Mycology – Introduction Student Lab Division of Medical Technology Carol Larson MSEd, MT(ASCP)

Mycoses Superficial Subcutaneous Systemic Opportunistic

Characteristics of fungi Eukaryotic Growth requirements Forms Mold Yeast

Hyphae Septate Aseptate

Hyphae Hyaline Dematiaceous

Mycelium Mass of branching intertwined hyphae Vegetative Aerial Fertile

Vegetative types Favic chandeliers Nodular organs Racquet hyphae Spiral hyphae

Reproduction Identify fungi by: Morphology of reproductive structures Spores from vegetative mycelium or aerial fruiting bodies

Asexual Reproduction Conidia Conidiophore Arthroconidia

Asexual Reproduction Blastoconidia Pseudohyphae Chlamydoconidia Chlamydospores

Asexual Reproduction Macroconidia Microconidia Phialoconidia Phialide

Asexual Reproduction Annelloconidia Annellide Sporangiospores Sporangium Sporangiophore

Sexual Reproduction Perfect Fungi – has a sexual stage Fungi Imperfecti – no know sexual stage Spores

Sexual Reproduction Ascospores Ascus Ascocarp Basidiospores Zygospores

In review … Mycoses – fungal diseases Characteristics of fungi Growth requirements Forms (mold, yeast) Structures Reproduction Asexual Sexual

Fungal Culture Process Specimen collection and transportation Direct examination of specimen Selection and inoculation of media Evaluation of fungal growth Serological testing Antifungal susceptibility testing

Specimen Collection Specimen types Collect from area most likely infected Use sterile technique Keep specimen moist Label container properly Transport right away Process right away

Direct Examination Provides preliminary report Guides MD in treatment of patient Observe yeast phase of dimorphic Gives clues to id causative agent Inoculate special media May require more than one direct examination method

Direct Examination Saline wet mount Lactophenol cotton blue wet mount 10% KOH preparation Gram stain Acid fast stain India ink stain

Direct Examination Calcofluor white stain Wright’s stain Gomori Methenamine Silver stain Periodic Acid Schiff stain

Specimen Processing Safety Tube media preferred over plate media Work in safety hood Wear gloves and lab coat Autoclave specimens and media Disinfect work area daily

Specimen Processing Primary isolation media Goal: isolate potential pathogens Use non-selective and selective media Proper ingredients Incubation temperature Incubation time Incubation atmosphere

Non-selective Media Sabouraud dextrose agar Brain heart infusion (BHI) with/without 5% blood and 1% glucose

Selective Media Mycosel agar Inhibitory mold agar Dermatophyte test medium

Subculture / Identification Media Neutral Sabouraud dextrose agar (Emmon’s) Cornmeal-Tween 80 agar Niger seed agar (Birdseed agar) Tween 80 / Oxgall / caffeic acid agar Potato dextrose agar

Examination of Culture Growth Potential pathogens Slow growers Growth on Mycosel Color: dull buff, brown, mousy gray Dimorphic

Examination of Culture Growth Growth rate Rapid growers: 1-5 days Intermediate growers: 6-10 days Slow growers: >10 days

Colony Morphology – Appearance Rugose Umbonate Verrucose Flat

Colony Morphology – Texture Cottony Glabrous Granular Velvety

Colony Morphology – Pigmentation Surface Reverse

Microscopic Morphology Definitive means of identification Evaluate: Shape Method of production Arrangement of conidia/spores Size and color of hyphae

Microscopic Techniques Tease mount Scotch tape preparation Slide culture

Serological Diagnosis Immunodiffusion Complement fixation EIA Latex agglutination

Antifungal Susceptibility Determine appropriateness Standardization of testing Methods Predictability in vivo Antifungal agents

In Summary … Specimen collection and transport Specimen processing and culture Direct examination of specimen Examination of culture Serological testing Antifungal susceptibility