Fungal Lab ID - Stain, Culture, Tests Flashcards
Visualization Tests for Fungi
10% KOH Lactophenol Cotton Blue (LPCB) Grocott-Gomori Methenamine Silver stain Periodic acid-Schiff -PAS (mucin stain) Gram Stain/Hematoxylin and Eosin India Ink
Fluorescence under UV light Tests Fungi
Calcofluor white
10% KOH method
Strong alkali; softens and clears keratin in hair, skin, nails so hyphae and conidia can be seen clearly
10% KOH purpose
Detects yeast and/or hyphae in hair, skin and nails; does not identify species
LPCB principle
The fungal spore cell wall is made up of chitin of which the components of the Lactophenol Cotton Blue solution stains for identification.
The solution is clear and blue in color and it is made up of a combination of three main reagents:
Phenol: It acts as a disinfectant by killing any living organisms
Lactic acid: To preserve the fungal structures
Cotton blue: To stain or give color to the chitin on the fungal cell wall and other fungal structures
The stain will give the fungi a blue-colored appearance of the fungal spores and structures, such as hyphae.
LPCB limitations
It can only be used as a presumptive identification method of fungi which should be followed up with other diagnostic tools such as biochemical and cultural examination.
Grocott-Gromori’s Methenamine Silver Stain (GMS)
The fungal cell wall is composed of polysaccharides that interact with chromatin Acid, undergo oxidation to form aldehydes.
The fungal species will stain black due to the reduction process of the silver nitrate solution (argentaffin reaction).
The fungal mycelium and hyphae, stain rose pink/pink-red, while the mucin stains dark grey.
The background will appear pale green by taking up the light green solution.
GMS results
Fungi,Pneumocystis jirevoci,Histoplasmaspp stainblack
Inner parts of mycelia and hyphae stainpink-red/ rose
Leishmania spp, Toxoplasma spp –negative
Mucin stain defined
-highlight carbohydrate portion of glycoproteins, not the protein component. PAS is used for this.
PAS (Periodic acid-Schiff) purpose
- Visualise fungi
It is used to demonstrate the fungal hyphae and yeast-forms of fungi in tissue samples to identifyCandida albicans,Aspergillus fumigatus, andCryptococcus neoformansinfections
PAS result
Periodic acid oxidizes the C-C bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color.
Red hyphae staining against a green/blue background
Best visualization technique for fungal elements
GMS has higher sensitivtity for detecting fungi
Gram stain not very effective in visualizing fungi
India Ink, negative stain. Why?
Stains background not fungi
India ink, positive test
Unstainedcapsule= ‘halo’ appearance around purple-stainedcells
India ink purpose
India ink capsule stain is used to demonstratecellcapsules.
-< used to detect presence of encapsualtedspecies, e.g.Cryptococcus neoformans, and Cryptococcus gatti.
Fluorescence under UV light
Calcofluor white
Calcofluor white
stains chitin-containing structures so that they fluoresce bright white under ultraviolet light in a fluorescent microscope.
Calcofluor white Interpretation
a fluorescent blue dye that binds to cellulose and chitin, which can be found in the cell walls of fungi.
In budding yeastCalcofluorWhite stains bud scars more strongly, because they have a higher concentration of chitin.
(All specimen types)
Media for culturing fungi
Sabourraud Dextrose Agar (SDA) Mycosel Agar Niger Seed Agar Brain-heart infusion agar Potato Dextrose agar Potato flake agar BHI biphasic culture bottle
SDA describe
Sabouraud Dextrose Agar contains digests of animal tissues (peptones) which provide a nutritious source of amino acids andnitrogenouscompounds for the growth of fungi and yeasts.
Adjustments to Sabouraud agar
pH changed to 5.6 to:
- enhance growth of fungi esp. Dermatophtes
- Slightly inhibit bacterial growth
SDA disadvantages
-Does not promote conidiation of filamentous fungi
Mycosel agar
Addition of Chloramphenicol and cycloheximide to SDA
- inhibit bacterial overgrowth allowing fungal isolation
Niger Seed Agar
- ID for Cryptococcus Neoformans
Brain-heart infusion (BHI) agar
- non selective; primarily for recovery of Dimorphic fungi
Potato Dextrose agar
Rich medium for wide range of fungi
Potato flake agar
BHI biphasic culture bottle
Potato flake agar
- Primary recovery of dimorphic fungi (dermatophyte), particularly fastidious and slow growing strains
BHI biphasic culture bottle
-enhances the recovery of fungi from blood and bone marrow.
Organisms that stain poorly
Treponema (Bacteria)
Ricksettia (Bacteria carried by ticks)
Mycobacteria (Bacteria resembling fungi-TB)
Legionella pneumophila (intracellular parasite causing legionellosis pneumonia)
Mycoplasma (Bacteria causing pneumonia-no cell wall)
Ureaplasma (Bacteria in urinary/genital tract, no cell wall)
Chlamydia (GNB; STD causing; intracellular; cell wall w/o peptidoglycan)
Treponema visualization
- too thin to be visualized
- use darkfield microscopy or fluorescent antibodies
Mycobacteria visualization
- high-lipid-content cell wall
- use acid-fast stain
Legionella pneumophila
-use silver stain
Colony morphology method
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Form
what is the basic shape of the colony? For example, circular, filamentous, etc.
Size
the diameter of the colony. Tiny colonies are referred to as punctiform
Elevation
side view of a colony. Turn the Petri dish on end
Margin/border
edge of a colony. What is the magnified shape of the edge of the colony?
Surface
how does the surface of the colony appear? For example, smooth, glistening, rough, wrinkled, or dull
Opacity
for example, transparent (clear), opaque, translucent (like looking through frosted glass), etc.
Colour (pigmentation)
white, buff, red, purple, etc.
Cellular morphology
unicelullar
multicellular
Unicellular
Yeasts - vegetative body is unicellular Saccharomyces cerevisiae (baker’s yeast) and Candida species (agents of thrush)
Multicellular
Dimorphic fungi can be either uni/multicellular depending on temp
Two stages of fungi
Vegetative and reproductive
The vegetative stage consists of a tangle (mycelium) of slender thread-like structures called hyphae
Reproductive stage can be more conspicuous.
Reproductive stage
Two haploid hyphae (+ and - genders) join together and their nuclei fuse, sporangia form then burst and release spores which grow into hyphae which fuse to start the reproduction process again.
Septate vs aseptate (Hyphal morphology)
Fungi hyphae can be divided by endwalls called septa.
Spore morphology
(1) Conidiospore
(2) Sporangiospore
(3) Arthrospore
(4) Chlamydospore
Conidiospore
Asexual spore; produced at the tip or side of hyphae; detach when mature -Penicillium
Sporangiospore
Asexual spore produced in sac (sporangium) - Mucor
Sporangiospores are either naked and flagellated (zoospores) or walled and nonmotile (aplanospores).
Zoospores release into water with rain
Arthrospore
Formed by the breaking up or disarticulation of fungal mycelia
-Seen in Yeast-like fungi -Trichosporon
Chlamydospore
- Dark-coloured, spherical, and have a smooth surface.
- Multicellular, with cells connected by pores in the septae between cells
- From asexual reproduction - Mycelia produces spores
- Ascomycota such as Candida, Basidiomycota - yeasts
Yeast morphology - size
Oval cells 1-5 µm wide by 5-30 µm long
Yeast morphology - Thickness of walls
Thick polysaccharide cell wall.
Yeast morphology - Capsule absence/presence
Cryptococcus neoformans, Candida albicans - capsulated yeast
Neoformans is the only one that has true polysaccharide capsule
Yeast ID
- Biochemical tests
- Behavior in broth and serum (germ tube formation)
- Behavior on cornmeal agar (pseudohyphae formation)
Biochemical tests - Carbohydrate assimilation
Carbohydrate assimilation test
- definite identification for yeast and yeast like organisms.
- testing yeast’s ability to utilize a particular carbohydrate
- carb free agar + filter paper disks infused with diff carbs
Positive assimilation test
Growth around the disk indicates the yeast can utilize that carbohydrate.
(glucose, maltose, sucrose, lactose, galactose, melibiose, cellobiose, inositol, xylose, raffinose, trehalose, and dulcitol.)
Biochemical tests - Urease test
- preliminary identification of cryptococcus neoformans
urea (substrate) -> ammonia and carbon dioxide (Urease-enzyme)
Result: alkaline environment in the medium
->not reliable for pink-colored yeasts, e.g. Rhodotorula
Positive Urease test
Positive: color change from yellow to pink in the phenol red indicator.
Negative: no pink to purple color (yellow colour)
C. neoformans- +ve, C albicans- -ve
Germ tube
Outgrowth produced by spores of spore-releasing fungi during germination.
The germ tube differentiates, grows, and develops by mitosis to create somatic hyphae.
Germ tube test
- presumptive identification
A germ tube is approximately half as wide and three to four times as long as the yeast cells.
Positive germ tube test
No point of constriction should exist where the germ tube arises from the mother cell.
C.stellotidae is germ tube positive.
Quality control:
C. albicans: +ve control; germ tube within 2 hrs
C.tropicalis: -ve control; no germ tube within 3 hrs
Cornmeal Agar with Tween 80
-primarily for the testing of Candida species for their ability to produce chlamydospores (arising from pseudohyphae).
distinguish the various species of candida (general purpose media) and other yeasts through examination of hyphae, blastoconidia, chlamydoconidia, and arthroconidia. - pseudohyphae
Cornmeal Agar with Tween 80 - Constituents
Cornmeal agar stimulates conidiation; Tween 80 (polysorbate) reduces surface tension and enhances the formation of hyphae, blastoconidia, chlamydoconidia, and arthroconidia.
Pseudohyphae vs hyphae
hyphae - elongated, thread-like filaments
pseudohyphae - newly-divided cells through budding. hyphae - occur in filamentous fungi
pseudohyphae - occur in the unicellular fungi such as yeast
Skin Tests - Immunological method
Demonstrate T-cell immunity (cellular) to a fungus
Serological Tests - Immunological method
Demonstrate B-cell (humoral) immunity to a fungus Acute + Convalescent (recovering person) samples Tests: Complement fixation Agglutination tests Precipitin tests Immunofluorescence Immunodiffusion techniques Counterimmunoelectrophoresis
Animal Inoculation
Establishing fungi pathogenicity + observing the fungal tissue phase
- Rabbits, mice, rats, guinea pigs
- Animal observed at regular intervals
