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Mycology > Fungal Lab ID - Stain, Culture, Tests > Flashcards

Fungal Lab ID - Stain, Culture, Tests Flashcards

1
Q

Visualization Tests for Fungi

A 
10% KOH 
Lactophenol Cotton Blue (LPCB)
Grocott-Gomori Methenamine Silver stain 
Periodic acid-Schiff -PAS (mucin stain)
Gram Stain/Hematoxylin and Eosin 
India Ink
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2
Q

Fluorescence under UV light Tests Fungi

A

Calcofluor white

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3
Q

10% KOH method

A

Strong alkali; softens and clears keratin in hair, skin, nails so hyphae and conidia can be seen clearly

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4
Q

10% KOH purpose

A

Detects yeast and/or hyphae in hair, skin and nails; does not identify species

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5
Q

LPCB principle

A

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.

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6
Q

LPCB limitations

A

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.

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7
Q

Grocott-Gromori’s Methenamine Silver Stain (GMS)

A

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.

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8
Q

GMS results

A

Fungi,Pneumocystis jirevoci,Histoplasmaspp stainblack

Inner parts of mycelia and hyphae stainpink-red/ rose

Leishmania spp, Toxoplasma spp –negative

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9
Q

Mucin stain defined

A

-highlight carbohydrate portion of glycoproteins, not the protein component. PAS is used for this.

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10
Q

PAS (Periodic acid-Schiff) purpose

A
  • 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
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11
Q

PAS result

A

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

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12
Q

Best visualization technique for fungal elements

A

GMS has higher sensitivtity for detecting fungi

Gram stain not very effective in visualizing fungi

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13
Q

India Ink, negative stain. Why?

A

Stains background not fungi

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14
Q

India ink, positive test

A

Unstainedcapsule= ‘halo’ appearance around purple-stainedcells

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15
Q

India ink purpose

A

India ink capsule stain is used to demonstratecellcapsules.
-< used to detect presence of encapsualtedspecies, e.g.Cryptococcus neoformans, and Cryptococcus gatti.

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16
Q

Fluorescence under UV light

A

Calcofluor white

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17
Q

Calcofluor white

A

stains chitin-containing structures so that they fluoresce bright white under ultraviolet light in a fluorescent microscope.

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18
Q

Calcofluor white Interpretation

A

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)

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19
Q

Media for culturing fungi

A 
Sabourraud Dextrose Agar (SDA)
Mycosel Agar 
Niger Seed Agar
Brain-heart infusion agar
Potato Dextrose agar
Potato flake agar
BHI biphasic culture bottle
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20
Q

SDA describe

A

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.

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21
Q

Adjustments to Sabouraud agar

A

pH changed to 5.6 to:

  • enhance growth of fungi esp. Dermatophtes
  • Slightly inhibit bacterial growth
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22
Q

SDA disadvantages

A

-Does not promote conidiation of filamentous fungi

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23
Q

Mycosel agar

A

Addition of Chloramphenicol and cycloheximide to SDA

- inhibit bacterial overgrowth allowing fungal isolation

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24
Q

Niger Seed Agar

A
  • ID for Cryptococcus Neoformans
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25
Q

Brain-heart infusion (BHI) agar

A
  • non selective; primarily for recovery of Dimorphic fungi
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26
Q

Potato Dextrose agar

A

Rich medium for wide range of fungi

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27
Q

Potato flake agar

BHI biphasic culture bottle

A

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.

28
Q

Organisms that stain poorly

A

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)

29
Q

Treponema visualization

A
  • too thin to be visualized

- use darkfield microscopy or fluorescent antibodies

30
Q

Mycobacteria visualization

A
  • high-lipid-content cell wall

- use acid-fast stain

31
Q

Legionella pneumophila

A

-use silver stain

32
Q

Colony morphology method

A

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33
Q

Form

A

what is the basic shape of the colony? For example, circular, filamentous, etc.

34
Q

Size

A

the diameter of the colony. Tiny colonies are referred to as punctiform

35
Q

Elevation

A

side view of a colony. Turn the Petri dish on end

36
Q

Margin/border

A

edge of a colony. What is the magnified shape of the edge of the colony?

37
Q

Surface

A

how does the surface of the colony appear? For example, smooth, glistening, rough, wrinkled, or dull

38
Q

Opacity

A

for example, transparent (clear), opaque, translucent (like looking through frosted glass), etc.

39
Q

Colour (pigmentation)

A

white, buff, red, purple, etc.

40
Q

Cellular morphology

A

unicelullar

multicellular

41
Q

Unicellular

A 
Yeasts - vegetative body is unicellular
Saccharomyces cerevisiae (baker’s yeast) and Candida species (agents of thrush)
42
Q

Multicellular

A

Dimorphic fungi can be either uni/multicellular depending on temp

43
Q

Two stages of fungi

A

Vegetative and reproductive
The vegetative stage consists of a tangle (mycelium) of slender thread-like structures called hyphae
Reproductive stage can be more conspicuous.

44
Q

Reproductive stage

A

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.

45
Q

Septate vs aseptate (Hyphal morphology)

A

Fungi hyphae can be divided by endwalls called septa.

46
Q

Spore morphology

A

(1) Conidiospore
(2) Sporangiospore
(3) Arthrospore
(4) Chlamydospore

47
Q

Conidiospore

A

Asexual spore; produced at the tip or side of hyphae; detach when mature -Penicillium

48
Q

Sporangiospore

A

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

49
Q

Arthrospore

A

Formed by the breaking up or disarticulation of fungal mycelia
-Seen in Yeast-like fungi -Trichosporon

50
Q

Chlamydospore

A
  • 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
51
Q

Yeast morphology - size

A

Oval cells 1-5 µm wide by 5-30 µm long

52
Q

Yeast morphology - Thickness of walls

A

Thick polysaccharide cell wall.

53
Q

Yeast morphology - Capsule absence/presence

A

Cryptococcus neoformans, Candida albicans - capsulated yeast
Neoformans is the only one that has true polysaccharide capsule

54
Q

Yeast ID

A
  1. Biochemical tests
  2. Behavior in broth and serum (germ tube formation)
  3. Behavior on cornmeal agar (pseudohyphae formation)
55
Q

Biochemical tests - Carbohydrate assimilation

A

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
56
Q

Positive assimilation test

A

Growth around the disk indicates the yeast can utilize that carbohydrate.
(glucose, maltose, sucrose, lactose, galactose, melibiose, cellobiose, inositol, xylose, raffinose, trehalose, and dulcitol.)

57
Q

Biochemical tests - Urease test

A
  • 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
58
Q

Positive Urease test

A

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

59
Q

Germ tube

A

Outgrowth produced by spores of spore-releasing fungi during germination.
The germ tube differentiates, grows, and develops by mitosis to create somatic hyphae.

60
Q

Germ tube test

A
  • presumptive identification

A germ tube is approximately half as wide and three to four times as long as the yeast cells.

61
Q

Positive germ tube test

A

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

62
Q

Cornmeal Agar with Tween 80

A

-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

63
Q

Cornmeal Agar with Tween 80 - Constituents

A 
Cornmeal agar stimulates conidiation; 
Tween 80 (polysorbate) reduces surface tension and enhances the formation of hyphae, blastoconidia, chlamydoconidia, and arthroconidia.
64
Q

Pseudohyphae vs hyphae

A

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

65
Q

Skin Tests - Immunological method

A

Demonstrate T-cell immunity (cellular) to a fungus

66
Q

Serological Tests - Immunological method

A 
Demonstrate B-cell (humoral) immunity to a fungus
Acute + Convalescent (recovering person) samples 
Tests:
Complement fixation 
Agglutination tests 
Precipitin tests 
Immunofluorescence 
Immunodiffusion techniques 
Counterimmunoelectrophoresis
67
Q

Animal Inoculation

A

Establishing fungi pathogenicity + observing the fungal tissue phase

  • Rabbits, mice, rats, guinea pigs
  • Animal observed at regular intervals

Mycology (6 decks)

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