MICRO - Introduction to Mycology

Mycology

The study of fungi

Fungi

Non-motile, non-photosynthetic eukaryotic organisms that possess a rigid cell wall. Utilized in food, beverages, and medicine production

Eukaryotic

A characteristic of fungi: Have true nucleus and membrane-bound organelles

Non-photosynthetic

A characteristic of fungi: Due to lack of chloroplast and chlorophyll, making them achlorophyllous

Molds

What is an example of filamentous fungi?

Yeast

What is an example of unicellular fungi?

Mushrooms

What is an example of fleshy fungi?

Capable of facultative reproduction

A characteristic of fungi: Able to reproduce sexually or asexually

Non-motile

A characteristic of fungi: Due to the presence of rigid cell walls, which are composed of chitin

Chemoheterotrophic

A characteristic of fungi: Secretes enzymes that degrade organic substrates into soluble nutrients which they consume and use as source of nourishment

Saprophytic

A characteristic of fungi: living in soils or decaying bodies

Symbiotic

A characteristic of fungi: Live with other organisms without causing harm

Parasitic

A characteristic of fungi: In disease-causing organisms; harms host organism

Phylum Basidiomycota

Rarely cause diseases in humans. Most important cause of mycotoxicosis and mycetismus

Malassezia sp.

Most well-known common fungal species. Causative agent of tinea versicolor

Mycotoxicosis

Consumption of food contaminated with mycotoxins; may produce chronic conditions (i.e., liver failure, hepatic carcinoma). Example: Consumption of peanuts contaminated by aflatoxin of Aspergillus fungi

Mycestimus

Consumption of inherently toxic mushroom; usually lethal. Example: Direct consumption of Amanita phalloides

Cell Wall

Protection from osmotic and other environmental stresses. Surface component functions as an attachment medium to host cell. May stimulate innate immune responses of the host. May provoke inflammatory reaction by activating the complement cascade of the host

Chitin, Glucans, Mannans

What are the component of the cell wall of fungi?

Dematiaceous

Cell wall contains melanin which makes the appearance brown

Hyaline

Cell wall has no melanin and appear as colorless in the microscope

Ergosterol

Regulates the fluidity of cell membrane

Cryptococcus species

The only fungal organism that possess a capsule for it to survive against extreme environments

Capsule

Composed of glucuronoxylomannan and galactoxylomannan. Protection for environmental stress

Yeast

Unicellular. Spherical to ellipsoid. Reproduce asexually (via budding; more common) or sexually (via conjugation)

Budding

A form of asexual reproduction utilized by yeast

Interphase

It is the preparation prior to mitosis and includes 3 subphases (G1, S, and G2 phases)

G1 phase

A sub-phase of interphase where the growth and development of the cell start

S phase

Actual budding starts before this phase, marked by the emerging bud. This is the phase where DNA replication takes place

G2 phase

Occurs after the DNA replication. Nuclear migration is observed from the parent cell to the daughter cell. Grouping will begin but separation has not yet occurred.

Mitosis

It is the process of cell division where the cell replicates its chromosomes and segregates, producing 2 identical nuclei. Chromosome segregation and nuclear division will happen here.

G0 phase

Also called the temporary resting phase

Pseudohyphae

The chain of daughter cells formed during fungal budding because the daughter cells do not separate from the parent cell

Molds

Growth of multicellular branching cylindrical tubules called hyphae

Aerial hyphae

Also called the reproductive spores

Vegetative hyphae

A type of hyphae that acts as structural support for the whole organism when they are in

Septate

A form of hyphae divided with septum (or is compartmentalized)

Coenocytic

A form of hyphae with no partition

Hyphae

Basic unit of fungal mold. Vary in diameter from 2 to 10 microns. Extended by apical elongation

Mycelium

Used when the hyphae intertwine due to active growth. Due to this continuous growth, they may be seen by the naked eye, appearing as cottony or velvety colonies in a medium

Thermal Dimorphism

Capability of several pathogenic fungi to reversibly grow as yeast or mold, depending on environmental temperature

Blastomyces sp., Coccidiodes sp., Histoplasma sp., Paracoccidiodes sp., Sporothrix sp., Talaromyces sp., Emergomyces sp.

What are the fungi that possesses thermal dimorphism?

Mitosis

Via budding, fragmentation, or nuclear fission — producing haploid (1n) spores

Germination

Haploid spores germinate to become mycelium (1n)

Plasmogamy

Haploid cells from 2 different mycelia fuse to form heterokaryotic cell with 2 or more nuclei (dikaryotic stage)

Karyogamy

Nuclei will fuse to form a diploid (2n) zygote

Meiosis

Zygote undergoes meiosis to form haploid (1n) spores

Germination

Haploid spores germinate to form mycelium

Spores

Sexual unit of fungal organisms. Represent microscopic dispersal or survival propagules produced by most species of fungi. Usually dormant, readily dispersed, more resistant to adverse conditions, and germinate to form vegetative cells under favorable conditions. Often contain substantial amounts of nutrient reserves which may take the form of lipids, trehalose, and glycogen

Zygospore

A telemorphic spore found under Order Mucorales. Large, thick-walled. Formed by fusion of haploid mating hyphae

Acrospore

A telemorphic spore found under Phylum Ascomycota. Contained within sac-like structures called “ascus”. Each ascus usually contains 4 to 8 meiospores

Basidiospore

A telemorphic spore found in species under Phylum Basidiomycota. Formed as swellings at the tip of sterigma of basidium. Sterigma - outgrowths of basidium. Structural connection between basidiospore and basidium

Sporangiospore

A anamorphic spore found in species under Order Mucorales. Endogenous - contained within a structure called “sporangium” (houses sporangiosphore). This sporangium is supported by an aerial hypha called “sporangiophore”

Zoospores, Aplanospores

What are the two main types of sporangiospore?

Conidia

A anamorphic spore found in most pathologic fungi belonging to Phylum Ascomycota and Basidiomycota. Exogenous - supported by an aerial hypha called “conidiophore”

Thallic Conidia (Arthroconidia), Blastic Conidia (Blastoconidia)

What are the two main types of conidia?

Macroconidia

Another form of conidia that is large (15.0 - 60.0 um x 6.0 - 10.0 um) or multicellular

Microconidia

Another form of conidia that is small (2.0 - 4.0 um x 4.0 -7.0 um)

Chalmydoconidia

Another form of conidia that is large, thick-walled, usually spherical produced from terminal or intercalary hyphal cells (e.g. Candida albicans). Also called “resting conidia” due to its dormant state

Phialoconidia

Another form of conidia that is produced by vase-shaped conidiogenous cell called phialide (e.g. Aspergillus fumigatus)

Mycosis

Infection caused by fungi. Not reportable. Acquired through environmental source or through auto-inoculation (own flora)

Superficial Mycosis

Infects the stratum corneum of the skin and the hair. Does not penetrate underlying tissues. Does not lead to inflammation

Cutaneous Mycosis

Confined to the outer layers (keratinized) of the skin, hair, and nail. May involve the entire epidermis. May lead to inflammation. Rarely disseminates systematically

Subcutaneous Mycosis

Normally reside in soil or vegetation. Acquired by traumatic inoculation, subsequently infecting the exposed dermis and underlying tissues. Rarely becomes disseminated

Systemic Mycosis

Infective agents normally reside in soil or vegetation. Usually acquired through inhalation or dissemination of existing mycosis. Can be classified as Endemic or Opportunistic

Virulence factor

A phenotype that enables a microorganism to thrive into a host cell and potentially cause a disease

Determinants of fungal virulence

Morphologic transformation, Genetic switching of metabolic processes, Production of surface adhesins, Secretion of enzymes to attack host substrates, Cell wall components that resist phagocytosis, Ability to form biofilms are called?

Yeast cells

Are potentially infectious and can cause mild to severe illness. Malassezia spp., Candida spp., Cryptococcus neoformans

Hyphae (Hyphal elements)

The primary disease-causing agents of organisms that cause superficial or cutaneous mycosis. Malassezia spp., Dermatophytes, Piedraia hortae

Asexual spores

Can cause endemic mycosis. Sporangiospore are agents of mucomycosis. Conidia (Histoplasma capsulatum, Aspergillus spp, Blastocytoma dermatitidis)

Mycestismus

The ingestion of inherently toxic mushroom. Usually lethal within the first few days after ingestion of the toxic mushroom without medical intervention

Mycotoxcosis

The ingestion of food contaminated by a mushroom toxin (mycotoxin); may produce chronic conditions. Ex. Consumption of peanuts contaminated by aflatoxin. Causes acute but mild symptoms that may develop into chronic conditions if left untreated

Direct Microscopic Examination

Most readily available technique for diagnosing mycosis

KOH Solution

Default reagent. Best used for aqueous or serous specimens (i.e. urine, sputum, aspirates). Dissolved any tissue cells; highly refractive fungal cell wall becomes visible. Can also be used to examine skin scraping, minced tissue samples, pus, and viscous exudates by heating the slide to dissolve excess tissue debris. Heat slide first to dissolve excess tissue

Swartz-Lamkins Stain

Increases sensitivity of KOH. Composed of black ink, paraphenylenediamine solution, benzoic acid. Additional step needed in order to isolate fungal organisms if you submit a skin scaping

India Ink

Used to visualize the capsule of Cryptococcus sp. in immunocompromised patients with meningitis. The capsule appears as a peripheral clearing around the transparent yeast cell. Not used in histopathologic studies; just stains background

Gomori Methenamine Silver

Used to visualize fungal cell wall in histopathologic biopsy specimens. Stains the cell wall black

Periodic Acid Schiff (PAS) Stain

Used to visualize fungal cell wall in histopathologic biopsy specimens. Stains the cell wall red

Fluorescence Microscopy

More sensitive but more expensive technique; fluorescent stain for rapid detection of fungi

Calcofluor White

Most commonly used fluorescence dye. Dye binds to polysaccharide cell wall → fluorescence emitted under UV light enhances fungal detection. Only used in fluorescent microscopy

Culture Media

More sensitive than microscopic examination. A portion of the collected specimens for microscopic examination should be cultured

With antibiotics

What type of culture media is used for non-sterile specimens like urine and sputum?

Without antibiotics

What type of culture media is used for sterile specimens like blood and CSF?

Sabouraud’s Dextrose Agar (SDA)

Standard medium for most fungi

Mycobiotic Agar

Primary recovery of dermatophytes; has antibiotics, normal bacterial flora of skin are eliminated

Potato Dextrose Agar (PDA)

General isolation medium for molds; induces spore formation and pigment production

Brain Heart Infusion Agar

Primary recovery for dimorphic and fastidious fungi (e.g., those with thermal dimorphism)

Corn Meal Agar

Identification of C. albicans by production of chlamydospores

Agar Plates

Provide better isolation and aeration for colonies, but more hazardous to handle

Screw-Capped tubes

Less isolation and aeration for colonies, but less hazardous to handle. All mold cultures must be inoculated and examined within a biosafety cabinet

Slide Culture

Best method to preserve and allow observation of morphologic features of fungi. Ideal for definitive identification, but reserved only for instances when identification cannot be done on adhesive or lactophenol blue (LPCB) mounts. LPCB is default stain. Least practical. Never performed on dimorphic pathogens

Yeast colonies

Colony appearance is elevated, round-ovoid colonies in medium, similar to bacteria

Mold colonies

Colony that have cottony/velvety/hairy appearance

Lactophenol Cotton Blue (LPCB) Stain

Default stain used for microscopy of fungal organisms. Stain used for staining and making semi-permanent wet mounts of fungi from cultures for microscopic examination. Stains chitin and cellulose

Serology

Detection of fungal antibodies in samples. Positive antibody tests may confirm diagnosis in immuno-competent patients only

PCR

Utilize oligonucleotide probes that emit a signal amplified by fluorescence, chemical reagents, or enzyme immunoassay

Matrix-Assisted Laser Desorption Ionization-Time of Mass Spectrometry (MALDI-TOF-MS)

Extracts microbial proteins to identify pathogenic fungi. More accurate and faster than conventional culture methods

Antifungal Agents

Three major modes of action: Direct damage to fungal cell membrane, Inhibition of cell wall synthesis, Inhibition of protein synthesis

Azoles

Inhibit the synthesis of the ergosterol

Allylamines

Inhibit the synthesis of ergosterol by blocking squalene epoxidase