MYCOLOGY

Mycology:

a branch of botany dealing with fungi (greek-mykes: fungus).

Mycosis:

a disease caused by fungi; a fungal infection in or on a part of the body.

Fungi:

A kingdom of plantlike spore-forming organisms that grow in masses with out roots, stems, leaves, or photosynthetic pigments.

Sporangium:

(large sac-like structure) - a closed sac-like structure where sporangiospores are formed. 725

Eukaryotic:

A single celled or multicellular organism whose cells contain a distinct membrane-bound nucleus.

Eukaryotic protists, possess:

Nucleus

Nuclear membrane

Endoplasmic reticulum

Golgi apparatus

Mitochondria

Rigid cell wall

Blastoconidia/Blastospores:

A spore formed by budding, as in yeasts; thin-walled and water-balloon-like. 772

Chlamydoconidia/Chlamydospore:

a spore formed by the rounding-up of a cell; thick-walled; intercalary or terminal position; it is not shed.

Saprophytic:

A plant that derives its nourishment from dead or decaying organic matter.

Septate:

Hyphae that are subdivided into individual cells by transverse walls.

Aseptate:

Those without walls.

Hyphae:

Basic structural units of mold, tube-like projections.

Pseudohyphae :

Elongated buds that have failed to separate and are connected together to form a link-of-sausage appearance. 772

Mycelium:

Loose network of hyphae.

1. Vegetative - nutrient absorbing and water exchanging portion

2. Aerial - extends above the substrate

Yeast

unicellular fungus that reproduce by budding (2.5 to 6 microns )

GENERAL CHARACTERISTICS OF FUNGI

Yeast - single cell forms

Multiple cells that form filaments - molds

Unicellular / multi-cellular

Lack chlorophyll

Heterotrophic

Saprophytic and/or parasitic

Many are ubiquitous

Reproduce by spores, either sexually asexually stage

- May be derived directly from mycelium or fruiting bodies

CELLULAR MORPHOLOGYVegetative Structures

Hypha / hyphae

Tubular filament or threads branched or

unbranched

Septate: possessing crosswalls

Aseptate (coenocytic): lacking crosswalls

Vegetative Structures: Mycelium:

Mass of hyphae

Vegetative mycelium

Growth in or on a substrate

Aerial or reproductive mycelium

Projects above the substrate and may be comprised of or support elaborate spore bearing, fruiting bodies

Vegetative Structures Characteristics:True hyphae:

Filamentous, flat-ended cells form transitional cells

Do not show points of constriction

Vegetative Structures Characteristics:pseudohyphae:

Regular points of constriction (link sausages)

Produced in nutritionally poor environment

May bud to form blastospores (yeast cells) with lesser diameter than true hyphae

3 types of clinically significant hyphae:

Coenocytic - sparsely septate

Pigmented/dark - septate of the dematiaceous fungi

Septate - non pigmented hyphae of the hyaline molds

Colonial Morphology (Gross): Molds

Cottony, wooly, powdery, or fluffy

Optimum temperature: 25-30C

Colonial Morphology (Gross): Yeasts

Smooth, pasty, mucoid, butyraceous

Optimum temperature: 35-37C

Colonial Morphology (gross):Dimorphic Fungi:

Possess both mold and yeast phases

Can be temp dependent (thermal)

Medically important dimorphic fungi

H. capsulatum

B. dermatidis

P. marneffei

C. immitis - non-thermally dimorphic

P. brasiliensis

S. schenkii

Colonial Morphology (gross):Dimorphic Fungi:

-Mold/mycelial/saprophytic phase

Produce delicate hyphae, <1-2 mm, mold form of colonies have cobweb or hair like appearance

-Yeast/tissue/parasitic phase

Can grow on media with cyclohexamide or anti fungal

-Pigmentation - Noteworthy characteristic

Dematiaceous: dark

Hyaline: absence of color

Growth rate is significant

CULTURE MEDIA

- Normal tubes / plates optional (BAP, Choc, etc.)

- Battery recommendations

With or without blood

With or without cycloheximide

With Antibacterial Agents

-Large culture tubes recommended (150 x 25mm)

Poured in thick slants

Do not screw down cap

CULTURE MEDIA: Large Culture Tubes

ADVANTAGES:

Easily stored, less space

Easily handled, less hazardous

Lower dehydration

DISADVANTAGES:

Poor isolation

Reduced surface for growth

CULTURE MEDIA: Petri-dishes

ADVANTAGES:

Provide larger surface of growth

Mixed culture easier to separate

Provide maximum aeration

DISADVANTAGES:

Tendency to dehydrate during incubation

Hazardous for cultivation of certain systemic mycoses

Histoplasma

Blastomyces

Coccidioides

Media used for primary Recovery

BHI Agar w/out & with antibiotics

Chromogenic agar

Dermatophyte test medium

Inhibitory mold agar

Mycophil agar

Potato flake agar

Mycosel agar

SABHI agar

Yeast extract phosphate

Selective Media

-Antibiotics added

Chlorampenicol

Broad spectrum

Bacteriostatic

- Cycloheximide

Inhibits most saprophytic fungi

- Gent and Cipro

Selective Media: BHI Agar

Available with antibiotics or blood

For dimorphic

Mold / fungi @ 20C

Yeast @ 35C

- For recovery of saprobic and pathogenic fungi

Selective Media: SABHI Agar

For isolation of significant fungi from mixed flora specimen (i.e. sputum)

Saprobic and pathogenic fungi

Selective Media: Mycosel Agar

Inhibits bacteria and saprophytic fungi

Cycloheximide, chlorampenicol, dextrose

- For dermatophytes

Selective Media: Dermatophyte Test medium (DTM)

Selective for dermatophytes

Infects skin, hair, or nails

For screening purposes only:

Subculture to a medium w/out antibiotics for maintenance

Selective Media: Corn Meal Agar

Morphological studies of Candida

Nutritionally deficient, C. albicans produces chlamydospores

Selective Media: Czapek's Agar

Differential ID of Aspergillus species

Laboratory Safety in Mycology

All mold cultures and clinical specimens must be handled in a class II BSC

Keep pathogenic isolates sealed

Direct Microscopic Examination:Gram Stain:

Spore and hyphae stain Gram positive

Candida albicans appears black

Large blastospores / pseudohyphae

Direct Microscopic Examination: Potassium Hydroxide (KOH)

10-40% KOH

For demonstrating fungal hyphae and spores in clinical material

Skin, hair, nails, other tissue

Clears opaque material and hydrolyzes the keratin in epithelium

Diagnosis of Cutaneous Mycoses

Wood's black light

Detecting infected hairs by fluorescing a bright, yellow-green

Wash affected skin sites with 70% isopropanol

- Obtain small fragments / scales

Skin or nail

Scrape with scalpel or slide

Mince as needed

- Add KOH and coverslip

- Heat may accelerate clearing process (may be overdone!)

Nails may require 30 minutes.

Press coverslip gently, express bubbles

- Clearing apparent as change in preparation to turbid

Thick, opaque samples

Unsatisfactory for examination

Obscured hyphae and spores

Interpretation

Hyphae demonstrate uniformity in size and symmetry

Reporting:

Depends on Lab SOP

(+) Dermatophyte

Branching hyphae, occasional arthrospores

(+) Tinea versicolor

Short, stubby hyphal elements and grape-like clusters of spores

(+) Candida:

Pseudohyphae & chlamydospores/blastospores

Avoid Reporting False Positives

Mosaics and other artifacts such as cholesterol deposits

Cell wall skeletons

Hyphae do not grow geometrically (e.g. sharp acute or right angles)

Lactophenol Cotton Blue (LCB) Stain

- Temporary wet mounts for staining inocula from culture

- Fungal morphology in LCB mounts

Basis of textbook descriptions

- Fungicidal and sporicidal

LCB Procedure

- Work under a biological safety hood

- Special care in obtaining inoculum with needle

- Care taken to acquire small portion of medium which contains mycelium

- Transfer to slide, apply coverslip

-Apply few drops of stain

Gently heat

Examine:

Fungal mycelium and fruiting structures take on delicate light blue color

Negative Staining

Reagent:

India ink or nigrosin stain

-Routinely utilized for staining CSF

Demonstrate large capsules enveloping blastospores of Cryptococcus neoformans

Germ Tube Test

ID Candida albicans

Germ tube:

Long, narrow tubes projecting outward from blastospores

Not pseudohyphae, not constricted at their point of origin

Germ Tube Test: Procedure

Reagent: 0.5 ml serum (human, rabbit, or bovine)

Inoculate serum

Incubate 2 hrs, 35C

Place a drop to slide / cover

- Examine:

Germ tube appear as short hyphal like extensions

Half the width, 3-4 times the length of yeast cell

-Test not valid if examined after 2 hours

Yeast Assimilation Test: Principle

-Yeast species are differentiated based on Carbon and Nitrogen usage (assimilate)

- If (+) will show growth or turbidity

- Considered along with morphology and reproductive structures

- Commercial kits:

API 20C

Vitek Yeast card

YT microplate

Yeast Fermentation Test: Principle

- Consists of an organism suspension + carbohydrate

- If (+) will show gas bubbles

- Rarely used (long incubation), replaced with assimilation test

- Used as back-up test

Special Culture Techniques: Slide Techniques

- Designed for microscopic examination

Fungi in natural state

Used on dermatophytes only

- Materials:

SAB-DEX medium

Long coverslip (sterile)

- Cut 1 cm square block of SabDex agar out of plated medium

Place on sterile slide

Inoculate each corner block

Place heated coverslip glass directly on surface agar block

- Incubate in Petri-dish

30 C until obvious growth occurs

Examine:

Gently lift coverslip from surface of agar block

Portions of mycelium adhere to underside of coverslip

-Place on slide containing drop of LCB stain

- Examine microscopically

Tease mount

- Equipment:

Dissecting needles or pointed applicator sticks (spuds)

- Procedure:

Dig out small portion of colony to be examined

- Include some subsurface media

Place on slide in drop of LCB stain

Tease colony apart

Overlay w/ cover slip

- Examine:

10X

40X

Oil immersion

Teasing colony disrupts delicate fruiting structures of filamentous molds

Scotch Tape Preparation

- Better suited to preserve spore arrangements

- Especially delicate filamentous molds

Scotch Tape Preparation Procedure: Culture

- Press sticky side of tape to surface of colony

Picking up portion of aerial mycelium

- Place drop LCB stain on slide

- Place sticky side down on slide

Stretch tape over stain

Lower gently

- Examine

Scotch Tape Preparation Procedure: Skin

- For diagnosis of Tinea versicolor

- Tape is applied directly to skin

- Scales adhere to the tape

- Place drop LCB stain (or KOH) on slide

- Place sticky side down on slide

- Stretch tape over stain

Lower gently

- Examine

Periodic Acid Schiff (PAS)

Stain used to detect fungi

Stains fungal elements well

Nocardia spp. do not stain well

Calcocluor white

Stain used to detect fungi

Used with KOH; KOH clears specimen while calcofluor shows fluorescence

Use fluorescence microscope

Fungal elements show apple-green or blue white fluorescence

Cutaneous Mycoses (superficial)

Superficial scaling

Rarely invades deeper tissues

Forms demonstrate hyphae and arthrospores only

Culture: forms hyphae and arthrospores only

Cutaneous Mycoses (superficial): Clinical characterization:

Habitat from geophilic to zoophilic to anthrophilic

Clinical types

Designated by Latin binomial

Tinea capitis

Tinea pedis

Microsporum audouii

most important cause of Tinea capitis in school children

Tinea capitis

Spread by direct contact with infected hairs on caps, hats, combs, clippers

Causes hair to fluoresce

M. canis

causes an inflammatory Tinea capitis

Zoophilic

Usually acquired from puppys or kittens

Trichophyton mentagrophytes

Most common species isolated

Causes T. barbae, T. capitis, T. corporis, T. pedis, and onychomycosis

T. rubrum

2nd most common spp

T. pedis, T. corporis, T. cruris, onychomycosis

T. tonsurans

T. capitis, T. pedis, T. corporis and onychomycosis

Malassezia furfur

Tinea versicolor

Microsporum gypseum

T. corporis and T. capitis

Recovered from hair and skin

Epidermophyton flocossum

Tinea cruris, onychomycosis

Tinea pedis

Candida albicans

Cutaneous candidiasis/moniliasis

Systemic dx in immune compromised

Thrush

Subcutaneous Mycoses: Clinical significance:

-Caused by fungi inhabiting soil/decaying vegetation

- Usually induced by trauma

- Disseminated forms

- Some individuals predisposed to systemic infections

Sporothrix schenckii

Causes sporotrichosis

Hazard to gardeners, florists

Causes "rose gardener's dx"

Also pulmonary, osteomyelitis

Systemic (deep) Mycoses: Clinical significance:

Soil fungi usually involved

Infections due to inhalation of spores

Disseminated forms invade organs

Each spp has a favorite organ

Certain individuals predisposed

Coccidioides immitis

Causes coccidiomycosis "Valley fever"

Skin infection

Osteomyelitis

Meningitis

Arthritis

Histoplasma capsulatum

Causes histoplasmosis

Confused with Leishmania (similar morphology and found in RE system)

Causes "Spelunker's dx"

Found in bat and pigeon droppings

Blastomyces dermatitidis

Causes blastomycosis pp. 745

Paracoccidoides brasiliensis

Causes paracoccidiomycosis pp. 746

Opportunistic Mycoses: Clinical significance:

Nonpathogenic fungus that cause subcutaneous and disseminated infection

In immunosuppressed or debilitated patients

HIV and Diabetes mellitus

Treatment with corticosteroids, cytotoxic drugs and antimicrobials

Zygomycetes

Produces large, ribbon-like hyphae

ID by presence/absence of rhizoids, structure and position

3 commonly encountered:

Rhizopus, Mucor, Absidia

Cryptococcus neoformans

Causes Cryptococcosis

Aspergillus

Causes Aspergillosis

Most frequently encountered fungus in lab

A. fumigatus - most common spp.

Penicillium marneffei

From mucocutaneous infection to disseminated infection