MYCOBACTERIA (LAB)

• Specimen Collection


  • Sterile, wide-mouth cup with a tightly fitted lid
• Sputum and Other Respiratory Secretions

  • Early-morning specimen collected on three consecutive days
  • At least two of the first three sputum direct smears are positive
  • When none, or only one, of the first three sputum smears is positive, additional specimens are needed for culture confirmation
  • 5 to 10 mL of sputum produced by a deep cough of expectorated sputum or induced by inhalation of an aerosol of hypertonic saline
• Other Specimen Types


  • Bronchial washing, bronchoalveolar lavage (BAL), or transbronchial biopsy specimens
  • Brushings appear to be more commonly diagnostic than washing or biopsy
  • Gastric aspirates should be obtained in the morning after an overnight fast
  • Urine: first morning midstream specimen, minimum of 15 mL
  • Stool: patients with AIDS, who may be at risk for developing disseminated mycobacterial disease resulting from Mycobacterium avium complex
  • Blood for mycobacteremia
  • Tissue or body fluids: 10 to 15 mL of sterile saline should be added to prevent dehydration

  
• Digestion and Decontamination of Specimens

  1. Liquefy the sample through digestion of the proteinaceous material

  2. Allow the chemical decontaminating agent to contact and kill the nonmycobacterial organisms

  3. Liquefying the mucin enables the mycobacteria to contact and use the nutrients of the medium to which they are inoculated
• Decontamination Methods


  • Sodium Hydroxide (2%, 3%, or 4%)
  • N-Acetyl-L-cysteine (NALC) plus NaOH
  • Benzalkonium Chloride (Zephiran) combined with trisodium phosphate (Z-TSP)
  • Oxalic Acid (5%)
• Staining for Acid-Fast Bacilli

  1. Apply heat with the carbolfuchsin stain (Ziehl-Neelsen)

  2. Use a cold stain (Kinyoun acid-fast)

  3. Examine slides using a ×100 oil immersion objective on a light microscope for 15 minutes, viewing a minimum of 300 fields before a slide is called negative

  
• Mycobacteria


  • Strictly aerobic and grow more slowly
  • Generation time of mycobacteria is longer than 12 hours, with M. tuberculosis having the longest replication time at 20 to 22 hours
  • Most pathogenic mycobacteria require 2 to 6 weeks of incubation
  • Growth of M. tuberculosis is enhanced by an atmosphere of 5% to 10% CO2
  • Mycobacteria require a pH between 6.5 and 6.8
• Culture Media and Isolation Methods

  • Egg-Based Media: Löwenstein-Jensen (LJ), Petragnani, and American Thoracic Society (ATS) media
  • Agar-Based Media: Middlebrook 7H10 and 7H11 agars
  • Liquid Media: Middlebrook 7H9 broth and Dubos Tween albumin
• Preliminary Identification of Mycobacteria

  • Colony Morphology: smooth and soft or rough and friable appearance
  • Growth Rate: range from 3 to 60 days
  • Niacin Accumulation: most commonly used biochemical test for the identification of MTB
  • Nitrate Reduction: differentiates M. tuberculosis from the scotochromogens and MAC
  • Catalase: most M. tuberculosis complex organisms do not produce heat-stable catalase
  • Hydrolysis of Tween 80: some mycobacteria possess a lipase that can split the detergent Tween 80
  • Iron Uptake: some mycobacteria can convert ferric ammonium citrate to an iron oxide
  • Arylsulfatase: most members of the genus Mycobacterium possess the enzyme arylsulfatase
  • Pyrazinamidase: hydrolyzes pyrazinamide to pyrazinoic acid and ammonia in 4 days
  • Tellurite Reduction: reduction of colorless potassium tellurite to black metallic tellurium in 3 to 4 days is a characteristic of MAC
  • Urease: detection of urease activity can be used to distinguish M. scrofulaceum from M. gordonae

  
• Inhibitory Tests


  • NAP (p-nitroacetylamino-β-hydroxypropiophenone): selectively inhibits the M. tuberculosis complex
  • Thiophene-2-Carboxylic Acid Hydrazide (T2H): M. bovis is susceptible to lower concentrations than MTB
  • Sodium Chloride Tolerance: high salt concentration (5% NaCl) inhibits the growth of most mycobacteria, except M. flavescens, M. triviale, and most rapidly growing Mycobacterium spp.
  • Growth on MacConkey Agar: the Mycobacterium fortuitum-chelonae complex can grow, whereas most other mycobacteria cannot
• The CDC currently recommends that when isolated, M. tuberculosis be tested for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin
• Skin Testing


  1. Protein extracted and purified from the cell wall of culture-grown M. tuberculosis is used as the antigen (i.e., purified protein derivative; PPD)

  2. A standardized amount of antigen is injected intradermally into the patient's forearm

  3. Reactivity is read at 48 hours; in immunocompetent individuals, the presence of a raised firm area (induration) 10 mm or larger is considered reactive

  4. A reactive tuberculin skin test indicates past exposure to M. tuberculosis; other Mycobacterium spp. generally result in an induration smaller than 10 mm