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

• Aseptate

Hyphae

• Septate

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