MYCOLOGY

SPECIMEN COLLECTION &

HANDLING

Important considerations:

Proper collection

Rapid delivery to the laboratory

Prompt and correct processing

Inoculation into proper and appropriate medium

Incubation at a suitable temperature

Specimen collection & transport

Transport of Specimen:

Antibiotics may be incorporated in body fluid specimens to prevent proliferation of bacteria:

50, 000 units of Penicillin

100,000 units of Streptomycin

0.2 mg of Chloramphenicol

Specimen collection & transport

Transport of Specimen:

Storage temperature of specimen for fungal culture:

Blood & CSF: 30 – 37 OC

Dermatological: 15 – 30 OC

Others: 4 OC

Specimen collection & transport

SPUTUM first early morning sample

Deep cough specimen; may be induced by:

Aqueous aerosol

Bronchial tap

Volume: 5 – 10 ml

Specimen collection & transport

BLOOD and BONE MARROW

Transport medium: at 1:10 proportion

TSB or TSA (biphasic agar or broth)

BHI transport medium

Thioglycollate broth

Volume: 10 ml

Specimen collection & transport

CEREBROSPINAL:

Transport immediately. Do NOT refrigerate.

For suspected Cryptococcus, Coccidioides infections, containers must be leak proof and lab manipulations should be done under a hood

Specimen collection & transport

DERMATOLOGICAL SPECIMENSSKIN LESIONS

Sterilized area with 70% alcohol or sterile water

Collect at the the active border

Specimen collection & transport

NAILS

Clean with 70% alcoholIf: Dorsal plate:

scrape the deeper portion

Nail plate:

scrape beneath the nail plate

Whole nail or clippings

Specimen collection & transport

HAIR Collect from:

Areas of scaling

Alopecia

Hair that fluoresce under Wood’s lamp

Specimen collection & transport

EXUDATES & PUS

Undrained or unruptured abscess

Aspirate using sterile syringe, recap needle and transport to lab immediately

Failed aspiration, do skin biopsy

Specimen collection & transport

URINE First early morning

Transport and perform test ASAP within 2 hours

If not possible, refrigerate specimen.

Specimen collection & transport

VAGINAL SECRETIONS

Sterile swabs Put in transport medium or

primary isolation broth immediately (ex: TSB)

Specimen collection & transport

TISSUES & Biopsy specimens:

Collect aseptically at the center and edge of the lesion

Place in between sterile gauze wet with sterile NSS or transport medium.

Specimen collection & transport

MYCOLOGY

METHODS OF IDENTIFICATION

A. DIRECT FUNGAL MICROSCOPY

Clinical significance: Provide an immediate presumptive

diagnosis Aid in the selection of appropriate

culture media Aid in decision of what’s best

inoculation technique to use It will provide evidence of infection despite

negative culture

Macroscopic Examination (physical exam): Note for:

caseous material Purulent exudate Necrotic material Granules Punch biopsies Layers of skin that are broken vertically

(fissures) Obtain specimens for microscopy and culture

fro

A. DIRECT FUNGAL MICROSCOPY

Preparation for Microscopic examination: Mince or grind hard specimens Centrifuge for 3-5 minutes fluid specimens Pulvorize nail clippings Volume for fluid specimens: 0.5 ml Assemble a wet chamber for incubation

A. DIRECT FUNGAL MICROSCOPY

REAGENTS used for DIRECT MICROSCOPIC STUDY KOH 10-20%

Routinely used 10% = skin and soft tissues, body fluids 20% = nail and hard tissues

Calcoflour white Green flourescense

India ink “Dark field” microscopy for Cryptococcus

neoformans

A. DIRECT FUNGAL MICROSCOPY

STAINS for MICROSCOPIC STUDIES: Lactophenol Blue

very popular for quick evaluation of fungal structures stains the chitin in cell walls of fungi blue Use for following up fungal culture growths

Wright’s/Giemsa stain (Diff quick) For rapid staining of blood and bone marrow fungi (ex:

Histoplasma capsulatum) Modified Acid-Fast Stain

used to differentiate the acid-fast Nocardia from other aerobic Actinomyces

Gram Stain generally fungi are gram positive Actinomyces and Nocardia are gram variable

A. DIRECT FUNGAL MICROSCOPY

STAINS for MICROSCOPIC STUDIES:Stains for tissue mycoses: Periodic Acid - Schiff Stain (PAS)

stains certain polysaccharide in the cell walls of fungi Fungi stain pink-red with blue nuclei.

Gomori Methenamine Silver Stain silver nitrate outlines fungi in black due to the silver

precipitating on the fungi cell wall. The internal parts of hyphae are deep rose to black, and the background is light green.

Gridley Stain Hyphae and yeast stain dark blue or rose. Tissues stain

deep blue and background is yellow.

A. DIRECT FUNGAL MICROSCOPY

Stains for tissue mycoses … Fluorescent Antibody Stain

simple, sensitive, and extremely specific method of detecting fungi in tissues or fluids. Applications for many different fungal organisms.

Mayer Mucicarmine Stain will stain capsules of Cryptococcus neoformans

deep rose. Papanicolaou Stain

good for initial differentiation of dimorphic fungi Works well on sputum smears also

A. DIRECT FUNGAL MICROSCOPY

KOH Wet Mounts Principle:

KOH softens most tissues, dissolves fat droplets, bleaches many pigments and dissolves the “cement” that holds keratinized cells together; glycerine clears tissue debris, thus making it easier to demonstrate presence of fungal elements.

Reagents: 10 – 20 % KOH:

KOH pellets 10 – 20 grams Glycerine (optional) 10 ml Distilled water 90 ml

KOH Wet Mounts Procedure:

Place a small amount of specimen on a clean glass slide place 1-2 drops of KOH on the specimen and overlay a cover

slip Allow the preparation to stand for 10-30 minutes in a wet

chamber. You can gently heat preparation to hasten the action of

KOH Do not over heat for it may crystallize the KOH

Examine preparation under low then high magnification. Take note for the presence of fungal elements (hyphae and/or spores)

INDIA INK PREPARATION aka: Nigrosin stain Principle:

Specimen placed in a drop of India ink becomes darkly colored because of the carbon particle in the ink. Hyaline structures such as capsules and cell walls will be highlighted against a dark background of inked colored specimen creating an illusion of darkfield microscopy.

Reagent: 1:1 dilution of the ink

India Ink Preparation Procedure:

Place a drop of the specimen (body fluid or from culture) on a clean glass

Put a drop of India Ink, mix and overlay a cover slip Examine under low power and high power with a bright

field microscope Result:

India ink creates a dark background against which hyaline fungal cell wall and capsules can se seen

Limitation: wbc may be confused as fungi

Lactophenol Cotton Blue Principle:

The morphology of fungal elements are preserved and stained better.

Reagents: Lactic acid & Phenol

Kills the organism Glycerin

Prevents easy dehydration Cotton blue

Dye or stain

DIAGNOSIS OF MYCOSES by UNSTAINED & STAINED

MICROSCOPY

A. SKIN or DERMATOMYCOSIS

KOH for superficial involvement, look for:

Spaghetti & meat balls (lung aspirate) Malassezia furfur

Pseudohyphae and yeasts (vaginal secretions) Candida species

A. SKIN or DERMATOMYCOSIS

KOH & LPCB for superficial involvement, look for:

Hyaline septate hyphaeex: Dermatophytes

Dematiaceous septate hyphaeex: Tinea nigra

Alternaria

A. SKIN or DERMATOMYCOSIS

H & E stain for Oral Candidiasis on Skin biopsy of tongue, look for: Pseudohyphae yeasts

B. DRAINING SINUS for MYCETOMAS & ACTINOMYCOSIS

KOH, look for Various colored

granulesActinomycosis/

Nocardiosis

GMS stain, look for Various granules

Mycetoma

C. EYE SCRAPINGS & ASPIRATE for KERATOMYCOSIS

KOH & LPCB, look for Septate hyaline hyphae

Aspergillus species Fusarium species

Coenocytic hyaline hyphae Mucor species

Pseudohyphae and yeasts Candida species

D. NASOPHARYGNEAL ASPIRATES f for RHINOSPORIDIOSIS

KOH, look for

Large sporangium with spores (lacrimal gland aspirate) Rhinosporidium

species

E. HAIR for DERMATOMYCOSES & ALOPECIA

KOH, look for Endothrix spores/hyphae

Trichophyton

Ectothrix spores/hyphae Trichophyton

mentsgrophytes

E. HAIR for PIEDRA

KOH, look for:

Hard, brown, compact nodules (Black piedra) Piedraia hortae

Soft, off-white, concretions/nodules(White piedra) Trichosporon beigeli

F. NAILS for ONYCHOMYCOSIS KOH & LPCB, look for

Septate, hyaline hyphae Dermatophytes

Epidermophyton Trichophyton Microsporon

Pseudohyphae and yeast cells Candida species

G. SYSTEMIC MYCOSESSpecimens: blood, CSF, sputum, other body fluids

KOH & Mucicarmine stain systemic involvement, look for: Pseudohyphae and yeast

cells (CSF) Candida species

Broad based buds(brain and CSF) Blastomyces species

SYSTEMIC MYCOSES

GMS for systemic involvement, look for: Spherules/sporangia

(CSF)Coccidioides immitis

PAS stain for systemic involvement, look for: Dematiaceous septate

hyphae (brain tissue)

SYSTEMIC MYCOSES

H & E stain for systemic involvement, look for:

Endospores (CSF brain tissue) Coccidioides species

Fission/sclerotic bodies Chromomyces species

SYSTEMIC MYCOSES

Wright’s/Giemsa stain (Diff quick) & LPCB for systemic involvement, look for: Small, intracellular budding

yeast (CSF) Histoplasma species

Small, intracellular yeast dividing by fission (CSF) Penicillin species

SYSTEMIC MYCOSES

Mucicarmine stain & India Ink for systemic involvement, look for: Encapsulate yeast

(CSF) Cryptococcus

neoformans

SYSTEMIC MYCOSES

LPCB & Fluorescent Antibody stain for systemic involvement, look for Large yeast with multiple

buds called “mariner’s wheel” Paracoccidioides

braziliensis

SYSTEMIC MYCOSES Calcoflour mounts for

systemic mycoses , look for (flourescence) Pseudohyphae and

yeasts (blood) Candida species

Septate, hyaline at right degrees angle (bronchial lavage) Aspergillus species

Interpretation of Direct Microscopic Findings (Summary)

Observation Diagnostic Possibility

Yeast and pseudohyphae Candidiasis

Fine, hyaline, septate hyphae Dermatomycosis

Clear, hyaline, septate, branching hyphae

Aspergillosis

Clear, hyaline, coenocytic, branching hyphae

Mucor or Phycomycosis

Dematiaceous, septate hyphae

Tinea nigra or Phaeohypomycosis

Spaghetti and meatballs (spores and hyphae)

Tinea versicolor (Malassezia furfur)

Observation Diagnostic Possibility

Large sporangia (>300 microns) and spores

Rhinosporidiosis

Granules: varied shapes, colors, shapes and sizes

Mycetomas; Actinomycosis

Brown fission bodies Chromomycosis

Broad-based yeast cells with thick walls

Blastomycosis

Large, thick-walled yeast with multiple small buds (“mariner’s wheel)

Paracoccidioidomycosis

Spherules and releasing endospores Coccidioidomycosis