Lesson 2

• • Skin
  • Nails
  • Hair


  Most common specimen for diagnostic mycology
• • Take into consideration the clinical presentation
  • Swabs are generally rejected for fungal culture
  • Placed in a sterile container or petri dish
  • Not frozen nor allowed to dry; left at room temperature
  • Transported and process/examined ASAPand in BSC


  General Specimen Considerations
• -Skin scrapings - scrape using sterile scalpel

  -Hair Scalp or beard - Cut/clipped or pulled from the areas of infection using sterile scissors or forceps

  -Nails - must be disenfected with alcohol prior to collection


  Cutaneous Samples
• 1. Aseptic collection (microbiological samples) - Crusts, Pus, Aspiration of fluids
  2. Tissue - must be placed on sterile moistened gauze


  Subcutaneous and Tissue Sample
• -respiratory specimens collected as with other microbiological samples


  Samples of endemic/primary systemic mycoses
• 1. Cryptococcal meningitis
  2. Rhinocerebral mucormycosis
  3. Invasive aspergillosis
  4. Candidemia and candidiasis


  Samples of Opportunistic Mycoses
• -early/first morning specimen

  -gargle of mouth wash

  -directly examine using: India Ink, Gram Stain, Acid Fast Stain


  Sputum
• -inoculate for culture

  -Centrifuged

  -Sediment is directly examined using: India Ink, Gram Stain, acid fast stain


  Cerebrospinal Fluid
• -direct examination: smear

  -Culture: 3-4mL aspirate in a sterile container with 0.5mL to 1:1000 heparin and brain heart infusion blood (BHI) blood agar


  Bone Marrow
• -Atleast 10mL, 2-3 blood samples

  -Incubated at 30'C for 30days

  -check for growth on day 1,2,7, the weekly


  Blood
• 1. Direct Examination: microscopy, Wood’s light/lamp
  2. Culture
  3. Stained smears - BM
  4. Histological sections
  5. ImmunoSero
  6. Molec diagnosis
  7. Radiological/imaging studies - Xrays/CT scan/MRI/Ultrasound


  Methods of Diagnosing Mycoses
• 1. Direct microscopy
  2. culture isolation: 25-30’C, BSC, 4-6 weeks, 2 weeks for dermatophytes, 4 weeks for systemic fungi
  3. Gross examination of culture
  4. LPCB - Lactophenol Cotton Blue
  5. Microscopy of isolates (Biochemical Test: VITEK/CHO/Assimilation)


  microbiology laboratory identification
• rapid diagnosis and information

  detects fungi that do not grow in vitro

  KOH Prep, Calcofluor white, India ink, Gram stain, Giemsa/Wright

  Direct Microscopy
• • 10% KOH - skin
  • 20% KOH - nail
  • Heat is used to increase the rate of clearing
  • yeasts, hyphae, spores

  KOH preparation / examination
• fluorescent dye binds to chitin

  yellow/green fluorescence

  higher sensitivity

  Calcofluor White
• negative stain

  Cryptococcus neoformans in CSF

  low sensitivity, high specificity

  India Ink
• hucker’s modification

  all fungi gram positive


  Gram Stain
• giemsa/wright stain

  Histoplasma capsulatum in WBC

  Blood/Bone Marrow Smear
• stain used after culture

  AKA Aman method/stain

  not for direct microscopy

  • Phenol: will kill the microorganisms
  • Lactic Acid: to preserve the structures of fungi
  • Cotton Blue: the stain

  LACTOPHENOL COTTON BLUE
• • Development of treatment plan
  • Investigation of outbreaks
  • Determination of antifungal susceptibility
  • saphrobes/opportunists

  Purposes of Identifying Fungi
• Saboraud’s Dextrose Agar (SDA)


  4% dextrose, 1% peptone, 2% agar pH: 5.5/5.6

  selective for fungi/general/nonselective media for fungi
• can not support cryptococcus

  2% glucose, 1% neopeptone, 2% agar + cycloheximide + chloramphenicol

  Mycosel / Mycobiotic
• phenol red


  Dermatophyte test medium (DTM)
• dimorphic fungi

  brain heart infusion agar (BHIA)
• sporulation and pigmentation

  potato dextrose agar (PDA)
• chlamydospores for C. albicans

  Cornmeal Tween 80 agar
• T. rubrum produces red pigment


  Cornmeal agar w/ 1% glucose
• C. neoformans


  Staib’s niger seed/ bird seed agar
• isolation of Aspergillus spp

  Czapek’s medium
• M. canis (+) from M. audouinii. (-)


  Rice medium
• • C. neoformans (+) from Candida and other yeasts (-)
  • T. mentagrophytes (+) from T. rubrum (-)


  Urea agar
• B. dermatitidis


  Cotton seed agar