Mycobacterium

• MYCOBACTERIUM - Slow growers [takes weeks to culture or create mature colonies of mycobacterium] except M. fortuitum and M. chelonei
• Cell wall OF MYCOBACTERIUM - high lipid content and mycolic acid
• Carbol fuschin of Kinyoun – mataas nag phenolic compound
• MYCOBACTERIUM Resist decolorization: AFB Retain ang primary stain; remain red upon staining with either hot or cold method of acid-fast staining
• "Much Granules” – M. tuberculosis
• 2 Groupings of mycobacterium

  1. Rounyon’s classification [4 groupings]
  2. Pathogenesis [consists of the 3 groups below]
• 3 Groups of mycobacterium

  • M. tuberculosis complex: causing Tb [M. tuberculosis, M. bovis, M. africanum]
  • M.O.T.T (Mycobacterium other than tuberculosis) or NTM (Non tuberculosis mycobacteria)
  • M. leprae: Hansens disease
• MYCOBACTERIUM TUBERCULOSIS

  
  

  AKA: KOCH BACILLUS

  • Characteristics:
  • Robert Koch in 1882
  • Obligate aerobe or strict aerobes
  • Require CO2 for growth (5-10%)
• • Virulence Factor of MYCOBACTERIUM TUBERCULOSIS Cord factor & sulfatides: sticky response
• specimen for mycobacterium?

  sputum [pulmonay]
• Bartlett’s classification

  • >10 EC (per LPO) with <25 PMN (per HPO): reject (saliva – not suitable for culture)
  • <10 EC (per LPO) with >25 PMN (per HPO): accept (sputum)
• Specimens of mycobacterium

  • Sputum (3 consecutive days)
  • Secretions obtained by bronchoscopy
  • Blood
  • Urine
  • CSF
  • Pleural, pericardial, peritoneal fluid
• Sputum collection for Direct Microscopic Examination

  1. 1st day: before going to the laboratory, the patient after waking up [after bed], collect sputum [first morning specimen]; 2nd specimen will be collected upon arrival in the laboratory

  2. 2nd day: First morning specimen [3rd specimen]
• Sputum


  • Contains mucin and organics debris
• Secretions obtained by bronchoscopy are for patients that have difficulty in releasing sputum
• Tuberculosis can occur in different organs [lungs, kidneys [once spread], not just in the pulmonary system]
• CSF: pellicle of web-like clot is an indication of tuberculosis in the CNS
• Pleural, pericardial, peritoneal fluid: increased ADA [Adenosine Deaminase: test that can be used to rule out tuberculosis]
• Methods for decontamination and digestion of mycobacterium for culture

  1. Remove normal flora and other contaminating organisms [decontamination]

  2. Break disulfide bond in mucus trap MTb [digestion]
• 1. 4% NaOH


  Digestant and decontaminating agent
• N-Acetyl-L-cysteine (NALC)


  Liquefying agent
• Decontamination and digestion methods

  • 2-4% NaOH
  • NALC or dithiothreltol + NaOH
  • 2-4% NaOH + dithiothreitol
  • Benzalkonium Chloride (Zephiran)
  • Benzalkonium chloride + trisodium phosphate (Z- TSP)
  • 5-6% Oxalic acid
• Benzalkonium chloride + trisodium phosphate (Z- TSP)

  • Shortens exposure time [benzalkonium chloride]
  • Liquefies sputum [requires longer time of exposure] – [trisodium phosphate]
• 1. 6% Oxalic acid is used to decontaminate specimens contaminated with Pseudomonas aeruginosa
• Ordinary staining


  1. Spread [not overlapping; to avoid disarrangement of bacteria]

  2. Circular or horizontal
• Sputum staining


  Coil [madadaanan lahat; no digestant prior to staining; physical force exerted in coiling will break down mucin]
• Staining methods for mycobacterium

  • Gram Stain
  • Acid Fast stains
  • Fite-Faraco's
  • Auramine-rhodamine fluorochrome stain (Truant's)
  • Spengler's
  • Pappenheim's
  • Baumgarten's
• Gram Stainof mycobacterium

  Gram ghost or neutral [gram positive]
• Acid Fast stains

  • Carbol fuschin (1° stain), acid-alcohol (decolorizer) and methylene blue or malachite green (counterstain)
  • Ziehl-Neelsen: heat
  • Kinyoun stains: tergitol
• Fite-Faraco's


  Hematoxylin instead of methylene blue as counterstain
• Auramine-rhodamine fluorochrome stain (Truant's)


  • More sensitive than the carbolfuschin stains
  • Bright, yellow-orange bacilli against a dark background
• Spengler's


  For color blind individuals (MTb: black)
• Pappenheim's

  MTb- red; M. smegmatis: blue
• Baumgarten's

  MTb- blue; M. leprae: red
• AFB Smear Size: Coiling by 2 cm x 3 cm
• CULTURE of mycobacterium

  • Requires increased protein
• AGAR BASED

  • Duboi’s Oleic Acid Albumin Medium
  • Mitchison’s Medium
  • Middlebrook 7H10-7H11: Anti-susceptibility Test
• EGG BASED

  • Inhibitor: Malachite Green
  • Petragnani medium: used or heavily contaminated specimen (increased conc. Of MG)
  • Lowenstein-Jensen medium
  • American Thoracic Society
  • Dorset Egg medium
• LIQUID MEDIA

  • For automated machines
  • Once blood is cultured, hindi agad nilalagay sa plate; blood culture media [approximately 5L of blood: average body structure] [blood: no normal flora; infection once nagkaroon ng kahit isang bacteria] [small amount of blood = small chance of presence of pathogen = propagate (paramihin muna para may ma-isolate)
  • Blood à blood culture bottle [propagate bacteria in the blood and detect if the growth of bacteria in the blood is enough] [within 7 days]
  • Bactec 128, Septi-Check, Middlebrook 7H9
• ANTI-TUBERCULOSIS AGENTS of mycobacterium

  1. PRIMARY DRUGS [RIPES] - Rifampicin, Isoniazid, Pyrazinamide, Ethambutol, Streptomycin
  2. SECONDARY DRUGS - Ethionamide, Capreomycin, Ciprofloxacin, Ofloxacin, Kanamycin, Cyclosrine, Rifambutin