in the dark, and pigmented colonies after exposure to light and re-incubation. Scotochromogens produce deep-yellow-to-orange colonies when grown in either light or darkness. Non-photochromogens are non-pigmented in light and darkness or have a pale-yellow, buff or tan pigment which does not intensify after light exposure. [3]

Picrate tolerance

Grows on Sauton agar containing picric acid (0.2% w/v) after three weeks [1]

Pigmentation

Some mycobacteria produce carotenoid pigments without light; others require photoactivation for pigment production (see photoreactivity, above). [3]

Pyrazinamide sensitivity (PZA)

The deamidation of pyrazinamide to pyrazinoic acid (assumed to be the active component of the drug pyrazinamide) in four days is a useful physiologic characteristic by which M. tuberculosis-complex members can be distinguished. [1]

Sodium chloride tolerance

Growth on LJ medium containing 5% NaCl [1]

Thiophene-2carboxylic acid hydrazide (TCH) sensitivity

The growth of M. bovis and M. africanum subtype II is inhibited by thiophene-2carboxylic acid hydrazide; growth of M. tuberculosis and M. africanum subtype I is uninhibited. [1]

Polysorbate 80 hydrolysis

A test for lipase using polysorbate 80 (polyoxyethylene sorbitan monooleate, a detergent). 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 polysorbate 80; when the latter 80 is hydrolysed, the phenol red changes from yellow to pink. [1]

Urease (adaptation to mycobacteria)

With an inoculation loop, several loopfuls of mycobacteria test colonies are transferred to 0.5 mL of urease substrate, mixed to emulsify and incubated at 35 °C for three days; a colour change (from amber-yellow to pink-red) is sought. [1]

Related Research Articles

<i>Mycobacterium</i> Genus of bacteria

Mycobacterium is a genus of Actinomycetota, given its own family, the Mycobacteriaceae. Over 190 species are recognized in this genus. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy in humans. The Greek prefix myco- means 'fungus', alluding to the way mycobacteria have been observed to grow in a mold-like fashion on the surface of cultures. It is acid-fast and cannot be stained by the Gram stain procedure.

<span class="mw-page-title-main">Pyrazinamide</span> Medication

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Mycobacterium branderi is a slowly growing, nonchromogenic Mycobacterium first isolated from patients in Finland. Etymology: of Brander, referring to Eljas Brander, the former head of the Tuberculosis Laboratory of the National Public Health Institute, Finland, who collected the strains.

Mycobacterium brisbanense is a member of the Mycobacterium fortuitum third biovariant complex. They are rapidly growing ubiquitous environmental organisms that normally inhabit soil, dust and water. These organisms frequently are human pathogens that cause a wide spectrum of clinically significant disease. It is important for practitioners to be aware of these organisms as possible etiological agents, as they are resistant to most first-line anti-tuberculous agents.

Mycobacterium brumae is a rapidly growing environmental mycobacterial species identified in 1993. Aside from one 2004 report of a catheter related bloodstream infection no other infections by this organism have been reported. It was first isolated from water, soil and one human sputum sample in Spain.

Mycobacterium canariasense is a rapidly growing, non-pigmented mycobacterium first isolated from blood samples obtained from 17 patients with febrile syndrome. Etymology: canariasense; referring to the Latin adjective of the Spanish islands where all strains were isolated.

Mycobacterium conspicuum is a species of the phylum Actinomycetota, belonging to the genus Mycobacterium.

<i>Mycobacterium cosmeticum</i> Species of bacterium

Mycobacterium cosmeticum is a rapidly growing mycobacterium that was first isolated from cosmetic patients and sites performing cosmetic procedures.

Mycobacterium elephantis, a bacterium of the family Mycobacteriaceae, was discovered and isolated from a deceased elephant near India and may be linked to respiratory dysfunction. Organisms in the genus Mycobacterium are known to be aerobic and non-motile. Organisms within Mycobacterium belong to either the rapid growing group or the slow growing group. M. elephantis is classified as a rapid grower and relates most closely to Mycobacterium confluentis and Mycobacterium phlei.

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<i>Mycobacterium kansasii</i> Species of bacterium

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<span class="mw-page-title-main">Löwenstein–Jensen medium</span> Growth medium used to culture Mycobacterium species

Löwenstein–Jensen medium, more commonly known as LJ medium, is a growth medium specially used for culture of Mycobacterium species, notably Mycobacterium tuberculosis.

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<span class="mw-page-title-main">Niacin test</span> Bacterial lab test

The niacin test has been widely used since the 1960s to identify mycobacteria at the species level in the clinical laboratory. The niacin test detects niacin in aqueous extracts of a culture. M. tuberculosis strains that test negative for the niacin test are very rare. Redox reactions happening in Mycobacterium species produce niacin as a part of energy metabolism. Even though all mycobacteria produce niacin, M. tuberculosis accumulates an excess of niacin because of its inability to process niacin, excreting the excess niacin into the culture media, thus allowing it to be detected using the niacin test. The niacin test is typically only conducted on slow-growing, granular, tan colored colonies, as these are the morphology characteristics of M. tuberculosis on an agar plate. Because of its affordability compared to expensive identification methods like pyrosequencing or MALDI-TOF MS that require expensive machines and reagents.

Diagnostic microbiology is the study of microbial identification. Since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. Using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies. New studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining.

References

  1. 1 2 3 4 5 6 7 8 9 10 11 12 13 Koneman, E. (1988). Diagnostic Microbiology. Philadelphia: J.B. Lippincott.
  2. ACHARYA, TANKESHWAR. "Key biochemical methods used to distinguish Mycobacterial group" . Retrieved 21 November 2014.
  3. 1 2 3 Metchock, B.G; Nolte, F.S.; Wallace, R.J. (1999). "Mycobacterium". In Murray, P.R.; Baron, E.J.; Pfaller, M.A.; Tenover, F.C.; Yolken, R.H. (eds.). Manual of Clinical Microbiology. Washington, D.C.: ASM Press. pp. 399–427.
  4. Deutsches Institut für Normung (1993). "Part 9: minimum requirements for the identification of tubercle bacilli". Medical Microbiology: Diagnosis of Tuberculosis. Berlin: Beuth Verlag (DIN 58943-9).
  5. M. Tsukamura. "Adansonian classification of mycobacteria". Journal of General Microbiology. 45: 252–273. doi: 10.1099/00221287-45-2-253 .