Culture: Several types of media are used for the cultivation of mycobacterium.

1.

Solid media: Two types of solid culture media are available for the isolation of mycobacterium. a) Egg-based media: This is solid culture media used for primary isolation of mycobacterium. It has coagulated egg as a base.

American Trudeau Society (ATS) Medium: ATS Medium is an egg-based medium containing a small amount of Malachite Green. Due to the low concentration of Malachite Green, this formulation permits the relatively early detection of mycobacteria colonies.

Dorset Egg Medium: Dorset Egg Medium is a modification of the whole egg medium. It is a nonselective medium well suited for the growth and maintenance of pure cultures of mycobacterium. Beef extract and peptone provide nutrients such as amino acids and organic phosphates. Whole egg mass contains complex nutrients necessary for mycobacterial growth and additionally, neutralizes toxic compounds..

Lowenstein Jensen medium: Lowenstein-Jensen media (L-J) is an egg-potato base solid media containing malachite green as an inhibitory agent. The use of L-J media is excellent for the recovery of M. tuberculosis from sterile-site specimens as well as decontaminated-digested sputum specimens.

Ogawa medium: This medium is cheaper than Lwenstein-Jensen because it is made without asparagine. The formulation contains mineral salts like Potassium dihydrogen phosphate anhydrous, sodium glutamate and whole eggs as protein sources. It also contains malachite green to eliminate other contaminating organisms.

Petragnani Medium: Petragnani Medium is an egg-based medium. The formulation contains whole milk, whole eggs and egg yolks as protein sources. Potatoes and Potato Flour are starches that provide a carbohydrate source. Glycerol is a carbon source. Malachite green is present in large amount to inhibit the contaminating organisms. b) Agar-based media: Agar based media are solidified with agar, they are not liquefied by contaminating proteolytic organisms.

Dubos oleic acid-albumin agar: The formulation contains peptone and asparagines as sources of nitrogen. Disodium phosphate and monopotassium phosphate are sources of phosphates; help maintain the pH of the medium. Polysorbate 80, an oleic acid ester, supplies essential fatty acids for the replication of mycobacterium. Bovine albumin acts as a protective agent by binding free fatty acids that may be toxic to mycobacterium. The albumin is heat-treated to inactivate lipase, which may release fatty acids from the polysorbate 80. Agar is used as solidifying agent.

Middle brook 7H-10 agar: It contains a variety of inorganic salts essential for the growth of mycobacterium. The sodium citrate, when converted to citric acid, serves to hold certain inorganic cations in solution. Glycerol is an abundant source of carbon and energy. Oleic acid, as well as other long chain fatty acids, are utilized by mycobacterium and play an important role in the metabolism of organism. Catalase destroys toxic peroxides that may be present in the medium. The primary effect of albumin is to protect mycobacterium against toxic agents and therefore, it enhances their recovery on primary isolation. Partial inhibition of contaminating bacteria is achieved by the presence of the malachite green dye. Colony Morphology: M. tuberculosis bacilli are slow-growing mycobacteria. In primary isolation, they hardly show any visible growth during the first week of culture. On egg-based media they produce characteristic non-pigmented colonies, with a general rough and dry appearance resembling breadcrumbs. On agar based media, the colonies appear flat, dry and rough with irregular edges.

2.

Liquid media:

a) Middlebrook 7H10 media: It is a liquid based media containing salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, and glucose. This media enhances the recovery of mycobacterium. b) TB Broth Base w/o Tween 80: It contains proteose peptone and yeast extract provide nitrogenous nutrients like amino acids and peptides, vitamin B complex and other essential nutrients. The medium is well buffered by

phosphates. The salts present in the medium supply ions required for the mycobacterial metabolism. Sodium citrate inhibits gram-positive organisms and coliforms. The medium can be used without additives and supplements; however sterile dextrose and sterile serum can be added for enrichment. Glycerol addition helps in the cultivation of Mycobacterium tuberculosis. This medium provides dispersed growth of tubercle bacilli which is free of excessive clumps and so it can be used to prepare a uniform suspension of Mycobacteria. c) Dubos Broth: It is enriched with enzymatic digest of casein and an amino acid L-asparagine, as sources of nutrients. A variety of inorganic salts provide ions required for the metabolism of mycobacteria. Polysorbate 80, an oleic acid ester, supplies essential fatty acids for the replication of mycobacteria. Bovine albumin acts as a protective agent by binding free fatty acids that may be toxic to mycobacteria. The albumin is heat-treated to inactivate lipase, which may release fatty acids from the polysorbate 80. Phosphate buffers maintain the pH of the medium

3.

Other non conventional methods: a) BACTEC 460 TB system: It is a technique for semi-automated detection of metabolism of bacteria by measuring the 14CO2 liberated during the growth and decarboxylation of 14C-labeled substrate incorporated in the growth medium. This radiometric technique was widely used for blood culture using the BACTEC 460 instrument. In 1980, this technique was introduced commercially for mycobacterial recovery from clinical specimens and drug susceptibility testing. The high efficiency of the BACTEC TB System is due to the use of liquid medium. The BACTEC 460 TB System has been reported to yield 15-

20% increased culture positivity of clinical specimens as compared to conventional solid media such as LJ medium, with an average time-todetection of positive growth from 8 to 14 days as compared to 3 to 5 weeks on solid media. The introduction of the BACTEC 460 TB System revolutionized laboratory testing for mycobacterium and has established itself as the gold standard for culture and susceptibility testing.

b) BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 System: The MGIT (Mycobacteria Growth Indicator Tube) consists of liquid broth medium that is known to yield better recovery and faster growth of mycobacteria. The MGIT contains 7ml of modified Middlebrook 7H9 broth base. This medium is terminally sterilized by autoclaving. An enrichment, MGIT 960 Growth Supplement is added to make the medium complete. MGIT tube contains an oxygen-quenched fluorochrome, tris 4, 7-diphenyl-1, 10-phenonthroline ruthenium chloride pentahydrate, embedded in silicone at the bottom of the tube. During bacterial growth within the tube, the free oxygen is utilized and is replaced with carbon dioxide. With depletion of free oxygen, the fluorochrome is no longer inhibited, resulting in fluorescence within the MGIT tube when visualized under UV light. The intensity of fluorescence is directly proportional to the extent of oxygen depletion and growth of bacteria.

c) MB/BacT Detection System: The MB/BacT detection system utilizes a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO2) dissolved in the Middlebrook 7H9 broth base. If microorganisms are present

in the test sample, carbon dioxide is produced as the organisms metabolize the substrates in the culture medium. When growth of microorganisms produces carbon-di-oxide, the gas permeable sensor installed in the bottom of each culture bottle changes its color from blue-green to yellow. The lighter color results in an increase in reflectance units as monitored by the system. Bottle reflectance is monitored and recorded by the instrument every 10 minutes.