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A comparatively slow growth rate is characteristic of the mycobacteria, with generation time ranging from 14-20 hours.
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Colonies appear only in about two weeks and sometimes may be delayed upto 6-8 weeks. Optimum temperature is 37°C and pH is 6.4 –7.0.
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Mycobacterium tuberculosis is an obligate aerobe while Mycobacterium bovis is microaerophilic. Growth is stimulated by 5-10% CO2.
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Tubercle bacilli do not have exact growth requirements. But they are highly susceptible to even traces of toxic substances like fatty acids in culture media.
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The toxicity is neutralized by addition of serum, albumin or charcoal.
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Several media, both solid and liquid, are available. The egg based Lowenstein Jensen medium and Stone Brink's medium are most commonly used.
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Malachite green dye (0.025 g/100ml) is commonly used as the selective agent.
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Mycobacterium tuberculosis, Mycobacterium avium and many of the atypical mycobacteria require glycerol for growth. However glycerol is inhibitory to Mycobacterium bovis, while sodium pyruvate enhances its growth.
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On Lowenstein Jensen medium (i.e. glycerol containing media), Mycobacterium tuberculosis giving the characteristic rough, tough and buff colonies – is known as eugonic.
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The growth of Mycobacterium avium in this medium is also described as eugonic. Mycobacterium bovis has sparse, thin growth on glycerol containing media that is called dysgonic.
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Mycobacterium bovis however grows well on pyruvate containing media without glycerol (i.e. Stone brink's medium).
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Pigment formation is tested with young, well-developed colonies on Lowenstein Jensen medium.
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The cultures are exposed to a 100-watt, clear electric bulb, at a distance of 50 cm, for atleast an hour and then incubated again in darkness for a further 1-3 days.
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After this treatment the photochromogens will develop pigement.
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Many of the mycobacteria produce yellow/orange pigments while Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium are non-chromogenic.
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In liquid media, the growth begins at the bottom, creeps up the sides and forms a prominent surface pellicle (mould like pellicle) that may extend along the sides above the medium.
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Virulent stains tend to form long serpentine cords on liquid media, while avirulent strains grow in a more dispersed fashion.
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Supplementation of media with mycobactin (extracted from non-mycobactin dependant isolates of M.avium subsp. paratuberculosis) is required for M.avium subsp. paratuberculosis.
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