Method of Staining Flashcards
Most widely used; 1 min. Lactic acid/ glycerol; slides can be made permanent. This method dislodges fungal structures.
Lactophenol Cotton Blue (LPCB)
LPCB component that preserve structures.
Lactic Acid/Glycerol
Clears specimen using 10-20%; 5 min to 15 min. Add time if more clearing is needed. Add 0.1% thimerosal to preserve specimen. Requires experience because of artifacts.
Potassium Hydroxide (KOH)
Stains chitin (cell wall) of fungi. Can be mixed with KOH. Detect fungi rapidly due to bright fluorescence. Requires fluorescence microscope.
Calcofluor White
Detects cryptococcus in CSF. Diagnostic of meningitis. Not so sensitive.
India Ink (Nigrosin)
Mucopolysaccharide for capsule of Cryptococcus. Detect encapsulated yeast. Blastomyces and Rhinosporidium may also be positive.
Alcian Blue or Mucicarmine
Rapid detection of P. jirovecii from lung biopsy/BAL. Quick, easy, rapid results, cost-effective. Trophozoites are not discernable.
Toluidine Blue
Examination of bone marrow and blood smear. Detects Histoplasma capsulatum and Cryptococcus spp. Quality of stain should be monitored.
Wright’s Stain
Detects fungi; add 5 min time if with counterstain. Hyphae of molds and yeasts can be readily distinguished. Nocardia do not stain well. Time-consuming.
Periodic Acid-Schiff
Melanin in fungal cell wall. Differentiates melanin from hemosiderin. If only rare granular staining is present, difficult to interpret.
Masson Fontana
Detects fungi in histologic sections. Best stain for fungal elements (black) against pale yellow/green background. Requires specialized staining method.
Methenamine Silver
CSF uses:
India ink, cryptococcal antigen
Direct examination of tissues
Histology stain, fluorescent antibody (selected organism)
Direct examination of skin, hair, nails, and other selected specimens
KOH/KOH with calcofluor white
Hyphal elements - width, hyaline vs dematiaceous; septate vs sparsely septate; special supplementary media
Culture - 22 to 25 deg C growth = yeast/mold
Culture at 30 deg C
Suspect dimorphic
Periodic acid–Schiff color of fungi and background
Magenta against pink/green
Gomori methenamine silver color of fungi and background
Black against green
Giemsa color of fungi and background
Purple-to-blue yeast with clear halo (capsule), Pink to Purple
India ink (negative staining) color of fungi and background
Yeast with clear halo (capsule), Black
KOH (clearing agent) color of fungi and background
Refractile, Clear
KOH–calcofluor white color of fungi and background
Fluorescent, Dark
Masson-Fontana color of fungi and background
Brown, Pink to Purple
Two needles to release mycelia from middle-third of colony. Easy but disrupts conidia.
Tease mount method
Clear tape, touch only surface, not colony. Retains conidial arrangement but can contaminate culture.
Cellophane Tape Preparation
Agar block (1x1 cm) on slide with coverslip, cultured in petri dish with moistened filter paper. Shows natural morphology but is tedious.
Slide culture method
