MYCOVIRO - INTRO TO MYCOLLGY (II)

• Skin specimens should be cleaned with 70% alcohol to remove dirt, oil and surface saprophytes.
• Same procedure must be done if the specimen is a nail but it should be clipped and needs to be finely minced before inoculating to media.
• hair can be obtained by plucking, brushing, or with sticky tape.
• Normal sterile must be done if the specimen is a body fluid (sputum, etc.) (alcohol, iodine, alcohol)
• Hair and nails sent in a dry envelope, inside a proper container.
• Other specimens are usually sent frozen or on dry ice.
• Specimen must be inside a packaging with biohazard regulations.
• Any growing cultures must be on tube media, not in plates. (it is in tube media because it has a cap which can seal the media preventing inhalation of spores)
• Aluminum screw- capped inner with outer cardboard mailing tube is usually the container of fungal specimen.
• Inside labeling information must contain:

  • Patient ID
  • Specimen Source
  • Suspected organism (for the medtech to properly prepare which biosafety procedure is to be followed 1,2,3)
• Outside labelling information must state

  WARNING: POTENTIAL PATHOGEN
• SKIN, NAIL & HAIR

  • Direct exam following KOH (Histoplasma) preparation
  • KOH – Potassium Hydroxide > this can dissolve keratin which can easily look for the fungi
  • Skin, nail, and hair all contain keratin
• PLEURAL FLUID, SPUTUM & BRONCHIAL ASPIRATION

  • Specimen must be collected fresh due to possible growth of saprophytes over pathogens such as Histoplasma capsulatum
  • This is for respiratory samples.
• GASTRIC WASHING

  Same as pleural fluid
• GENITO – URINARY SPECIMENS

  • First morning specimen is preferred
• BLOOD/ BONE MARROW

  • Inoculated directly to BHI broth and BHI slant
• WOUND ABSCESS OR DRAINAGE

  • Culture anaerobically, especially if actinomycosis is suspected
• CSF

  • Centrifuged examine sediment microscopically then inoculate media
• TISSUE SPECIMENS

  • Examine for pus, caseous material or ganules; minced aseptically can used small sterile saline supernatant inoculated.
• Fungi can be identified by Directly observing their distinctive morphological features on direct microscopic exam of specimen.
• MedTechs usually look for spores, hyphae, mycelial elements, budding yeast, and mycotic granules.
• fungal element seen in urine - yeast
• WET MOUNT PREPARATION

  • Good for yeast examination, loss of fragile structure is minimized
  • Use of NSS + slide + sample + cover slide
• 10% KOH mount

  Good for skin scrapings, hair, nails, sputum, vaginal specimens etc. KOH clears the specimen’s tissue cells, mucous without destroying the cell wall so fungal elements can be seen
• Fungal stains

  direct examination can be aided by the use of stains/dyes that can enhance the visualization of fungal structures
• Fungal Culture (gold standard)

  • Culture is more sensitive than direct examination and a portion of the specimen used in microscopy should be cultured
• Cultures must be held for 21 days at room temperature 25- 30DegC
• Yeast grows better at 37C and Molds at 30C
• Least pathogenic fungi - mold
• LACTOPHENOL COTTON BLUE (LPCB)

  • Quick evaluation of fungal structures; stains chitin in cell walls of fungi (blue fungi)
• Periodic acid – Schiff stain (PAS)

  • Stains polysaccharide in the cell walls of fungi; Fungi stain purplish-red with Blue nuclei
• • Gomori Methenamine Silver stain (GMS)
  • Outlines in black due to silver precipitating on the fungi cell wall. Internal structures are deep rose to black; Background is light green
• GRIDELY STAIN

  • Hyphae and yeast stain dark blue or rose. Tissues stain deep blue and background is yellow
• MAYER MUCICARMINE STAIN

  • Stains capsules of Cryptococcus neoformans deep rose
• FLUORESCENT ANTIBODY STAIN

  Simple, sensitive and specific. Applicable for many different fungi (fluoresce in color green)
• PAPANICOLAU STAIN

  • Good for initial differentiation of dimorphic fungi. Works well on sputum smears
• GRAM STAIN

  Most fungi are gram positive (purple/violet)
• GIEMSA STAIN

  • Used on blood and bone marrow specimens (blood smear = very rare)
• INDIA INK

  Demonstrates capsule of Cryptococcus neoformans in CSF specimens
• BIRDSEED AGAR

  • Isolation and preliminary identification of Cryptococcus neoformans.
  • Appears as Black colonies