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
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