COLONIAL CHARACTERISTICS Flashcards by Arriane Cyrel (MTI)

Age:

young or maturity

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Rate of Growth:

Rapid Growers

Intermediate Growers

Slow Growers

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Matures in less than 5 days

Rapid Growers

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Saprobes: uses organic matter but doesn’t harm the host

Rapid Growers

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Examples: Aspergillus and Ganoderma

Rapid Growers

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6-10 days

Intermediate Growers

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Dermatophytes and most opportunistic fungi

Intermediate Growers

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Examples: Candida, Microsporum, Trichophyton

Intermediate Growers

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11 days or more

Slow Growers

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Systemic and subcutaneous fungi

Slow Growers

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Example: Blastomyces, Coccidioides, Histoplasma

Slow Growers

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both the obverse (front) and reverse (back)

PIGMENT

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most are dematiaceous (olive green) - causes an infection in both
immunosuppressed and immunocompetent individuals

PIGMENT

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how colonies look

TEXTURE

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leathery/ waxy

Glabrous

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with little mycelia

Glabrous

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short aerial hyphae

Velvety/Suede

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bacteria-like in appearance

Yeast-like

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(coagulase negative Staphylococcus)

Yeast-like

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no aerial mycelium

Yeast-like

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wooly, floccose

Cottony

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long aerial hyphae

Cottony

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Powdery

Granular

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fungi that conidiate heavily

Granular

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colony surface arrangement

TOPOGRAPHY

“lay of the land”

TOPOGRAPHY

o with radial grooves from center to the rim

Rugose

o like a bicycle wheel

Rugose

o parallel at right angles

Folded

o volcano-like

Crateriform

o wart-like

Verrucose

o rough knobs at the surface

Verrucose

o with elaborate folds and convolutions

Cerebriform

M. MACROSCOPIC EXAMINATION NOTE FOR:

 Caseous material  Purulent exudate  Necrotic material  Granules  Punch biopsies  Layers of skin that are broken vertically (fissures)

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

PREPARATION FOR MICROSCOPIC EXAMINATION

 Mince or grind hard specimens  Centrifuge for 3-5 mins fluid specimens  Pulverize nail clippings  Volume for fluid specimens: 0.5 ml  Assemble a wet chamber for incubation

- distorts colonial morphology

TEASE MOUNT

- rapid method

TEASE MOUNT

- may cause widespread dispersion of conidia

CELLULOSE TAPE SWAB

- morphology is preserved

CELLULOSE TAPE SWAB

- cannot be preserved for long periods

CELLULOSE TAPE SWAB

- substitute for wet mount

CELLULOSE TAPE SWAB

- 95% ethanol

CELLULOSE TAPE SWAB

- Applicator stick and tape (disadvantage: not sterile)

CELLULOSE TAPE SWAB

- needs tease-mount first

MICROSLIDE/AGAR BLOCK

- used for fungi with “delicate linkages” between conidium and conidiophore

MICROSLIDE/AGAR BLOCK

- used when (-) conidia

MICROSLIDE/AGAR BLOCK

- agar height: 4 millimeters

MICROSLIDE/AGAR BLOCK

- 10% for skin and soft tissues

KOH 10-20%

- 20% for nails and hard tissues

KOH 10-20%

- Clearing of specimens to make fungi more readily visible

KOH 10-20%

- Glycerol (glycerin: clearing solution) prevent crystallization

KOH 10-20%

- stand 10 to 15 minutes or heat gently (5 to 30 mins; 10 to 30 mins)

KOH 10-20%

- methylene blue may also be added (2:1)

KOH 10-20%

o methylene blue :

KOH

- use in the detection of fungi

Calcofluor White

- binds with chitin and cellulose

Calcofluor White

- detects fungi rapidly because of bright fluorescence (green fluorescence)

Calcofluor White

- may also be added to KOH (1:1)

Calcofluor White

- not for hair or thin scales of skin (specimen may dissolve)

DMSO

- Detection for encapsulated yeast

India Ink

- Used in dark-field microscopy

India Ink

- To detect capsule of Cryptococcus neoformans (agent of fungal meningitis)

India Ink

- With limitations since WBCs may be confused as fungi

India Ink

- Also known as Nigrosin stain

India Ink

- Most often used to detect H. capsulatum and C. neoformans complex in disseminated disease.

Wright’s Giemsa

- Used in differential count

Wright’s Giemsa

- Rapid staining of blood and bone marrow

Wright’s Giemsa

- stains the chitin in cell walls of fungi=blue

Lactophenol Blue

- popular for quick evaluation of fungal structures

Lactophenol Blue

- for rapid staining of blood and bone marrow fungi

Wright’s/Giemsa Stain

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

Modified Acid - Fast Stain

- Actinomyces and Nocardia are gram variable

Gram Stain

- Fungi are gram positive

Gram Stain

- stains certain polysaccharide in the cell walls of fungi

Periodic Acid – Schiff Stain (PAS)

- pink to red or purple with blue nuclei and green background

Periodic Acid – Schiff Stain (PAS)

- silver nitrate outlines fungi in black with lavender-gray areas due to the silver precipitating on the fungi cell wall

Gomori Methenamine Silver Stain

- internal parts of hyphae are deep rose to black, and the background is light green

Gomori Methenamine Silver Stain

- used in tissue sections

Gomori Methenamine Silver Stain

- to isolate aspergillus

Gomori Methenamine Silver Stain

- Tissues stain deep blue and background is yellow

Gridley Stain

- hyphae and yeast stain dark blue to rose

Gridley Stain

- simple, sensitive, and extremely specific method of detecting fungi in tissues or fluids

Fluorescent Antibody Stain

- dyes: tags with specific antibodies

Fluorescent Antibody Stain

- Cryptococcus neoformans: deep rose to red with nucleus

Mayer Mucicarmine Stain

- Stains capsules of Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis

Mayer Mucicarmine Stain

- differentiation of dimorphic fungi

Papanicolaou Stain

- for cervical and sputum specimen

Papanicolaou Stain

– mounting fluids

KOH and DMSO

– most commonly used in the lab

KOH, India Ink, LCPB

softens most tissues, dissolves fat droplets, bleaches many pigments and dissolves the “cement” that holds keratinized cells together

KOH WET MOUNTS PRINCIPLES

KOH WET MOUNTS REAGENTS: 10-20%

- KOH 10-20 g - Glycerin 10 mL - Distilled Water 90 mL

KOH WET MOUNTS PROCEDURE 1. Place a small amount of specimen on a clean glass slide 2. Place (?) of KOH on the specimen and overlay a cover slip

1-2 drops

KOH WET MOUNTS PROCEDURE 3. Allow the preparation to stand for (?), in a wet chamber a. You can gently heat preparation to hasten the action of KOH b. Do not over heat for it may crystallize the KOH

10-30 mins

KOH WET MOUNTS PROCEDURE 4. Examine preparation under (?). Take note for the presence of fungal elements (hyphae and/ or spores)

low then high magnification

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

INDIA INK PRINCIPLE

INDIA INK REAGENT

1:1 dilution of the ink

INDIA INK PROCEDURE 1. Place a drop of the specimen (body fluid or from culture) on a clean glass 2. Put a drop of India ink, mix and overlay a coverslip 3. Examine under (?) with a bright field microscope

low power and high power

The morphology of fungal elements is preserved and stained better

LACTOPHENOL COTTON BLUE PRINCIPLES

LACTOPHENOL COTTON BLUE REAGENT

- Lactic acid & Phenol (kills organisms) - Glycerin (prevents easy dehydration) - Cotton blue (dye or stain)

LACTOPHENOL COTTON BLUE PROCEDURE 1. Place a drop of the specimen (body fluid or from culture) on a clean glass 2. Put a drop of India ink, mix and overlay a coverslip 3. Examine under (?) with a bright field microscope

low power and high power

- for the rapid definitive identification of Candida albicans

GERM TUBE PRODUCTION TEST

GERM TUBE PRODUCTION TEST Incubate for (?)

2-3 hrs at 35 degrees celsius

GERM TUBE PRODUCTION TEST Place one drop on a slide after incubation o (+) result: (?)

germ tube

- most basic and easiest to perform for the identification of yeast

An appendage 1⁄2 the width and 3⁄4 the length

- may use plasma from expired blood bags

An appendage 1⁄2 the width and 3⁄4 the length

Formaldehyde - (?) of formaldehyde absorbed by cotton then placed in a tube - Formaldehyde-soaked cotton in a jar together with the tubes

10 drops

Storage - by slowing the metabolism of fungi a. (?) b. (?)

decreasing oxygen decreasing nutrients

Sealing with Parafilm

o 6 to 12 months interval of culture

o 24 months interval

Mineral Oil

o disadvantage: messy

Mineral Oil

o at –200 C to –700 C

Freezing

o 1 to 2 years interval

Freezing

o 2 mL of distilled water in culture

Sterile Water