Basic Mycology 1 - 5 PDF

Basic Mycology Francis Ian L. Salaver, RMT, MD  Mycology Branch of biology concerned with the study of fungi, including their biochemical, physical and microscopic properties, their use to humans and as well as the human diseases they can cause, such as toxin producti...

Basic Mycology Francis Ian L. Salaver, RMT, MD  Mycology Branch of biology concerned with the study of fungi, including their biochemical, physical and microscopic properties, their use to humans and as well as the human diseases they can cause, such as toxin production or infection.  Mycology Mykos (fungus) Logus (study) Study of fungi which include Molds Yeasts  Fungi: General characteristics Eukaryotic Contains chitin in their cell walls Peptidoglycan for bacteria Cellulose for plants Can be single-celled or multicellular Can reproduce sexually or asexually Heterotrophs Fungi are Eukaryotic cells  Heterotrophic organism can be (1) Saprophytic – feeds on dead organic material (2) Symbiotic – fungi is living together with other organisms and establishes mutualism (3) Parasitic – fungi living in another organism and causes harm to the latter Saprophytic fungi Fungi that are responsible for breaking down and recycling dead plant and animal material. Symbiotic fungi: Mycorrhiza Mycorrhizae fungi germinates in soil. Its hyphae penetrates the roots of the plant. The plant roots provide essential nutrients for the growth of the fungi. Symbiotic fungi: Mycorrhiza In return, the large mass of fungal hyphae acts as a virtual root system for the plants, increasing the amount of water and nutrients that the plant may obtain from the surrounding soil. Symbiotic fungi: Mycorrhiza Watch this link  https://www.youtube.com/w atch?v=v88gbtKBTv4 I will ask questions from video Parasitic fungi Fungi that attack living organisms, penetrate their outer defenses, invade them, and obtain nourishment from living cytoplasm, thereby causing disease and sometimes death of the host. Watch this link  https://www.youtube.co m/watch?v=o57imEfknM Q Ill ask questions from the video Fungi can be a yeast or a mold  Yeast Unicellular circular or oval-shaped forms of fungi Optimum temperature: near body temperature “Parasitic” Yeast Molds Multicellular Filamentous, branching form Room temperature “Saprophytic” Molds  Dimorphism Fungi that can exist in the form of both mold and yeast. This is usually brought about by change in temperature Yeast form at 37 C Mold form at room temperature Sporothrix schenckii Rose thorns will demonstrate presence of molds Skin nodule biopsy will demonstrate yeast cells Reproduction: Sexual/Asexual Asexual reproduction by the release of spores into the air Fungal cultures Optimum temperature of growth for saprophytes (molds) is 20-30 degree Celsius and 30-37 degree Celsius for parasitic fungi (yeast) The standard temperature for incubation of fungi is 30ºC and cultures should be incubated in a humidified environment for 21 days. Bacterial cultures require incubation temperature of 37 degree Celsius  Fungal cultures Fungi grow best at pH of 4-6; average of 5.0 Fungi are more resistant to osmotic pressure than bacteria Fungi requires less nitrogen and moisture than bacteria  Fungal cultures Fungi can be aerobic or anaerobic Molds are aerobic Yeasts are facultative anaerobes Fungi are capable of metabolizing complex carbohydrates such as lignin in wood. Harmful effects: Allergy Some spores of molds can trigger allergic rhinitis  Harmful effects: Mycoses a direct fungal infection of animals, including humans.  Harmful effects: Mycotoxicoses Disease cause by the natural toxin produced by fungi Aspergillus flavus can produce aflatoxin that can cause hepatocellular carcinoma  Beneficial effects: Preparation of bread (1) Yeast cells are used in preparation of bread Bread = sugar + gluten + yeast cells + water Gluten forms the framework of bread Yeast converts the sugar into carbon dioxide  Gluten Yeast cells Sugar  Gluten Yeast cells Sugar  Gluten Yeast cells Sugar CO2  Gluten Yeast cells Sugar CO2  Gluten Yeast cells Sugar CO2 35 Pesos = 10 pesos sugar plus CO2  Beneficial effects: Preparation of bread (2) Yeast cells are used in induce alcoholic fermentation in grape juice/barley grain to produce wine and beer; respectively.  Beneficial effects: Preparation of bread (3) Human hepatitis B virus vaccine is prepared using antigen produced by recombinant technology in yeast (Saccharomyces) Beneficial effects: Preparation of bread Beneficial effects: Preparation of bread (4) Sources of drugs  Beneficial effects: Preparation of bread (5) Higher fungi may be eaten directly as mushrooms Beneficial effects: Preparation of bread  Beneficial effects: Preparation of bread (6) Saprophytic fungi in soil produce degradative enzymes essential for the biologic recycling of organic matter Beneficial effects: Preparation of bread (7) Parasitic fungi can also help regulate the number of other species to maintain ecological balance 4 out 5 animals are NEMATODES Arthrobotrys conoides  Arthrobotrys conoides https://www.youtube.com/watch?v=OHgxM2HVnkQ  Review Yeast – unicellular, grows best at 37 degree Celsius Molds – multicellular; grows best at room temperature  Yeast Unicellular form of fungi Spherical or elliptical in shape Reproduces by: (1) Budding – most common (2) Binary fission (3) Formation of pseudohyphae Budding Asymmetric division of yeast cells A new yeast cell (BLASTOSPORE) is formed through mitotic cell division and remains attached as a bud on the old cell until it splits and becomes independent. Binary fission A yeast cell duplicates its genetic material, or deoxyribonucleic acid (DNA), and then divides into two parts (cytokinesis), with each new organism receiving one copy of DNA.  Pseudohyphae Buds fail to detach producing elongated yeast cells (pseudohyphae) Hyphae Pseudohyphae Septum not constrictions Yeast colonies  Yeast colonies Colonies are moist, pasty, creamy and opaque Yeasts are less colorful compared to molds, some yeast colonies can be pigmented May be mistaken as bacterial colonies Yeast colonies should be biochemically differentiated from bacterial colonies Yeast colonies should be biochemically differentiated from bacteria  Mold colonies Produce multicellular, filamentous, cottony & dry colonies Consist of branching cylindrical tubules with diameter called hyphae Mold colonies  Mold colonies Hyphae grow to form a filamentous mass of intertwining strands called a mycelium Mold colonies: 2 portion Reproductive or aerial hypha grow on the top of the agar's surface of the culture media and contains the reproductive structures such as spores Mold colonies: 2 portion Vegetative portion or thallus portion of the mycelium that anchors the mold and absorbs nutrients grows in or on a substrate and absorbs water and nutrients Hyphae can be septated or nonseptated  Hyphae can be septated or nonseptated Septated hyphae have dividers between the cells, called septa (singular septum). The septa have openings called pores between the cells, to allow the flow of cytoplasm and nutrients throughout the mycelium.  Hyphae can be septated or nonseptated Nonseptate/Coenocytic hyphae lack septum and cell membranes between the cells Septa can be seen in branching points Zygomycetes: Mucor, Rhizopus, Absidia Sporangium Sporangiospores Columella Sporangiophore Nonseptated hyphae Rhizoids Morphology of Zygomycetes  Hyphae Hyaline if fungal structures are colorless Highly refractile  Hyphae Dematiaceous are group of fungi that produce melanin in their cell walls, giving them a characteristic brown colour when grown on agar. group of fungi with dark colonies and pigmented fungal elements  Dematiaceous Fungi Agents causing Chromoblastomycosis (BROWN CAPE) Curvularia Alternaria Phialophora Exophiala EX nya si PHIA, naghanap sya ng ALTERnative na may CURVes Lesion infected by a dematiaceous fungus should appear to be pigmented Infection of plants  Fungal structures: Capsule An extracellular layer which lies outside the cell wall and it is usually composed of polysaccharides. It protects the cell from different environmental dangers such as phagocytosis, desiccation and harmful chemicals Capsule of Cryptococcus neoformans has antiphagocytic properties and is associated with virulence Fungal meningitis among immunocompromised patients India ink staining  India Ink Wet Mount Used to identify the capsule of Cryptococcus neoformans CSF is directly examined by adding one drop of India ink Capsule appear as a clear halo against a dark background (negative staining) Negative staining: White capsule against a black background  Fungal structures: Cell wall The cell wall is a characteristic structure of fungi and is composed mainly of glucans, chitin and mannan. As the components of the fungal cell wall are not present in humans, this structure is an excellent target for antifungal therapy. Fungal cell wall Fungal cell wall  Fungal structures: Cell wall Major component (molds) : chitin (polymers of N- acetylglucosamine) Major component (yeast): glucan (polymers of glucose)  Fungal structures: Cell wall Fungal cell wall is poorly stained with routine Hematoxylin and Eosin Fungal cell wall stains Periodic acid Schiff Methenamine silver stain Calcofluor white is a fluorescent stain Gram stain is useful for Candida and Cryptococcus Gram stain is only for bacteria except Candida and Cryptococcus Gram stain: Cryptococcus neoformans ”Sunburst appearance” Methenamine silver stain - black Periodic acid schiff - red  Fluorescence Test Calcofluor white stain Calcofluor binds to chitin in cell wall giving a brilliant fluorescence Calcoflour white stain Human cell membrane Fungal structures: Cell membrane  Fungal structures: Cell membrane Bilayered membrane composed of several phospholipids Contain sterols which are essential for the viability of fungi Principal fungal sterol is ergosterol  Fungal structures: Cell membrane Nuclei, mitochondria, ER, storage vacuoles containing hydrolytic enzymes, ions, metabolites such as amino acids Laboratory Identification of Fungi  Laboratory Methods Microscopic Methods Cultivation Biochemical tests Serologic Test  Microscopic Methods Wet Mounts KOH Lactophenol cotton blue (LPCB) Staining India ink Special stains for fungal cell walls 10% Potassium hydroxide  10% Potassium hydroxide Direct microscopic study of the fungi in infected tissues Softens the tissue KOH acts as clearing agent Dissolves keratin Eliminates debris Dissolves fat droplets 20% KOH is used for nails (highly keratinized tissue)  10% Potassium hydroxide One - two drop of KOH is added to specimen on a slide Cover with a coverslip Allow specimen to clear for 20 minutes (10-30minutes). Gentle heating may hasten the action of the KOH Fungal elements appear as hyaline structures. Some may be slightly pigmented. Disadvantage: poor contrast 10% Potassium Hydroxide Lactophenol Cotton Blue  Lactophenol Cotton Blue Lactic acid preserve the fungal structures Phenol is a killing agent Cotton blue imparts blue color to the fungal structures Advantages Structures are readily seen microscopically Has glycerine component  Fluorescence Test Wood’s lamp (UV light) Emits wavelength 320–450 nm (peak 365 nm) The light is held over an area of skin in a darkened room. Infected hair and skin will fluoresce when examined in the dark  Culture Media Culture media must include sources of Nitrogen, Nitrate, Amino acids, Carbon, Vitamins and minerals  Sabouraud Dextrose Agar Primary isolation media Used for isolation of most fungi Has a low pH which can inhibit growth of bacteria Disadvantages May not allow yeast phase of the fungi to grow Too many fungi (including saprophytes) can grow on the agar  Potato Dextrose Agar Composed Potato Infusion and Dextrose that encourage fungal growth. Agar is added as the solidifying agent. Cheaper than SDA but not as good in terms of quality  Mycosel SDA-CC Antimicrobial supplements Cyclohexamide: inhibit contaminating saprophytic fungi Chloramphenicol: inhibit gram bacterial growth Chloramphenicol can be substituted with Gentamicin or Tetracycline  Brain Heart Infusion Media Recommended for the culture of yeast phase of dimorphic fungi at 35-37 degree Celsius Made from pig heart and calf brain Blood, Peptone, sulfur, carbon, nitrogen, vitamins and dextrose are added to promote growth of fastidious fungi… Basic Mycology 2 Francis Ian L. Salaver, RMT, MD  Reproduc)on Yeast cells mul+ply by binary 2ssion, budding, and pseudohyphae forma+on Molds mul+ply by sexual and asexual means The nuclei inside the fungal hyphae are haploid, unlike the diploid cells of most plants and animals.  Molds Sexual Produc)on of sexual spores Asexual (1)fragmenta)on of hyphae (2) Producing asexual spores Asexual Reproduc)on: Fragmenta)on of hyphae Fragmenta)on of hyphae Asexual Reproduc)on: Asexual spores FUNGAL SPORES 09/30/2020 9  Asexual spores Chlamydospores Blastospore/Blastoconidia Arthrospores Macroconidia and microconidia Conidiospores Sporangiospore  Asexual spores Chlamydospores Blastospore/Blastoconidia Arthrospores Macroconidia and microconidia Conidiospores Sporangiospore  Chlamydospores Thick-walled resistant spores formed by Candida species if exposed to adverse environments Produced by rounding up and enlargement of hyphal segments Candida albicans MOLDS  Candida yeast Blastospores and Pseudohyphae  Candida albicans Candida albicans is a dimorphic fungus, which means that it grows as a yeast form in a carbohydrate medium and forms molds (hyphae) when the medium is low in nutrients. The yeast-to-mold transi+on (2lamenta+on) is a process induced by environmental factors.  Candida albicans Yeast cells seem to be beHer suited for the dissemina+on in the bloodstream while hyphal cells have been proposed as a virulence factor. Hyphal cells are invasive and speculated to be important for +ssue penetra+on, coloniza+on of organs and surviving plus escaping macrophages When C. albicans cells are grown in a medium that mimics the physiological environment of a human host, they grow as 2lamentous cells (both true hyphae and pseudohyphae).  Asexual spores Chlamydospores Blastospore/Blastoconidia Arthrospores Macroconidia and microconidia Conidiospores Sporangiospore  Arthrospores Produced by the fragmenta+on of hyphae into compartments separated by septa Box-like in shape Coccidioides immi+s  Asexual spores Chlamydospores Blastospore/Blastoconidia Arthrospores Macroconidia and microconidia Conidiospores Sporangiospore  Macroconidia and Microconidia Macroconidia Exospore Large, septated could spindle-shaped or club-shaped Microconidia Smaller than macroconidia Round, elliptical. pyriform, tear-shaped Born singly or in grape like clusters along the hyphae  Dermatophytes Microsporum Trichophyton Epidermophyton Dermatophytes are fungi that require kera+n for growth. These fungi can cause super2cial infec+ons of the skin, hair, and nails.  Dermatophytes The genus Trichophyton is capable of invading the hair, skin and nails (All three so tri). The genus Epidermophyton involves the skin and nails only (Not Hair). The genus Microsporum involves only the hair and the skin (Not Nail).  Microsporum Large mul)cellular spindle shaped macroconidia with few microconidia  Trichophyton Predominant forms are microconidia with few or no macroconidia If present, macroconidia is cylindrical in shape Do not Juoresce on Wood’s lamp  Epidermophyton Club-shaped Macroconidia only; microconidia not produced  Dermatophyte test Medium Recovery of dermatophytes from hair, skin and nails Useful as a screening medium Dermatophytes produce alkaline metabolites which raise the pH and change the color of medium into RED  Asexual spores Chlamydospores Blastospore/Blastoconidia Arthrospores Macro- and microconidia Conidiospores Sporangiospore  Conidiospores Conidiospores – spores produced in a chain at the )p of a conidiophore Phialides -an elongated and Jask shaped projec)on rising from the vesicle or conidoiophore Also known as Sterigma or Sterigmata  Conidiospores Aspergillus Penicillium Phialophora Exophiala  Aspergillus Conidiospores Phialides Vesicle Conidiophore Septated Hyphae *  Aspergillus Septated hyphae which bears terminal conidiophore *  Aspergillus Conidiophore expands to large inverted Sask shaped vesicle Vesicle is covered with phialides Phialides bear the chain of conidiospores * *  Penicillium Septated Hyphae with conidiophore that exhibit branching to form metulae Brush-like conidiophore (metulae) give rise to phialides with conidiospores *  Penicillium Brush-like conidiophores that give rise to phialides from which chain of conidiospores *  Phialophora Septated hyphae with flask-shaped phialides with cup-shaped collarettes with conidiospores  Exophiala Septated hyphae with with long conidiophore that has conidiospores at its tapering end  Asexual spores Chlamydospores Blastospore/Blastoconidia Arthrospores Macro- and microconidia Conidiospores Sporangiospore  Sporangiospores Asexual spores contained in a sac- like or sporangium Unique among fungi with Nonseptated or coenocy)c hyphae Zygomycetes Rhizopus, Mucor Absidia  Mucor Nonseptated hyphae with no rhizoids The sporangiophores arise singly with sac called sporangium Sporangium contains sporangiospores * *  Rhizopus Nonseptated hyphae With Rhizoids Sporangiophores Sporangium * * Sexual Reproduc)on Sexual Reproduc)on 3 phases Plasmogamy – fusion of haploid fungi resulting to one organism with two nuclei (dikaryotic fungi) Karyogamy – fusion of nuclei to form diploid organism Meiosis – diploid nucleus become haploid  Ascospores Clusters of four or eight spores within a sac called the ascus Ascospores Ascospores Analyze this  Ascomycetes Reproduce Sexually by forming Ascospores Reproduce Asexually by forming Conidiospores/ /microconidia/ macroconidia  Zygospores Fusion of 2 identical cells arising from fused hyphae  Zygomycota Aseptate hyphae Asexual spores: sporangiospores Sexual spores: zygospores Clinically important members: Rhizopus Mucor Absidia  Basidiospores Contained in a club-shaped basidium MUSHROOMS  Basidiomycetes Reproduce Sexually by forming Basidiospores Reproduce Asexually by hyphal fragmenta:on  Imperfect Fungi (Deuteromycetes) Those fungi that do not show any sexual stage Penicillium Basic Mycology 3 Francis Ian L. Salaver, RMT, MD  Mycoses Super