Neisseria and Mycobacteria

Cards (30)

  • 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