Lecture 6

Question 1

Mycology

Answer 1

Study of fungi
Include yeasts and molds
Eukaryotes
True nucleus, nuclear membrane, eukaryotic cellular organelles
(bacteria are prokaryotes: no nucleus)
Multicellular except for yeasts
Infection caused by fungi is known as dermatomycosis

Question 2

Fungi appear in one of two forms

Answer 2

Yeasts : 3-10 micrometers in diameter - Single celled
single celled organisms that reproduce by budding
produce moist, opaque, creamy colonies on culture media

Molds (with hyphae)  - Multi-celled
thread or tube-like filaments 
Give the mold its fuzzy or wooly appearance
Typical “mold” appearance
Granular, fluffy, downy surface
Variable colour
Fast to slow growers
More complex than yeasts

Question 3

Asexual cycle of molds

Answer 3

hyphae - the vegetative bodies of most fungi, constructed of tiny filaments
mycelium -an interwoven mat of hyphae

Question 4

Two types of Hyphae:

Answer 4

Vegetative hyphae:
submerged
on surface

Aerial hyphae:
Above surface of medium (gives fuzzy appearance)
Produce conidia (asexual spores)
macroconidia and / or microconidia

Question 5

2 sizes of reproductive cells

Answer 5

(both asexual)
Macroconidia
Microconidia
Distinct size and shape

Question 6

4 groups of infection caused by fungi

Superficial

Answer 6

Dead layer of skin

Question 7

4 groups of infection caused by fungi

Cutaneous and example

Answer 7

Penetrate all keratinized tissues
e.g. hair, skin, nails
Cannot live in deeper tissues

Dermatophytosis - “ringworm” disease of the nails, hair, and/or stratum corneum of the skin caused by fungi called dermatophytes
Disease referred to as “ringworm” or “Tinea”
Circular lesions, redness and inflammation at the outer edge (advancing edge of infection)
Each one a site of inoculation
Some infections result in dry, scaly lesions only without inflammation
Mild infections
Non-dermatophyte agents
Rarely isolated in routine clinical laboratories

Question 8

4 groups of infection caused by fungi

Subcutaneous

Answer 8

Localized, subcutaneous tissues /or bone

Question 9

4 groups of infection caused by fungi

Systemic

Answer 9

Blood, bone, internal organs, CSF

Question 10

Dermatophyte Infections

Answer 10

Dermatophytes rarely infect other body sites
Yeasts can also produce cutaneous infections of the nail and skin
Some yeasts can also cause systemic infections
(e.g. diaper rash)
Candida albicans is a commonly encountered yeast in vaginal infections

Question 11

Saprophytic fungi

Answer 11

Saprophytic fungi (those found in soil and organic matter) –dismissed as not clinically important
Found as clinical contaminants
Laboratory contaminants

Question 12

Specimen Handling Collection Specimens for Dermatophytes

Answer 12

stored at RT
They are collected in a dermatophyte Kit.
Blood and CSF collected in sterile containers held at 35°C-
Others can be refrigerated (4°C)
Hair: collect 10
Nail: disinfect with 70% alcohol
Scrape away outer surface
Collect deeper portions and debris under nail
Clippings or entire nail
Skin: disinfect area with 70% alcohol
Scrape active borders with scalpel
Above specimens should be submitted in black paper, folded and placed in biohazard bag.
Submit to laboratory

Question 13

Specimen Handling: Laboratory

Answer 13

All work must be carried out in biological safety cabinet
Direct inoculation
Generally applied directly to the media
Larger fluid volumes may be concentrated by centrifugation and the sediment used for inoculation
Tissues must be homogenized (ground)
Plates not usually streaked (especially for dermatophytes)
Inoculum in center of plate (nail/hair imbedded into media)

Question 14

Yeasts Molds: Growth Conditions

Answer 14

Obligate aerobes
Require pre-formed carbon source
E.g. decaying matter
Some are thermally dimorphic
Yeasts at 35°C (in the tissue of the host)
Molds at RT (in the environment)

Mold 25-30°C, Yeast in tissue or 35°C

Question 15

Laboratory Investigation

Answer 15

Laboratory Investigation will include both 1.Direct microscopic examination of the sample and
2. Both microscopic and macroscopic examination of the culture

Question 16

Direct Microscopy Examination (DME):

Answer 16

KOH or Cellufluor
India Ink
Gram stain
Examine for evidence of fungal elements
Microscopy can detect evidence of fungal infection much earlier than culture,as cultures typically require 7-14 days of incubation
Faster diagnosis and treatment!

Question 17

Direct Microscopy: KOH**

Answer 17

most commonly used method for skin, hair, nails and tissue
10-15% KOH- breaks down the keratin an cellular materials
fungus is not broken down because of the chitin in the cell walls of the fungi
On a glass slide a drop of 10-15% KOH is mixed with the specimen and a cover slip is applied and the slide examined
Cellufluor –fluorochrome label added to KOH
Viewed using fluorescent microscope

Question 18

Direct Microscopy: India Ink

Answer 18

India Ink –used for *CSF, urine or body fluid
Looking for Cryptococcus neoformans
Capsule repels ink particles and appear as a halo around the yeast cells
India ink has now been replaced in some labs with direct antigen detection tests for Cryptococcus
A drop of India ink is mixed with a drop of centrifuged CSF sediment and observed at 40X for, encapsulate yeasts Cryptococcus neoformans

This is a negative staining method
the budding yeast will be seen surrounded by a large clear area against a black background

Question 19

Cryptococcus neoformans

Answer 19

India Ink Prep for Cryptococcus neoformans

Ink does not penetrate capsule resulting in “halo”

Question 20

Direct Microscopy: Gram Stain

Answer 20

Gram stain:
Especially useful for yeast
Appear as Gram positive
Much larger than cocci

Question 21

Culture Media FOR Mycological samples.

Answer 21

Many of the specimen collected for fungal identification will be contaminated with bacteria.
For this reason the media used should contain antibiotics to inhibit these organisms and allow the pathogens to grow.
SAB- Sabouraud dextrose agar with antibiotics,is the most common medium for fungi.
Antibiotics like Cycloheximide and Chloromphenicol are added to the medium which, inhibits bacterial growth
Selective media not required for properly collected specimens

Question 22

Culture Media

Answer 22

Incubate at room temperature (at 25 to 30oC)
Require 3-4 weeks for culture, longer for some dimorphic fungi
examined weekly or twice a week for growth, before being reported as negative.