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Important Points

  1. Straight or slightly curved rods; Filamentous or branching forms may occur
  2. Mould like pellicle in liquid cultures therefore called mycobacterium
  3. Gram positive but stain poorly, acid fast (20%); mycolic acid + integrity of cell wall
  4. ZN; Kinyoun; Fluorochrome staining (Auramine & Rhodamine; most sensitive staining method)
  5. Aerobic or microaerophilic, nonmotile, nonsporing 

A. Mycobacterium tuberculosis complex

  1. M tuberculosis
  2. M bovis
  3. M bovis BCG
  4. M africanum
  5. M microti
  6. Recent additions to M. TB complex- M. canetii, M. caprae, M. pinnipedi 

B. Diagnosis:

  1. Microscopy: > 10,000 bacilli/ ml; 20-80%
  2. Culture: can detect: 10-100 bacilli/ ml 

C. Culture:

  • M tuberculosis: obligate aerobe and M bovis: microaerophilic

1. Solid media:

  1. L J; Petragnini; Dorset, American thoracic society medium : egg based
  2. Tarshis: blood based
  3. LSS: serum based
  4. Pawlowsky: potato based
  5. Middlebrook 7H11 and 7H10-agar based

2. Liquid media:

  1. Dubo’s, Middlebrook’s, Sula & Sauton
  2. Cord factor in virulent strains: serpentine cords in liquid media 

D. Newer culture methods : - (Recent Advances):

  1. BACTEC: C 14 labelled palmitic acid
  2. PANTA (polymixin, amphotericin B, nalidixic acid, trimethoprim, azlocillin)
  3. Growth index >50 considered positive
  4. NAP (paranitro-α-acetylamino-βhydroxy-propiophenone) containing media: growth rules out MTB complex
  5. (Mycobacterium growth indicator tube system) MGIT
  6. 7H9 broth with fluorescent compound in silicone sensor. Oxygen diminishes/quenches the fluorescent output of the sensor. Oxygen consumption leads to increase in fluorescence
  7. SeptiChek AFB system-manual detection method
  8. Biphasic culture system (7H9 broth and three sided paddle [chocolate, egg-based & 7H11 agar])
  9. ESP culture system-7H11 broth, cellulose sponge
  10. Pressure changes due to oxygen production or gas production detected
  11. BacT/Alert-an automated colormetric method which uses a gas permeable sensor that changes color when Co2 is generated by growing bacteria.
  12. Luciferase reporter phage system
  13. Molecular methods (most sensitive): PCR (target amplifying IS 6110); Ligase chain reaction (LCR); Transcription mediated amplification (TMA)
  14. DNA probes: can distinguish between species 

E. Sensitivity testing:

  1. Absolute concentration (MIC)
  2. Resistance ratio-Test strain & known standard strain (H37Rv)
  3. Lowest concentration showing 20 colonies taken as end point
  4. Ratio of MIC of test strain to MIC of known strain
  5. Ratio of 8: resistant and Ratio of 2: sensitive
  6. 1% proportion method-WHO recommended
  7. Standard inoculum of drug containing & drug free medium
  8. Resistant: more than 1% bacteria grow in the presence of the drug
  9. Radiometric automated systems- Based on 1% proportion method
  10. Phage based assays and MODS
  11. Molecular methods for detection of mutations associated with resistance 

F. Drug resistance in tuberculosis:

  1. MDR-TB: Resistance to INH & Rifampicin with/ without resistance to other drugs
  2. XDR-TB: Strain of M. tuberculosis resistant to INH, Rifampicin and three or more than three classes of second line drugs
  3. Recently XDR is changed to MDR + resistance to any fluoroquinolone and one of the 3 injectable drugs used in ATT, i.e,Capreomycin, Kanamycin and Amikacin.
  4. Primary drug resistance: patient who have never taken drug in the past & get infected with strain originating from patients who had acquired resistance
  5. Acquired resistance: Resistance acquired after intake of drug by patient
  6. Selection of pre-existing mutant because of inaccurate chemotherapy
  7. Natural resistance: Neither the patient with naturally resistant bacilli nor his source of infection has had chemotherapy in the past
    1. Spontaneous mutations                            
    2. Single drug
    3. INH & Rcin: 1/108 - 109 replications               
    4. SM: 1/106 replications
    5. Ethionamide, cycloserine, Thiacetazone: 1/103 replications
INH kat G
inh A
ahp C
Rcin rpo B
SM rps L, rrs, str A, S12
EM emb A, B, C
Pzide pnc A
Flouroquinolones gyr A, B





Straight, slightly curved, singly or in pairs or small clumps;

Beaded or barred forms are seen

Straighter, stouter, shorter

Uniformly staining

Growth requirement

Obligate aerobe


5-10% CO2

Growth enhanced

Not enhanced

Glycerol at 0.75%



Glycerol at 0.5%



Pyruvate enhancement



Type of growth




Rough, tough, buff

Smooth, flat, moist

Pzide sensitivity



Tween 80 hydrolysis



TCH sensitivity









Phage type




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