Introduction to Fungi

General
Microbiology
 The Characteristics of Fungi

• Mycology is the study of fungi

• Mycologists study fungi
• A fungicide is a chemical used to kill
fungi
• Grow best in warm, moist environments

Fungicide kills leaf

fungus

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 Introduction
• Mykes (Greek word) : Mushroom
• Fungi (Yeast and Mold) are eukaryotic

1. Cell walls containing chitin (rigidity & support), mannan &

other polysaccharides
2. Cytoplasmic membrane contains ergosterols
3. Possess true nuclei with nuclear membrane & paired
chromosomes.
4. Divide asexually, sexually or by both
5. Yeast…Unicellular or mold…..multicellular
 The Characteristics of Fungi

• Fungi are NOT plants

• Non-photosynthetic
• Eukaryotes
• Non-motile
• Most are saprobes (live
on dead organisms)

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 How the fungus are nourished
• All are chemo heterotrophs
• Fungi typically depends temperature: At 37oC:
Yeast form. At 25oC: Mold form.

• Grow at pH-5
• Grow in high salt and sugar
• Metabolize complex CH2O like lignin in
wood-wood rot
Fungus Everywhere
Basic terms as they relate to mycology:

• Hypha (hyphae plural) - fundamental tube-like

structural units of fungi.
– Septate - divided by cross walls.
– Aseptate - lacking cross walls.
• Mycelium - a mass (mat) of hyphae forming the
vegetative portion of the fungus.
– Aerial - growing or existing in the air.
– Vegetative - absorbs nutrients.
– Fertile - bears conidia (spores) for reproduction.
• Sporulation & Spores - preferred terms when there is a
merging of nuclear material. Self-fertile are termed
homothallic. Mating types are termed heterothallic.
 Classification
A. Morphological classification

 Yeasts
 Yeasts like fungi
 Moulds
 Dimorphic fungi

B. Taxanomic classification

 Phyllum Thallophyta
Four classes of fungi
A. Morphological classification
(Yeasts)
• Unicellular fungi
which reproduce
by budding
• On culture -
produce smooth,
creamy colonies
e. g Cryptococcus
neoformans
(capsulated yeast)
 Yeast like fungi
• Grow partly as yeasts and partly as
elongated cells resembling hyphae which
are called pseudo hyphae. e.g. Candida
albicans
 Molds/ Filamentous fungi
• Form true mycelia &
reproduce by formation
of different types of
spores.
• Vegetative/ aerial
hyphae
e.g. Rhizopus, Mucor
 4. Dimorphic fungi
• Occur in 2 forms
Molds (Filaments) – 25 C (soil)
Yeasts – 37 C (in host tissue)
Most fungi causing systemic infections are
dimorphic:
– Histoplasma capsulatum
– Blastomyces dermatidis
 Thallophyta
Thallophyta
Irregular plant masses lacking definite root, stem and leaf structures
Fungi Algae

(No chlorophyll) (Chlorophyll)

4 classes

Zygomycetes Ascomycetes Basidiomycetes Deuteromycetes

or fungi imperfecti
• Lower fungi having • Septate hyphae • Septate hyphae • Septate hyphae
non-septate hyphae • Sexual spores •Sexual spores are •Lack a known sexual state.
• Forms asexual (ascospores) are basidiospores on a Most fungi medically
spores present within the basidium important fungi belongs to
sporangiospores sac or ascus this class
• Sexual spores
zygospores and
oospres
Zygomycetes Ascomycetes

e.g. Rhizopus, Mucor Penicillium, Aspergillus

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Basidiomycetes
(CLUB FUNGI)
 Reproduction in fungi
• Sexual - formation of Zygospore, ascospores
or basidiospores
• Asexual reproduction – budding or
fission
• Asexual spores are formed on or in
specialized
• structures.
Vary in size, shape & colour but these
characteristics are constant for a particular
species.
Role of fungi in the economy:
Industrial uses of fungi –

• Natural food supply for wild animals.

• Yeast as food supplement, supplies vitamins.
• Penicillium - ripens cheese, adds flavor (roquefort,
etc.).
• Fungi used to alter texture, improve flavor of natural
and processed foods.
Fermentation
• Fruit juices (ethyl alcohol).
• Saccharomyces cerevisiae - brewer's and baker's
yeast.
• Fermentation of industrial alcohol, fats, proteins,
acids, etc.
Antibiotics -
• First observed by Fleming; noted suppression of
bacteria by a contaminating fungus of a culture
plate.
 Laboratory Methods in Medical Mycology

Collection, Handling and Processing of Specimens

• Skin - cleaned with 70% alcohol to remove dirt, oil and

surface saprophytes
• Nails - cleaned same as for skin. Usually clipped; need to be
finely minced before inoculating to media
• Hair - obtained from edge of infected area of scalp. Use a
Wood's lamp (fluorescence) to help locate infected hair. Hair
can be obtained by plucking, brushing, or with a sticky tape
• Body fluids - normal sterile collection procedures
 Preparation of Specimens for Transport to Laboratory

• Hair & nails sent in a dry envelope, inside proper container

• Other specimens are usually sent frozen or on dry ice
• Packaging - biohazard regulations. Any growing cultures
must be on tube media (not plates). Aluminum screw-capped
inner tube with outer cardboard mailing tube
• Inside labeling information: Patient ID, specimen source,
suspected organism
• Outside labeling information - must state:
• WARNING: POTENTIAL PATHOGEN
 Appropriate Processing of Specimen to Recover Fungus
• Skin, nails, & hair - direct exam following KOH preparation
• CSF - centrifuged; examine sediment microscopically,
inoculate media
• Pleural fluid, sputum, and bronchial aspiration, Gastric
washings - Specimens may be refrigerated up to 2 hours
• Genito-urinary specimens - first morning specimen
preferred
• Blood/bone marrow - generally inoculated directly to Brain
Heart Infusion broth and BHI slant.
• Wound abscess or drainage - should be cultured
anaerobically, especially if Actinomycosis is suspected
• Tissue specimens - mince aseptically, can use small
amount of sterile saline and the supernatant inoculated
 Direct Examination of Specimens
Stains Used in Mycology

• Lactophenol Cotton Blue (LPCB) - very popular for

quick evaluation of fungal structures; will stain the
chitin in cell walls of fungi
• Periodic Acid - Schiff Stain (PAS)
• Gomori Methenamine Silver
• Gridley Stain
• Mayer Mucicarmine Stain
• Fluorescent Antibody Stain
• Papanicolaou Stain
• India Ink
 The Lactophenol Cotton Blue (LPCB)
• The Lactophenol Cotton Blue (LPCB) wet mount is most widely
used method for the staining and observation of fungi
Principle
• LPCB is a stain used for making semi-permanent microscopic
preparation of fungi. The LPCB stain has following three
components :
Phenol : Kills any live organism.
Lactic acid : Preserves fungal structures.
Cotton blue : Stains the chitin and cellulose of the fungal cell
wall intensely blue
Procedure
• Place a drop of 70% ethanol on a clean microscopic glass slide
• Immerse the specimen in the drop of alcohol
• Add one or at most two drops of the LPCB before the alcohol
dries out
• Holding the coverslip between the index finger and thumb,
touch one edge of the drop of mountant with a coverslip
edge and lower gently avoiding air bubbles
• This preparation is now ready for examination
• Make the initial examination using low power objective.
Switch to higher power (40X) objective for more detailed
examination of spores and other structures
 Microscopic Evaluation of Fungi

• Teased Preparations – part of the colony is

removed to a slide with inoculating needle.
Lactophenol cotton blue stain often used
• Slide Culture Techniques - provides a view of
undisturbed microscopic morphology
• Transparent Tape Preparation – clear
cellophane tape used to “lift” parts of a colony
to a microscope slide
 Media Used for Isolation of Fungi

• Sabouraud's dextrose agar (SDA)

• Sabouraud's dextrose agar with chloramphenicol
• Brain heart infusion slant (BHI)
• Potato-dextrose agar (PDA)
 Fungal Culturing

• Tubed media and plated media because:

– there is less chance for spore release into the environment
– less chance for dehydration
– ease of storage
• The agar in a tube is inoculated in a straight line.
• The agar on plates is Inoculated like a large "S" so that rapid
growing fungi can removed
• Incubation should be aerobic
• Incubate cultures at room temperature & also at 37ºC if
dimorphic fungi are suspected
• Cultures are kept for 4 weeks & should be examined every
other day
• Systemic pathogens often require 10 days to 2 weeks
 Colony Morphology (macroscopic features)

• Surface topography - some fungal colonies may cover the

entire surface of agar; others grow in a more restricted
manner
• Surface texture examples: cottony or wooly (floccose),
granular, chalky, velvety, powdery, silky, glabrous (smooth,
creamy), waxy
• Pigmentation - fungi may be colorless or brightly colored.
Color may be on fungus itself, on its sporulating apparatus,
on the agar, or on the bottom of the colony (reverse
pigmentation). Pigment color is due to the color of the
sporulating apparatus. Pigment can diffuse into the agar. It is
important to note the top pigment (obverse); the underside
pigment (reverse); and any discoloration of the medium
Fungus colonies on Media