Neisseria and Mycobacteria

• Family Mycobacteriaceae


  • Strictly aerobic
  • Slow growing on culture media
  • Requires 2-6 weeks of incubation
  • All are acid-fast
  • Contain large amounts of mycolic acid in their cell walls
  • Catalase positive
• Laboratory examination for the diagnosis of bacterial infections

  • Microscopy
  • Culture
  • Biochemical test
  • Serological test
  • Molecular techniques
  • Phage typing
• When handling specimens suspected with TB, always work under a biological safety cabinet
• Specimen collection for TB diagnosis

  • Sputum and bronchial aspirates
  • Gastric aspirates and washings
  • Urine
  • Stool
  • Blood
  • Tissue and other body fluids
• Sputum collection


  Early morning deeply expectorated sputum for 3 consecutive days
• How to process a sputum sample

  1. Digestion

  2. Decontamination

  3. Concentration
• Decontamination and digestion agents

  • 4% NaOH + NALC (N-acetyl L-cysteine)
  • Benzalkonium Chloride + trisodium phosphate
  • 5% Oxalic acid
• Process of decontamination and digestion

  1. Mix sputum and digestant

  2. Mix on vortex mixer for 15-30 seconds

  3. Stand at room temp for 15 mins.

  4. Add phosphate buffer and invert to mix

  5. Centrifuge for 15 mins.

  6. Decant supernatant and resuspend sediment in 2 ml sterile water
• Follow aseptic technique for bacterial smear preparation
• Heat-fix properly approx. 2-3 secs. per contact with flame. Never scorch the smear!
• 3 (+) AFB SMEARS à CONFIRMATORY ALREADY!
• IF ONLY 1 SMEAR IS POSITIVE à CULTURE!
• SCAN FOR 15 MINS. UNDER OIO X 300 FIELDS
• CDC Reporting Scale


  • 4+: >9/OIF
  • 3+: 1-9/OIF
  • 2+: 1-9 AFB/10 OIF
  • 1+: 1-9 AFB/100 OIF
  • DOUBTFUL/RESUBMIT SAMPLE: 1-2 AFB/300 OIF
  • 0: No AFB seen in 300 OIF
• Staining methods


  • Ziehl-Neelsen Staining or Acid Fast Staining
  • Cold Kinyoun Method
• Ziehl-Neelsen Staining


  1. Apply carbol fuchsin to smears and heat for 3-5 mins.

  2. Tilt the slide and wash with tap water.

  3. Decolorize with acid alcohol until a faint color flows off from the slide.

  4. Tilt and wash.

  5. Counterstain with Loeffler's methylene blue for 45 seconds.

  6. Tilt and wash.

  7. Air dry.
• Acid-fast bacilli
• Acceptable specimen for culture

  • Sputum – Decontaminate and digest, concentrate
  • Blood, bone marrow
  • Tissues
  • Urine
  • CSF, pericardial fluid, peritoneal fluid

  
• Culture media


  • Lowenstein-Jensen Medium – egg based
  • Gruft – modification of LJ
  • Middlebrook 7H10 and 7H11 – agar based
  • Petragnani – egg based
  • Dorset medium – egg based
• Composition of Lowenstein-Jensen Medium


  • Malachite green – inhibits bacteria other than M. tuberculosis
  • Glycerol
  • Asparagine
  • Potato starch
  • Coagulated eggs
  • Potassium dihydrogen phosphate
  • Magnesium sulfate
  • Sodium citrate
• M. tuberculosis culture
• Biochemical tests (for specie differentiation)

  • Niacin Accumulation
  • Nitrate Reduction
  • Catalase Test
  • Hydrolysis of Tween 80
  • Iron uptake
  • Arylsulfatase Test
  • Pyrazinamidase Test
  • Tellurite Reduction
• Niacin test


  The key identifying characteristic for M. tuberculosis is the accumulation of niacin due to absence of the enzyme that converts free niacin to niacin ribonucleotide.
• Mycobacterium leprae has never been cultured in artificial culture medium. It is grown in the 9 banded armadillo or in the footpads of mice. It takes 3-4 weeks to replicate.
• Specimen sources for pathogenic Neisseria

  • Swabs from urethral discharge and/or vaginal discharge (Neisseria gonorrheae)
  • Blood (Neisseria gonorrheae and Neisseria meningitidis)
  • Nasopharyngeal swab (N. meningitidis)
  • CSF (N. meningitidis)
• Specimen collection and processing

  1. Only use Dacron or rayon swabs

  2. Plate within 6 hours upon collection

  3. Direct plating at bedside is recommended or use Amies Charcoal Transport Medium

  4. SPS should not exceed 0.025%

  5. CSF specimens are centrifuged at 1000 x g for 10 - 15 minutes. Vortex the sediment then inoculate onto culture media.
• Selective culture media


  • Thayer Martin Medium: Chocolate Agar(CA) + Isovitalex + VCN
  • Modified Thayer Martin (MTM): CA + Isovitalex + VCN + Trimpethoprim
  • Martin Lewis (ML) Medium: similar to MTM except that anisomycin is substituted for nystatin and with increased concentration of vancomycin
  • GC-LECT Agar: for oropharyngeal specimens
  • New York City (NYC) Medium: lysed horse blood + horse plasma + Amphoterecin B (yeast dialysate) and VCNT
• Oxidase test


  Detects for the presence of cytochrome oxidase enzyme. If the organism contains the cytochrome oxidase enzyme, it catalyzes the oxidation of cytochrome C while reducing oxygen to water. Positive result is a color change to PURPLE within 60 to 90 secs.
• Characteristics


  • Neisseria gonorrhoeae: Gram negative intracellular/extracellular diplococci, Catalase (+), Oxidase (+), Small, gray to tan, transluscent, raised colonies, Poor growth on SBA, Autolyse, Carbohydrate Fermentation: G+ M- L- S-
  • Neisseria meningitidis: Gram negative intracellular/extracellular diplococci, Catalase (+), Oxidase (+), Medium sized, gray, convex, mucoid colonies, Good growth on SBA, Autolyse, Carbohydrate Fermentation: G+ M+ L- S-
• Differences between Neisseria gonorrhoeae and Neisseria meningitidis

  • Site of Infection: Anogenital tract vs Colonizes the Upper Respiratory tract
  • Pathogen: 100% pathogenic vs Not always considered a pathogen (10% of adults are carriers)
  • Prevalence and Mortality: High prevalence, Low mortality vs Low prevalence, High mortality
  • Disease: Opthalmia Neonatorium, Gonorrhea, PID vs Meningitis or meningcoccemia