Mycology Student Lab

Potassium Hydroxide (KOH) preation

10% Used for hair, skin, nails- dissolves keritin to make fungal visible Sputum/Vaginal secretion- used to dissolve cells to make fungal more visible Procedure: Add a drop to specimen on glass slide (or into a tube). Allow 15-30 minutes to dissolve and observes under low light or phase

Sexual sporulation

2 specialized fertile cell that have undergone meiosis to merge and have a nuclear recombination on the aerial hyphae

culturing dermatophytes

2 types of culture media: BHI or SAB and Mycosal incubate 25-30 (ex T. verrucosum require 35) Ambient pair Visible growth: 3-4 days, mature 1-3 Examine 2x a week ID base on colony and microscopic morphology Most just report genus level

yeast growth condition

2-3 days on sheep blood agar 22-37C, best at 30C

Fungal incubation temperature

28-30C is best (room temerpature is acceptble) 35-37 if converting dimorphic fungi to yeast phase

Zycomycetes fungi

Absidia: intranodal rhizoids Mucor: No rhizoids Rhizopus: nodal rhizoids Cunninghamella: lillipop sporangium Syncephalastrum: elongated meosporangium Circinella: long curved, coiled sporangiophores

Dematiaceous Septate Hyphae Fungi (14)

Alternaria- macroconidia with transverse and longitudianl septa Aureobasidium- older hypahe have "empty cells" Bipolaris- germ tube forms straight Cladosporium- brancehs with repeated forking chains of conidia Chaetomium- perithecia are flask with many straight or curly dark hairs Curvularia- crescent roll conidia, large central cell Dreshslera- gre mtube is perpendicular Epicoccum0 mutliseptate conidia, net like spetation Exophiala- profuse oval conidia Fonsecaea- schleortic bodies Helminthosporum velutinum- carrots phialophora- funnel shaped collarettes phoma- conidia ooze out of pycnidium through ostiole Ulocladium-broady elipitcal condia

Hyaline fungi names (9)

Aspergillus fumigates- common pathogen Aspergillus niger (swimmers ear) Aspergillus flavus Pencillium Penicillium marneffei- dimorphic, emerging pathogen Fusarium0 canoes, mycotic keratitis Sepedonium Paecilomyces- pointing phialides Scopulariopsis- lemons, white/tan Acremonium Chrysosporum- white, with yelow reverse

Inhibitory Mold Agar (IMA)

Best for isolating opportunistic fungi from non-sterile sites Primary recovery for dimorphic pathogens or saprophytes that are inhibited by cycloheximide Antibiotics (chlorapehicol) inhibit growth of bateria Primary isolation, nonselective Pathogenic fungi not dermatophices

Piedraia hortae

Black piedra- superficial infection of hair shaft characterized by black nodules on hair. Hair breaks Central/South America, Asia, africa Lab: i. Hyphae closely septate, dark and thick walled ii. Intercalary chlamydoconidium-like cell iii. Asci and ascospores may form in culture LAB: KOH of hair

Blastomycosis

Blastomyces dermatitidis Endemic in Mississippi and Ohio River basin Found in soil, leaf litter, etc Causes acute or chronic suppurative and granulomatous infection Infection begins in lung by inhaling conidia or hyphal elements Can spread and involve lungs, long bones, soft tissue and skin

Types of Fungal Collection

Bone marrow CSF Cutaneous (Hair, nail, skin scrapping) Respiratory Tissue biopsy Urine Vagnial, uterine, cervical, prostatic secreation Wound, subcutaneous lesions, mucocutaneous lesions, exudes Cyst and abscesses (aspirated)

Zygomycetes

Broad, asepate hyphae, often branching Rapid growing (lid lifter), very cottony Produce sporangiospores inside sporangium May produce rhizoirs

Respiratory Fungal Samples

Bronchial washing, sputum, throat and transtracheal aspirate Early morning is best 24 hrs specimens are not acceptable- bacterial over growth

Blastoconidia

Budding forms characteristically produced by yeast A scar often remains at the point where conidium detatches Daughter cells

Methods of Characterizing Fungi

By disease By class

Antifungal susceptibility test

CLSI has 3 methods: Yeast Testing, Mold Testing and Disk Diffusion (microtiter and Etest) Lack of established break points for most fungal agents Emerane of antifungal

Dematiaceous fungi clinical

Can cause a range of infections from paranasal sinus infections, visceral, and blood infection, cutaneous ro subcutaneous Important to detemine fi contaminant or disease causing

Germ Tube

Candida albicans .5 mL of serum, inculate with yeast (.5 m L) 35C-37C for 2-4 hrs Drop on slide and examine on low power Postive: hyphal extension that arises with no constriction . 1/2 width and 3-4x times the length of yeast cell. No nucleus.

Yeast infections

Candida albicans Blastoschizomyces Cryptococcus Hansenula Malasserzia Saccaromuves Trichosporon Geotrichum

ingredient required in fungal media

Carbon, Nitrogen, Vitamins, Minerals, and amino acid

Opportunistic mycoses

Caused by non-pathogenic fungi Usually seen in immunocompromised or debilitated patients Subcutaneous or disseminated

Dermatophytes clinical significance

Causes Tinea (ringworm) or infections that involve superficial areas of body including hair skin and nails Break down and utilize the keratin as source of nitrogen

Acremonium

Causes white grain mycetoma Hylanie. World wide. SAB Colony: 1-3 weeks, gray~ish brown to gray~ish violet, glabrous to downy. Pale reverse Microscopic: hyline sepate. Phialides solitary long and narrow, septum at base with scarcely visible collarestte and apex. Conidia oblong to ovoid in cluster and philalides

Phaeoannellomyces werneckii

Causes: Tinea nigra (darkly pigmented, non inflammed skin lesion) Superficial mycoses Lab: KOH prep of skin scraping Yeast-like double and single celled conidia Beige yeast like colonies that mature into black velvet-napped mold

Fungal Acid Fast Stain

Certain filamentous bacteria will stain acid fast (Nocardia) Yeast Saccharomyces cerevisiae will stain acid fast

Cladosporium type

Chains of dark staining elliptical conidia that are branching, conidia often show scars at points of attachment. Branched condidium-bearing cells appear like a shield due to having 3 scars

Fungi cell wall contains

Chitin, mannans and sometimes cellose

Fonsacaea pedrosoi/compacta

Chorombastomycosis Colony: 2-4 weeks. Black-browh, grey black or jet black with downy texture Microscopic: Dematiacious septate hyphae. Conidiophores cylindrical, slightly inflated at tip. Produces many types (acrotheca, cladosporium and phialophora)

Cladophialophora carrionii

Chromoblastomycosis Colony: 1-4 weeks. Dark olive brown to brown-black, velvet teture Microscopic: dematiacious sepate hyphae. Cladosporium sporulation

Phialophora verrucosa

Chromoblastomycosis Colony: 2-4 weeks. Dark grey, brown or black. Vevetly to wooly. Microscopic: diatomaceous hyphae, phialorphora type sporulation

Chromoblastomycosis

Chronic fungal infection via traumatic inoculation Involves skin and subcutaneous tissue (feet, legs) Develop papule and site of infection that slowly spreads to form warty or tumor like lesions (cauliflower) LAB ID: Brown sclerotic bodies, non budding structures occurring stingly or in cluster- seen tissue= diagnostic Secondary infection and ulceration may occur

Mycetoma

Chronic granulomatous infection: Triad of sympotoms: 1. Swelling of subcutatenous tissue causing tumor-like deformities 2. Multiple sinus tract (fistula) that tunnel through subcutaneous tissue to surface 3. Drainage contains sulfur granules Slow progresses to bone, muscle or other contiguous tissue

Sporotrichosis

Chronic subcutaneous infection Due to Sporothrix schenckii Rose Gardener's Disease Primary lesion: small, non-healing ulcer (hand/finger) Nodular lesions of skin and subcutaneous tissue at point of contact evelope Can involve lumphatic channels and lymph nodes draining at region Only rarely can disease disseminate Pulmonary infection can be seen

tinea favus

Chronic, severe dermatophytosis of scalp, thick crust and scarring occurs Host genetic sand inability to mount a good immune response are predisposing factors From Mediterranean/N Africa Trichophyton schoenleinii

Nail fungal culture

Clean with 70% alcohol Scape away and dispose of outer layer Sample from beneath nail plate to obtain softened material from nail bed or shaving from deeper protions

Skin fungal samples

Clean with 70% alcohol If lesion present- scrape actively growing edge (Can draw sharpie circle to find actively growing) Scrape most infected area

Coccidioidomycosis

Coccidioides immitis Also known as San Joaquin valley fever Endemic to San Joaquin Valley CA, Maricopa and Pima AZ and Sothwestern Texas 60% of infections are asymptomatic and have self-limited respiratory tract infection Infection beings in lung by inhaling arthroconidia (high infection, 10 can cause infection) Can disseminate (1%) with extension into visceral organs, meninges, bone, skin, lymph nodes and subcutaneous tissue

Spiral hyphae

Coiled or corkscrew-like turns in hyphae Trichophyton mentagrophytes

Fungal specimen collection Issues

Collect from infected area Use sterile technique, avoid contamination with hands Adequate size Specimen must be moist Properly labeled, exact source/site (identification aid) Delivered to lab promptly and quickly processed - Prevents overgrowth of bacteria and ubiquitious mold -Pathogenic molds can be slow growing - Yeast multiple quick- refrigerator if delay in setting up

Flat

Colonies are flat against the agar

Rugose

Colonies have deep furrows irregularly radiating from the center of the colony

Verrucose

Colonies with wrinkle, convoluted surface

Identification of dermatophytes

Colony morphology and microscopic morphology Hyaline septate hyphae, macro and/or microconidia Scotch tape preps or slide culture Generally only reported at genus level (what needed for treatment)

Yeast

Colony: Bacteria-like, moist, smooth, creamy colonies Cells- single, round to oval cells Reproduce asexually by budding or fission Gram stain positive (much larger than bacteria)

Mold

Colony: Grows hyphae that from a mat called mycelium Cells: multiple cells that from a filamentous mycelium Reproduce either asexually (vegetative sporulation or aerial) or sexually

Fungal pigmentation

Colors on both sides of surfaces (Front and reverse)

Acrotheca sporulation

Conidial heads with sympodial arrangement of conidia with primary conidia giving rise to secondary conidia

Sporangiospores

Contains a sac called sporangium Supported by base called columella Tip of the specialized hyphal segmented called sporangiophore

Ectothrix infection

Dermatophytic infection with arthroconidia around the shaft of hair Fungi stars inside the hair shaft, damages cuticle and then grows inside amd outside of hair More acute inflamed lesion, moist, boggy

Tinae barbae

Dermatophytosis of beard and moustache Highly inflamed, acute pustulur folliculitis Caused by: Trichophyton mentagrophytes

Sporothrix schenckii

Dimorphic Mold: 3-5 days, 25-30 fro 2-4 weeks Colony: white- black on surface and reverse, glabrous moist Microscopic: hyaline septate hyphae, conidia hyaline to brown, group in ROSETTES at tip of conidiophore Yeast: 1-5 days at 35 Cream to beige colonies with cream testure. Yeast ovoid, elongaed CIGAR-SHAPE producing 1 to several bunds

Basic characteristic of Systemic Mycoses

Dimorphic - 2 stages Mycelial at 25-30C Yeast at 35-37C Definitive diagnosis, must demostrate both stages

Systemic mycoses

Disseminated Caused by True pathogenic fungi Involves deep tissue and organs Spread widely through the body (Blood stream) Initial site of infection is the lungs

Scotch Tape Prep

Drop of LPAB on slide, touch transparent tape on surface of coloy and remove, place tape on slide (in stain) and added coverslip. Observe at 10 and 40x

Fungal Urine samples

Early morning Clean catch or catheterized specimens Centrifudge and inoculate media

Microsporum gypseum

Ectothrix tinea capitis (hair and skin) Grows: 3-5 days Flat, white colones that turn brown/reddish and granular as conidia are produced Reverse: Light tan Microscopic: Macroconidia: moderately thick-walled, rough surgace, ellipitical, rounded tip and mutli-cellular (6+) Microconidia: few to absences Does not fluoresce

Neutral Sabouraud Dextrose Agar

Emmon's modification Yeast subculture Less dextrose and neutral pH

Nocardia asteroides

Enviromental: soil Pulmonary nocardiosis, acute imflammation with pus, coughing and difficult breathing Flamentous projects around edges, musty order Weakly acid fast positive (Gram + rods with branching)

Streptomyces

Environment, soil Opportunistic pathogen Macr: bacterial colony, musty odor Micro: hyphae seen on GMS, Catatlse _ , Gram + rod, No acid fast positive

Fungi are classified as

Eukaryotes Have nucleus and organelles Rigid cell wall No chlorophyll Uni or multicellular

Granular/powdery

Flat and crumbly due to dense conidia production (Aspergillus)

Chlamydoconidia

Formed from pre-existing cells in hyphae which become thickened and enlarged, maybe be found within (inercalary), along side (sessile) or at the tip (terminal) Form during poor enviromental conditions which germinated and produce conidia when better climate occurs

Systemic mycoses epidemology

Found primary in North America (Except Paracoccidiodes) Soil, decaying vegetation, bird and bath droppings

Dimorphism

Fungi that have ability to exist in two forms depending on growth conditions Yeast/Tissue- 37C Mold- 20-30C

Pneumocystis carinii

Fungus and parasite properities: cholesterol in membrambe, anti-protozoals, trophozoite form, chitin in cell wall, fungal ribosomal Disease: PCP (pneumonia)=? HIV. Can be passed person to person and be disseminated Lab: Cyst in bronchial lavage, sputum, biopsy. Unable to culture. Fluorescent antibody, GMS stain

Oospore

Fusion of two 2 morphologically identical cells from 2 different hyphae phylum Oomycota.

Yeast ID methods

Germ tube Urease Carbohydrate assimulation and germentation Commerical Id systems

Cryptococcus neoformans Id

Gram stain: large round budding yeast India ink- polysaccharide capsule Urease- rapid positive Niger seed agar, brown pigment Cryptococal antigen test

Microsporum audouinii

Grey-patch tinea capitis in children (Hair/skin) 2 weeks Growth Plate: white to salmon pink Reverse: tan to salmon pin Microscopic: sterile hyphae with terminal chlaydospores, favic chandliers and pectinate bodies Rare macro and micro conidia Fluoresce under Wood's Lamp

Annelloconidia

Grown from inside a vase-shaped conidiogenous annellide. The tip of the annellide will extend and react when the conidia leave, leaving scars scopulariopsis

Microsporium canis

Hair and skin- tinea capitis, corporis (cat and dog) 3-5 day growth Flat w/ feathery edge. White and silky-> later yellow pigment in periphery Reverse: lemon yellow/yellow brown Macroconidia: spiney surface (echiniilated), thick wall, pointed curved ends Microconidia: rarely seen- tearshaped Hair fluoresce under wood lamp

Endothrix form

Hair dermatophyte Fungi infection starts inside hair shaft but does not destroy cuticle of hair Dry scaly brittle lesions, usually anthropophilic organism

Tricophyton rubrum

Hair, skin nails (corporis, pedis, cruris, capitis) 2 weeks growth Plate: white to pink, granular with rugal folds Reverse: whine red color (yellow when young) Microscopic: Macroconidina: usually absent (thin wall, pencil shape) Microconidia: birds on fence (tear drop shape) Urea hydrolysis -, hair perforation -

Trichophyton metagrophytes

Hair, skin, nail infection (Corpris, capitis, barbae) 3-10 days growth Plate: Vaires, granular and cottony, white to tan Reverse: buff to reddish browh Microscopic: Macroconidia: smooth, thin walls, cigar/penicl shape and multi-celluar. RARE Microconidia: globe-like/tear drop shape in grape like cluster or laterally along hyphe Urea hydrolysis + and hair perforation +

Cotton/wooly

High, dense aerial mycelium (zygomyces)

Wright Stain

Histological stain for fungus Used to look for intracellular yeast in tissue and bone marrow

Periodic acid-Schiff (PAS) stain

Histological/Tissue stain Fungal elements stain magenta against green backround Can be done on a swab/sputum/spine- must have tissue

Histoplasmosis

Histoplasma capsulatum Endemic in Missouri, Mississippi, Ohio River delta (increase moisture) Found in Bird and bat feces 95% of the cases are asymptomatic Start as mild respiratory disease and eventually invades retculoendothelial system Can disseminate to lymphatic tissue, liver, spleen, kidneys, etc

Fungal incubation time

Hold culture for 4-6 weeks Examine 2x weekly for growh Time of growth is dependent of media, temp and inhibitors in specimen

Tween 80/Oxgall/Caddeic acid agar (TOC)

Hybrid media observe brown pigment from C. neoforms Observe germ tube by C. albicans Better chlamydoconidia develpment than cornmeal-tween 90 (special id media)

conidiophore

Hyphae segment Pencillin/Aspergillius

Dermatiaceous fungi

Hyphae/conidia are dark due to melanin-like pigament with septation (septate) Colonies are dark gray, brown, black, wollu, hairy or velvety with smoky gray to jet black reverse Distinctive macrocondia

Risk factors for Fungal Disease

Immunocompromised patients are hightest risk Organ transplant Individual treated with corticosteroids, cytotoxic agents or prolong antibiotics Immunologic and metabolic disorder Occupational hazards involving direct contact and inhalation/ingestion of infected animals or contaminated matieral

Fungi without a sexual stage

Imprefect fungi

Fungal Tissue biopsy

Include normal and infected tissue. Infected tissue from both center and edges of lesion. Inspect for granules and areas of pus and necrosis

tinea corporis

Infect the body (ringworm) Circular lesions that start from central point and radiate outward, scaling, redness, Usually acquired from zoophilic Lesions on arm, face, neck- children Caused by trichomphyton metagophytes/rubrum or microsporum canis

Etiological Agent of Subcutaneous mycoses

Intermediate Growth rate: 1-4 week World wide (Saprobes- soil and plates) Aquired by mechanical mean: splinters, thrones, sticks Ability to cause disease depends on the type of tissue, immune system and amount of exposure Generally a lesion at site of pentration- ski, subcatueous tissue, fascia, tendons, muscle and bone Rarely can be disseminated into visercal organs 2 grous: dematiaceous septate hyphae and hyaline septate hyphae

Epidermophyton floccosum

Invades skin and nails (Tinea cruris, unguium) Grows 3-5 days Plate: flat, starts as grey-white an then develops to khaki-green with folded center. Periphery is yellow with feathered edges Reverse: Yellow/brown with folds Microscopic: Macroconidia- abuntant large, smooh, thin wallked, mutli-cellular, clavate and in cluster No microconidia

Superficial and Cutaneous mycoses

Involve top most layer of skin and hair Not life threatening, very little immune response Affects dead keratinizied cells (slough off) Often caused by Dermatophytes (Trichophyton and microsporum)

Yeast basic characteristics

Isolate form any specimen- generally normal floar, but can be opportunstic Unicellular Reproduce: asexual (blastoconidia- budding). Can give pseudo hyphase (elongated blastoconidia) or Sexual (ascorpores)

Niger Seed agar/Bird seed agar

Isolation of Cryptococcus neoformans C. neoformans has a phenoloxidase enzyme that breaks down caffeic acid to form a brown pigment

Coccidioides immitis

Lab diagnosis: Sputum, biopsy of affected organ Direct direction: KOH, calcofluor white stain. lokk for nonbudding THICK WALLED SPHERULE (30-60 um) containing ether granular material or number small endospores (Cultivation is not advised) Mold: 4 week at 22 C (will grow at 35) Rapid White-grey colones. Hyaline, spetate hyphae. conidiophores are absend ARTHROCONIDIA unicellular, rectangular to barrel shaped, oftend wider in diamer than hyphae- alternatng with empty cells (disjunctors)

Blastomyces dermatitidis

Lab uses cutaneous lesions, sputum, biopys Direct detection: KOH + calcoflour white Look for LARGE, refractile yeast with double-contoured wall and buds connected to parents cell with BROAD BASE Yeast: DIAGNOISTIC 1 week at 35, white-beige creamy, verrucose Mold 1-4, up to 6 weeks at 22C White, tan or brwon, reverse is pate Nondescript hyaline septate hyphae, conidia borne on short lateral branches that are ovoid to dumbbell shaped

Paracoccidioides brasiliensis

Lab: Yeast in Sputum Yeast: 35-37 C, Large, fairly thick with multiple buds. Connected with narrow connection giving ship wheel appearance Mod: 25-30C, slow growth (3 weeks) White, heaped, compact colones Septate, branch hyphae with come intercalary and terminal chlamdospores

Hyaline fungi

Light coloed with sepatation Morphology- vary from white to greens to tan to beige to colors (beiges on reverse) Clinical: Ear, lung, granulomatous infection Important to determine if contaminate or disease

Hyaline

Light colored hyphae and/or conidia due to no pigmentation or brightly pigmented

Hyphae

Long stand of tube structures 2 types: Aseptate and Septate

Molecular probes for fungal disease

Looking for fungal DNA in serum and urine Used for: Histoplasma capsulatum Blastomyces Coccidiodes Cryptococcus

Actinomyces israelii

Lumpy jaw Inhibits GI tract of humans and animals Molar tooth colony, musty odor Sulfur granules, gram + with branching rods

Molds that cause superficial mycoses

Malassezzia furfur Phaenoannellomyces Trichosporon Pierdraia hortae

Systemic mycoses clinical signficance

Man is incidental host Acquired by inhalation and develop respiratory infection Can become systemic- involvement of internal organs including lymph nodes, bone, subcutaneous tissue and skin May be asymptomatic Immunocompromised and long term corticosteroid therapy patients are more prone

Mycelium

Masses of intertwined, branching hyphae forming a mat of growth

Fungal Microscopic Morphology

Most definite means of identification Evaluate: Shape, method of production, arrangement of conidia/spores, size and color of hyphae

Fungi growth requirement

Must absorb nutrients from environment (agar with added nutrients) prefers neural pH but can tolerate a large range Optimal growth 20-30C. Dimorphic Yeast: 37C Obligate aerobes Need moisture for growth, but survive dry conditions with spores/conidia

Psuedallescheria boydii

Mycetoma Sexual state Asexual: Scedoporium apiospermum Colony: 1-2 weeks, White that become brown, wooly to cotoon. Pale reverse with brownish-black zobe Microscopic: hyaline spetate, conidiophores with annellides simple or branched. Brown cleistothecia present after 2-3 week

Candida

Normal flora: skin, mucous, intestinal Leading opportunistic infection in men Common spp: albicans (germ tube + glabrata, tropicalis, brusei, parapsilosis

Yeast direct observation

Observe for reporductive structire Gram stain +, large budding and psuedohyphae India ink- capsule

India Ink Preparation

Only useful to detect capsule Cryptococcus neoform Procedure: mix small drop of ink with specimen and and cover slip. Let sit for 10 minutes. Observe under light mircoscope

Paracoccidiomycosis

Paracoccidioides brasiliensis Found in South America, mostly Brazil and Columbia A chronic granulomatous disease characteristically begins in the lungs Can spread to mucous membrane of nose, mouth, GI tract Dissemination to skin, lymph nodes, other internal organs is common

Fungi with a sexual stage are called

Perfect Fungi

Anti fungal classes and agents

Polyenes: Amphotericin B (primary anti-fungal) Azoles: Fluconazoel (yeast), Intraconzaole, Voriconazole Candins: Caspofungin

Lactophenol Aniline/cotton Blue (LPAB)

Primary dye used for molds Phenol kills organism Lactic acid preserve fungal structure Aniline chains chitin walls of fungal cells Look for fungal elements

Brain Heart Infusion (BHI) w/w out 5% blood

Primary isolation, Nonselective media Saprophytic and dimorphic fungi Isolation of Histoplasma and Nocardia Useful to convert dimorphic fungus from modl to yeat phase when incubated at 35C Added antibiotics (cyloheximide and chloramiphenicol) can make it selective for dimorphic

Fungal Culture set up

Primary media: BHI w/ 5% blood or SAB Specia: SAB Emmons, Potato flake or dectrose Skin, hair, nails- KOH and then SAB and Mycosel- may need to press in with tweezer Sputum, Bronchial, Mucus- directly on to media, washers- centifudge-> SAB, Mycosel, BHI Tissue- grink, SAB, Mycos, BHI

Sabouraud's brain heart infusion agar (SABHI)

Primary recovery osf saprophytic and dimorphic fungi Greater recovery than SAB or BHI Non-selective, primary isolation

Purpose of Direct Examination of Fungal Culture

Provides a rapid preliminary and immediate presumptive diagnosis (mold= same treatment) Unique morphology characteristics may aid in ID Helps media selection Direct examination can show evidence of infection despite negative cultures See yeast phase of dimorphic fungi

Basic characteristic of opportunstic fungi

Rapid growers (2-3 days, mature in 4-5) Found in soil (saprobic) Conidia/spores are airborne Normally inhaled, dependent on body's response after inhalation Immunocompromised pt Treatment can be toxic- must determine if its a contaminant

Fungi Growth Rate

Rapid- 1-5 days Intermediate: 6-10 days Slow: 11-28 days

Fungal Immunodiffusion

Reference lab Aspergillus, Blastomyces and Histoplasma

Dermatophytes

Relatively rapid growing mods 6 spp cause 98% of dermatophytoses or tinea (or infection in superficial area of body such as skin, hair and nails) Break down and use Keratin as nitrogen Spp: Microsporum, Epidermophyton and Trichophyton

Rhiziods

Root like structures that may be located at the base of a sporangiophore or internodally along the hyphae Rhizopus: Nodal Absidia: Intranodal Mucor: No nodals

metulae

Secondary segment between the conidiaphore and phiadlides

Dermatophyte test agar

Selective, pirmary isolation media Screening for dermatophytes pH change cause phenol red to change from yellow to red (if dermatophytes is present)

Mycosel agar

Selective, primary media Selectis dermatophytes Chloarmphenicol inhibits bacteria and Nocardia Cycloheximide inhibits rad saprophytes and some yeast

Fluorsecent antibody

Seroligcal diagnostic Pneumocystis (digs into muscle and does NOT culture)

Fungal ELISA

Serologic diagnosis Aspergillus

Fungal complement fixation

Serologica diagnosis Reference lab Used for Blastomyces, Coccidiodies and Histoplamsa

Fungal EIA

Serological diagnostic Blastomyces and Candida

Fungal Latex agglutination

Serological diagnostic Cryptococcus and Candida

yeast colony morphology

Similar to bacteria glabrous, mucoid, wribkled, velvety White, cream, tan Catalase +, coag -

Microconidia

Single-celled, small conidia, borne on side of hyphase or supported b a hair-like conidiophore

Differentiating Pathogenic fungi from culture

Slow growers (10+ days) growth on mycosel agar Dull buff, brown, mousey grey in color Dimorphic, mold phase at room temperature (enivormental), yeast at body temp (37)

Fungi natural habit

Soil and Vegetation Found everywhere

Saline wet mount

Spine fluid, excudate Used to view hyphae, conidia (fungal elements), budding yeasts, spherules, capsule

conidia

Spores produced on the surface of an elaborate fruiting body supported by a hyphal segment called conidiophore Can branch into secondary segment called metulane which can become condia producing segment called phialides

Molds that cause subcutaneous infections

Sporothrix Phialophora Cladosporium

Histoplasma capsulatum

Sputum, blood, bone marrow, bioposis Detection: Giema/Write stain, cacloflour white, GMS, PAS, H&E on tissue Mold: DIAGNOSTIC form hyaline, septate hyphae. Macroconidia are unicellur, hyaline, thick-walled, smooth or warty (tuberculated) Microcondia simialr looking Yeast:: 2-4 weeks at 35C Cream cloly. Small yeast. Can been seen packed in macrophage in tissue

Fungal Gram stain

Stain Gram positive Look for yeast or fungal elements (Hyphae) 2-3x wider than GPR and not solidly stain Yeast capsule interfere with staining

Bone marrow fungal collection

Sterile tech and inoculated into biphasic agar-broth bottles (special for fungi)

CSF fungal culture

Sterile technique, centrifudge, and use sediment to make slide (gram stain) and inoculate media

Potato dextrose agar

Stimulates spore formation and pigmentation use for slide cutlure special identifcation media

Sabouraud Dextrose Agar (SDA)

Subculture/idendifcation media Supports growth of almost all fungi contains dextrose, peptone, agar and water pH- 5.6 = inhibits bacteria

columella

Supporting structure at the tip of a sporangiophore and base of a sporangium. Most, but not all, zygomycetous fungi produce columellae

Calcofluor white stain

Tells whether or not fungal elements are present Binds to polysaccharides in fungal cell wall Fluoresces when exposed to UV (Apple Green) Procedure: 1 drop to specimen and coverslip. Sit 3 minutes, Use florescent microscope. KOH can be added ahead to dissolve background

Favic chandeliers

Terminal hyphal branches that are irregular, broad, and antlerlike in appearance. Especially characteristic of Trichophyton schoenleinii

sporangiophores

The stalk that a sporangiosphore sits on

Malasseziosis

Tinea versicolor: hypo or hyper pigmented patches of scaly skin Folliculitis Seborrheic dermatitis and dandruff

Gomori Methanamine Silver (GMS)

Tissue/Histological stain Stains fungal elements black against green backround

Fungal Safety

Tube or Flask is preferred- longevity and reduced chance of contamination Never plate media when Coccidioides immitis is suspected as it extremely infectious and aerosol may be inhaled Always work under biological safety cabinet Wear gloves and lab coat Autoclave specimen and inoculate media when complete Disinfected work surface daily

Hair fungal samples

Use Wood lmap to see infected area Pull out hair by roots with sterile tweesers IF not fluorescence- pull out hairs that are broken and scaly

Slide culture

Use a small square of agar (SAB, Potato Dextrose, Mueller Hinton) on slide. Inculate with fungus then place cover slop and growh in humidified chamber Remove cover slip and place in LPAB on 10x and 40x Allows for observation of colony as it is growing with disturbing hyphal/conidial arragements

Trichosporon beigelii

White piedra- superficial infection on hair shaft characterizied by soft nodules on hair Central/south America, Japan. Endland, Asia Lab diagnosis: i. Observe hyphae and arthroconidia ii. techically a mold, but initial growth resembles yeast ID by ueas assimilation test and morphology on cormeal agar

Tease mount

With two sterile teasing needles, transder a portion of colony to the slide, gently tease mycelium apart, add LPAB and observe at 10x and 40x

Geotrichum candidum

Yeast (not a true yeast) ID: Negative Germ tube after 3 hours Arthocondia and true hyphae Urease negative

cornmeal tween 80 agar

Yeast morphological structures Promotes hyphal and blastoconidia Observe psuedohyphae and chlamydoconidia produced by Candida albicans

Zygomycetes clinical

Zygomycosis Infects nasal sinues, black discharge spreads to blood vessels, causes necrosis and blood clots Can migrate to brain an dmeninges which can lead to rapidly fatal meningoencephalitis (2-10 days)

cerebriform

brain like

Malassezia furfur

causes tinea versicolor superficial mycosis More common in tropical area culture from skin scappings Diagnostic: Organism is lipophilic- add olive oil to media surface for growth Spaghetti and meaballs appearance- oval to round year as well as short, spetate, branching hyphae

Velvety/silky

colonies produce low aerial mycelium (Cladophialophora, Trichophyton)

umbonate

colonies with a button-like central elevation

Phialoconidia

condindia that arises from a vase-shaped cell called a phialide

umbilicate

convex with depressed center

Dematiaceous (olivaceous)

dark colored (brown-black) hyphae and conidia due to presences of melanin in cell wall

Molds that cause cutaneous mycoses

dermatophytes (Trichophyton, Microsporum, Epidermophyton)

tinea pedis

dermatophytosis of feet Defect in Kertin in foot for fungi to adhere Sweet moisture cuases small fissure and allow organism to adhere Typically starts between 4th and 5th toe and extends white, dead epidermis, flaky Cuased by: Trichophyton rubrum

tinea cruris

dermatophytosis of the groin Red raised lesion, itchy, can extend to but, thighs Spread via towels and cloting caused by: Epidermophyton, Trichophyton rubrum

tinea unguium

dermatophytosis of the nails Deformed, chalky, brittle nails with striations across the nail Starts at nail bed and works inwards/deeper Caused by: Epidermophyton

Mycoses

diseases caused by fungi

Psuedohyphae

elongation of blastoconidia (spores) showing sausage like constriction between segment. True hyphae do not construct at ends (Canidida albicans)

Vegetative mycelium

extends into agar and is responsible for absorbing water and nutrients

Phialophora type

flask-shaped philides witha collarette at tip Conidia- oval, one-celled, occyring at the tips of phialides in balls

tinea capitis

fungal infection of the scalp, eyebrows and eyelashes Caused by: Microsporium canis/gypseum, Trichophyton metagrophytes/rubrum

Zygospore

fusion of 2 morphologically identical cells from same hyphae structure

ascospores

haploid spore/sexual spores produced in a sac like structure called ascus and my be protected by a cleistothecium

capitate

head shaped colony

Septate

hyphae with cross walls Divide cells into subdivision by transverse cross walls

Aseptate (coenocytic) hyphae

hyphae without septa or transverse crosswalls

Pectinate bodies

hyphal projections that look like broken combs

Subcutaneous mycoses

involve skin, muscle and connective tissue immediately below the skin Caused by puncture into skin (traumatic implantation into skin)

Nodular organs

knots of twisted hyphae

Macroconidia

large multi-celled conidia which are produced by dermatophytes in culture

Fungal Incubation atmosphere

moist- 40-50% humidity Ambient air

Saprobes

organisms that obtain food from decaying organic matter/decomposers

aerial mycelium

portion that projects above the agar surface Special spore or conidia bearing fruiting bodies derive from this portion

sporangium

sac or case in which fungal spores are produced

Glabrous

smooth surface due to no aerial mycelium (auerbasidium)

Racquet hyphae

tennis reqcuets with smaller end attached to large end of adjacent club-shaped hypahe

Arthroconidia

thick-walled barrel-shaped conidia produced by fragmentation of the hyphal strand. May form adjacent to each other or may be separated by alternating empty spaces

phialides

vase shaped cells that produce chains of conidia called phialospores

crateriform

volcano with central depression

Dermatophytes epidemiology

worldwide Anthropophilic (primary found in human) Zoophilic (animals, such as cats and dogs) Geophilic- found primary in soil