Lecture 6 Flashcards
Mycology
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
Fungi appear in one of two forms
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
Asexual cycle of molds
hyphae - the vegetative bodies of most fungi, constructed of tiny filaments
mycelium -an interwoven mat of hyphae
Two types of Hyphae:
Vegetative hyphae:
submerged
on surface
Aerial hyphae:
Above surface of medium (gives fuzzy appearance)
Produce conidia (asexual spores)
macroconidia and / or microconidia
2 sizes of reproductive cells
(both asexual)
Macroconidia
Microconidia
Distinct size and shape
4 groups of infection caused by fungi
Superficial
Dead layer of skin
4 groups of infection caused by fungi
Cutaneous and example
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
4 groups of infection caused by fungi
Subcutaneous
Localized, subcutaneous tissues /or bone
4 groups of infection caused by fungi
Systemic
Blood, bone, internal organs, CSF
Dermatophyte Infections
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
Saprophytic fungi
Saprophytic fungi (those found in soil and organic matter) –dismissed as not clinically important
Found as clinical contaminants
Laboratory contaminants
Specimen Handling Collection Specimens for Dermatophytes
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
Specimen Handling: Laboratory
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)
Yeasts Molds: Growth Conditions
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
Laboratory Investigation
Laboratory Investigation will include both 1.Direct microscopic examination of the sample and
2. Both microscopic and macroscopic examination of the culture
Direct Microscopy Examination (DME):
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!
Direct Microscopy: KOH**
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
Direct Microscopy: India Ink
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
Cryptococcus neoformans
India Ink Prep for Cryptococcus neoformans
Ink does not penetrate capsule resulting in “halo”
Direct Microscopy: Gram Stain
Gram stain:
Especially useful for yeast
Appear as Gram positive
Much larger than cocci
Culture Media FOR Mycological samples.
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
Culture Media
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.
