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