COLONIAL CHARACTERISTICS Flashcards
Age:
young or maturity
Rate of Growth:
Rapid Growers
Intermediate Growers
Slow Growers
Matures in less than 5 days
Rapid Growers
Saprobes: uses organic matter but doesn’t harm the host
Rapid Growers
Examples: Aspergillus and Ganoderma
Rapid Growers
6-10 days
Intermediate Growers
Dermatophytes and most opportunistic fungi
Intermediate Growers
Examples: Candida, Microsporum, Trichophyton
Intermediate Growers
11 days or more
Slow Growers
Systemic and subcutaneous fungi
Slow Growers
Example: Blastomyces, Coccidioides, Histoplasma
Slow Growers
both the obverse (front) and reverse (back)
PIGMENT
most are dematiaceous (olive green) - causes an infection in both
immunosuppressed and immunocompetent individuals
PIGMENT
how colonies look
TEXTURE
leathery/ waxy
Glabrous
with little mycelia
Glabrous
short aerial hyphae
Velvety/Suede
bacteria-like in appearance
Yeast-like
(coagulase negative Staphylococcus)
Yeast-like
no aerial mycelium
Yeast-like
wooly, floccose
Cottony
long aerial hyphae
Cottony
Powdery
Granular
fungi that conidiate heavily
Granular
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
