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General Characteristics :

  • nonmotile, slender, slightly curved, rod-shaped bacilli; non-sporeforming
  • cell wall has a high lipid content (mycolic acid)
  • resist staining with gram stain
  • require complex media
  • strictly aerobic
  • slow-growers; those associated with disease take two to six weeks to produce growth

Classification :

  • Pathogenicity – diseases they cause
    • tubercle bacillus complex : organisms that cause tuberculosis (Mycobacterium tuberculosis, M. bovis, M. africanum, M. canetti)
      • primary agents of tuberculosis
    • atypical mycobacteria or nontuberculosis mycobacteria (NTM) or mycobacteria other than tubercle bacilli (MOTT)
      • can cause tuberculosis-like illness but no evidence of person to person transmission
  • Physiology – how they grow and react to light; based on pigment production and rate of growth
    • Photochromogens (Runyon group I)
      • slow-growing (2-6 weeks)
      • nonpigmented when grown in absence of light
      • produce yellow or orange carotenoid pigment when exposed to light and reincubated (photoactivation)
      • ex : M. kansasii
    • Scotochromogens (Runyon group II)
      • slow growing species that produce yellow to orange pigment whether grown in light or dark
      • pigment produced in absence of light deepens to orange or dark red with exposure to light
      • ex : M. scrofulaceum
    • Nonphotochromogens (Runyon group III)
      • slow-growing organisms that may be lightly pigmented (buff, tan, or pale yellow)
      • pigment does not intensify when exposed to light
      •  ex : M. tuberculosis
    • Rapid Growers (Runyon group IV)
      • organisms that form mature visible colonies within 7 days of incubation
    • NOTE : further testing, using biochemical and molecular methods, is needed to differentiate to species level
  • Complexes – based on similar characteristics
    • share similar biochemical, serological and pathogenic characteristics
    • Mycobacterium tuberculosis (tubercle bacilli) complex
    • M. avium complex (MAC)
      • M. avium and M. intracellulare
    • M. fortuitum complex
      • M. fortuitum and M. chelonae

Specimen Processing :

  • Respiratory Specimen –
    • expectorated sputum
    • normal-saline-nebulized, induced sputum
    • transtracheal aspirate
    • bronchoalveolar lavage
    • laryngeal swab
  • Body Fluids –
    • pleural fluid
    • pericardial fluid
    • joint aspirate
    • gastric aspirate
    • peritoneal fluid
    • cerebrospinal fluid
    • stool
    • urine
    • pus
  • Body Tissues
    • blood
    • bone marrow biopsy/aspirate
    • solid organ
    • lymph node
    • bone
    • skin

Acid-Fast Bacilli (AFB)

  • AFB (or mycobacteria) are dangerous to work with…
    • requires unique methods of specimen collection, processing, culture identification and susceptibility testing

Mycobacterial Stains :

  • AFB Smears can be :
    • direct smears
      • specimens from normally sterile sites
    • concentrated smears
      • specimens may contain contamination bacteria
  • Carbol Fuchsin-Based Stains :
    • Ziehl-Neelson stain technique
      • “hot” stain
    • Kinyoun stain technique
      • “cold” stain
    • examine at least 300 fields with oil immersion lens
    • red bacilli are acid-fast positive
    • wipe oil immersion lens after each positive specimen to avoid cross-contamination and a false positive report

Specimen Processing :

  • Sterile specimens inoculated directly to liquid and solid mycobacterial culture media :
    • grinding of tissue specimens
    • body fluids concentrated by centrifugation
  • Nonsterile specimens decontaminated prior to media innoculation :
    • digestion-decontamination procedure
      • N-acetyl-L-cysteine (mucolytic agent) and sodium hydroxide (decontaminant) – most commonly used
      • 5 other procedures
    • overexposure to NaOH is toxic to mycobacteria too

Isolation of Acid-Fast Bacilli

  • Media
    • use liquid, agar-based, and egg-based
      • eg. LJ slant
    • provide nutrients for growth
    • add heme medium (eg. chocolate agar) to grow possible M. haemophilum
    • antibiotics or inhibitory substances used to inhibit contaminants
  • Egg-Based Media
    • Lowenstein-Jensen (LJ)
    • Wallenstein
    • Petragnani
    • American Thoracic Society (ATS)
  • Agar-Based Media
    • Middlebrook 7H10 and 7H10 selective
    • Middlebrook 7H11 and 7H11 selective
    • clear medium which is good for microscopic observation of microcolony morphology
  • Liquid Media
    • Middlebrook 7H9, 7H12, 7H13 broths
    • BACTEC 12 B
    • Dubois Tween albumin broth
  • Other TB Media
    • Septi-ChekAFB system
    • Mycobacterial Growth Indicator Tube (MGIT)
    • BACTEC 460 TB

Other Methods of Identification :

  • Conventional Biochemicals
    • 96% M. tuberculosis isolates identified but takes 3 weeks or more
    • presumptive ID M. tuberculosis
      • acid-fast positive (tight, serpentine cording)
      • buff, dry, corded, “cauliflower-like” colony
      • niacin and nitrate positive
      • 68°C catalase negative
  • Gas Liquid Chromatography (GLC) and High-Performance Liquid Chromatography (HPLC)
    • analysis of fatty acids by GLC and HPLC
  • Molecular Testing
    • highly accurate and rapid
    • identifies mycobacteria difficult to speciate because of relatedness
      • nucleic acid amplification
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