Basic Mycology 1-3 MIDTERMS Notes PDF

MICROBIOLOGY AND PARASITOLOGY BASIC MYCOLOGY 1-3 Dr. Francis Ian Salaver, RMT, MD | September 11, 2024 OUTLINE ○ glucan (thickest layer; highly I...

MICROBIOLOGY AND PARASITOLOGY BASIC MYCOLOGY 1-3 Dr. Francis Ian Salaver, RMT, MD | September 11, 2024 OUTLINE ○ glucan (thickest layer; highly I Mycology immunogenic) Fungi General Characteristics ○ Peptidoglycan for bacteria A. Modes of Nutrition Heterotrophs – lack chloroplasts; cannot produce its B. Yeast vs Mold own food C. Dimorphism vs. Monomorphic Can be unicellular (yeast) or multicellular (molds) II D. Harmful Effects Can reproduce sexually or asexually E. Beneficial Effects F. Colonies G. Hyphae (Septated vs. Non- septated/ Coenocytic) Yeast A. Reproduction B. Colonies (Yeast and Mold) C. Hyphae D. Morphology of Zygomycetes III E. Hyaline and Dematiaceous (Hyphae) F. Fungal Structures - Capsule Figure 1. Fungi - Cell wall Source: Dr. Salaver’s PPT - Cell membrane A. (MODES OF NUTRITION) Molds HETEROTROPHIC ORGANISM CAN BE a. Reproduction (Molds) b. Asexual reproduction Saprophytic – feeds on dead organic material - Fragmentation of Hyphae Symbiotic – fungi is living together with other Asexual Spores organisms and establishes mutualism 1. Chlamydospores Parasitic – fungi living in another organism and 2. Arthrospores causes harm to the latter 3. Macroconidia and Microconidia SAPROPHYTIC FUNGI 4. Dermatophytes Fungi that are responsible for breaking down and 5. Microsporum recycling dead plant and animal material. 6. Trichophyton 7. Epidermophyton SYMBIOTIC FUNGI: MYCORRHIZA IV Conidiospores Mycorrhiza fungus germinates in soil. Its hyphae 1. Aspergillus penetrates the roots of the plant. 2. Penicillium The plant roots provide essential nutrients for the 3. Phialophora growth of the fungi. 4. Exophiala In return, the large mass of fungal hyphae acts as a Sporangiospores virtual root system for the plants, increasing the 1. Rhizopus amount of water and nutrients that the plant may 2. Mucor obtain from the surrounding soil. 3. Absidia c. Sexual reproduction PARASITIC FUNGI d. Sexual spores Fungi that attack living organisms, penetrate their e. Imperfect Fungi (Deuteromycetes) outer defenses, invade them, and obtain Laboratory Methods: nourishment from living cytoplasm, thereby causing a. Microscopic Methods disease and sometimes death of the host. V b. Cultivation c. Biochemical tests (Undiscussed) B. (YEAST VS. MOLD) d. Serological tests (Undiscussed) FUNGI CAN BE A YEAST OR A MOLD I. MYCOLOGY Greek words ○ Mykos (fungus) ○ Logos (study) Forms of fungi ○ Molds ○ Yeasts Dr. Salaver: II. FUNGI: GENERAL CHARACTERISTICS Yeasts are unicellular. Eukaryotic Molds are multicellular and appear branching and filamentous. Cell wall: ○ chitin (inner wall) ○ mannan (to protect glucan) NMD2027  Lesson 8: Basic Mycology 1-3 NMD2027 Dr. Salaver: Most important factor that can affect the mold and yeast form is temperature. Fungal infection (Sporothrix schenckii) in rose thorn ○ Fungus exists as a mold in the thorn of the rose Figure 2. Yeast vs Mold Source: Dr. Salaver’s PPT Dr. Salaver: ○ If introduced underneath the skin, fungus exists as yeast in skin nodule biopsy. Dimorphism is affected by temperature. At room temperature (25°-30°C), a mold will grow. At body temperature (35°-37°C), a yeast will grow. Not all fungi are dimorphic. Bread at room temperature = mold colonies YEAST Candidiasis in oral cavity = yeast colonies Unicellular circular or oval-shaped forms of fungi CSF of Cryptococcal meningitis = yeast colonies Optimum temperature: near body temperature Epidermis = can be a combination of mold and yeast Not all fungi are dimorphic. MONOMORPHIC FUNGI Only exist in one form. Figure 3. Yeast Colonies Source: Dr. Salaver’s PPT MOLDS Multicellular Filamentous, branching form Room temperature Figure 6. Monomorphic Source: Dr. Salaver’s PPT Figure 4. Molds Colonies Source: Dr. Salaver’s PPT REPRODUCTION Sexual C. DIMORPHISM VS MONOMORPHISM Asexual DIMORPHIC FUNGI Fungi that can exist in the form of both mold and yeast. - This is usually brought about by change in temperature and other factors Yeast form at 37°C Mold form at room temperature - Thermally dimorphic fungi Figure 7. Fungal Reproduction Source: Dr. Salaver’s PPT FUNGAL CULTURES Optimum temperature for growth: ○ Saprophytes (molds) = 20-30°C (Room temp.) ○ Parasitic fungi (yeast) = 30-37°C (Body temp.) The standard temperature for incubation of fungi is 30°C Figure 5. Sporothrix schenckii and cultures should be incubated in a humidified Source: Dr. Salaver’s PPT environment for 21 days Bacterial cultures require incubation temperature of 37°C 2 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 FUNGAL vs. BACTERIAL CULTURES Table 1. Differences between Cultures Fungal Culture Bacterial Culture Figure 10. Mycotoxicosis Grow best at pH of 4-6 Grow best at pH 7.0 Source: Dr. Salaver’s PPT (average: 5; acidic) (neutral) Dr. Salaver: More resistant to osmotic Less resistant to osmotic Unlike mycoses, the fungi is not the direct cause of pressure pressure the disease, instead it is the toxin produced by the fungi. Requires less nitrogen and Requires more nitrogen Aflatoxin - DNA-reactive carcinogens and moisture and moisture cytotoxins Molds - Aerobic Can be aerobic or E. BENEFICIAL EFFECTS OF FUNGI Yeasts - Facultative anaerobic 1. Preparation of bread Anaerobes 2. Preparation of wine and alcohol Capable of metabolizing Capable of metabolizing 3. Vaccine preparation complex carbohydrates (i.e, simple carbohydrates 4. Source of antibiotics lignin in wood) 5. Food 6. Decomposition 7. Pest Control Dr. Salaver: Fungi are easier to grow than bacteria. 1. PREPARATION OF BREAD Yeast cells are used in preparation of bread ○ Bread = sugar + gluten + yeast cells + water D.HARMFUL EFFECTS OF FUNGI Gluten - forms the framework of bread 1. Allergy Yeast - converts the sugar into carbon dioxide 2. Mycoses 3. Mycotoxicoses ALLERGY Mold spores get into your nose and can cause allergy symptoms They can also get into your lungs and trigger asthma Some spores of molds can trigger allergic rhinitis Figure 8. Allergic reaction Source: Dr. Salaver’s PPT MYCOSES A DIRECT fungal infection that involves invasion of tissues, skin, or cells Figure 11. Preparation of Bread Using Yeast Cells Source: Dr. Salaver’s PPT 2. PREPARATION OF WINE AND ALCOHOL Yeast cells are used in induce alcoholic fermentation in: ○ grape juice to wine Figure 9. Mycoses ○ barley grain to beer Source: Dr. Salaver’s PPT MYCOTOXICOSES Disease caused by the natural toxin produced by fungi Aspergillus flavus ○ grows on corns and peanuts ○ can produce aflatoxin that can cause hepatocellular carcinoma Figure 12. Preparation of Alcohol and Wine Source: Dr. Salaver’s PPT 3 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 3. VACCINE PREPARATION 6. DECOMPOSITION Human hepatitis B virus vaccine - prepared using Saprophytic fungi in soil produce degradative enzymes antigen produced by recombinant technology in yeast essential for the biologic recycling of organic matter. (Saccharomyces). Figure 13. Vaccine Preparation Figure 16. Saprophytic Fungi Source: Dr. Salaver’s PPT Source: Dr. Salaver’s PPT 7. PEST CONTROL Parasitic fungi can also help regulate the number of other species to maintain ecological balance. Figure 17. Parasitic Fungi Source: Dr. Salaver’s PPT III. YEAST REPRODUCTION Yeasts - unicellular form of fungi ○ spherical or elliptical in shape Yeasts can reproduce in three ways: Figure 14. Vaccine Preparation Procedure 1. Budding - most common Source: Dr. Salaver’s PPT 2. Binary fission (equally divided) 3. Formation of pseudohyphae 4. SOURCE OF ANTIBIOTICS A. REPRODUCTION Yeast cells are source of some antibiotics: 1. BUDDING Table 2. Yeast Cells as Antibiotics Most common form of reproduction ANTIBIOTICS SOURCES USES Asymmetrical division of yeast cells (smaller daughter cell) Penicillin Penicillium Used against BLASTOPORE or BLASTOCONIDIA notatum gram+ bacteria. ○ New yeast cell is formed through mitotic cell Clinically division and remains attached as a bud on the old against cell until it splits and becomes independent. pneumonia, rheumatic fever, tonsillitis, tetanus, diphtheria, and other diseases. Griseofulvin Penicillium Used against Figure 18. Budding griseofulvum mycosis Source: Dr. Salaver’s PPT Cephalosporin Cephalosporium Used against 2. BINARY FISSION acremonium (a Gram+ and A yeast cell duplicates its genetic material, or marine fungus) Gram- bacteria; deoxyribonucleic acid (DNA), and then divides into two typhoid parts (cytokinesis), with each new organism receiving one copy of DNA. 5. FOOD Higher fungi may be eaten directly as mushrooms. Figure 15. Mushrooms Figure 19. Binary Fission Source: Dr. Salaver’s PPT Source: Dr. Salaver’s PPT 4 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 3. PSEUDOHYPHAE Reproduction/growing in terms of size Buds fail to detach producing elongated yeast cells or PSEUDOHYPHAE ○ These are long branched chains of yeast cells with constrictions at the interface of elongated blastoconidia that remain attached after multiplication Figure 22. Mold Colonies and its mycelium Mature buds can either become detached as Source: Dr. Salaver’s PPT discrete cells or remain attached to the parent cell, and give rise to chains or clusters of cells. The Dr. Salaver: tendency of some yeasts to form chains of cells Yeast colonies can be mistaken for bacterial results in the formation of pseudohyphae. colonies, while mold colonies cannot. Yeast colonies cannot be morphologically differentiated from bacterial colonies but can be biochemically differentiated. PORTION OF MOLD OF COLONIES REPRODUCTIVE or AERIAL HYPHAE Figure 20. Pseudohyphae (red arrow) → grow on top of the agar’s surface of the culture media Source: Dr. Salaver’s PPT → contains the reproductive structures such as spores Dr. Salaver: VEGETATIVE PORTION or THALLUS Budding - One (bigger) mother cell and a (smaller) → portion of the mycelium that anchors the mold & daughter cell absorbs nutrients (embedded in culture media) - not successful all the time; buds fail to → grows in or on a substrate and absorbs water and detach thus presenting an elongated nutrients appearance (Pseudohyphae) Binary Fission - splitting into two equal parts Pseudohyphae - elongation; found in yeasts True hyphae - found in molds B. COLONIES (YEAST AND MOLD) Figure 23. Portion of Mold Colonies YEAST COLONIES Source: Dr. Salaver’s PPT 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 Figure 24. Bread Mold Source: Dr. Salaver’s PPT Dr. Salaver: Blue arrow → Reproductive Portion Portion that is embedded in the bread → Figure 21. Yeast Colonies Vegetative Portion Source: Dr. Salaver’s PPT SUMMARY: YEAST VS MOLD COLONIES MOLD COLONIES Produce multicellular, filamentous, cottony, & dry colonies Consist of branching cylindrical tubules with diameter called hyphae Hyphae grow to form a filamentous mass of intertwining strands called mycelium 5 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 C. HYPHAE D.MORPHOLOGY OF THE ZYGOMYCETES Hyphae grow to form a filamentous mass of intertwining strands called mycelium Describe as Hyaline if fungal structures are colorless Highly refractile Dr. Salaver: Hypha (singular); Hyphae (plural) Hypha - each of the branching filaments in a mold Mycelium - group of hyphae HYPHAE CAN BE SEPTATE OR NONSEPTATE Figure 28. Morphology of the Zygomycetes Source: Dr. Salaver’s PPT Dr. Salaver: Nonseptate hyphae or Coenocytic ○ smooth like appearance Rhizoids ○ root-like structure ○ found in vegetative portion Sporangiophore ○ long stalk ○ structure that arises immediately from nonseptate hyphae Columella Figure 25. Septate vs Nonseptate Hyphae ○ dilated part; source of nutrients Source: Dr. Salaver’s PPT Sporangium ○ sac-like structures 1. SEPTATE HYPHAE Sporangiospores ○ spores inside the sporangium Have dividers between the cells called septa (singular: ○ spores within the sac septum) ○ found in reproductive portion The septa have openings called pores between the cells → to allow the flow of cytoplasm and nutrients throughout the mycelium ZYGOMYCETES Table 3. Zygomycetes Rhizopus species Nodal rhizoids Rhizoids immediately below sporangiophore Embedded in bread molds Figure 26. Septate Hyphae Mucor species Source: Dr. Salaver’s PPT No rhizoids 2. NONSEPTATE HYPHAE or COENOCYTIC Lack septum and cell membranes between the cells Septa can be seen in branching points Absidia species Zygomycetes - important species that are nonseptate Internodal rhizoids → Mucor Rhizoids in → Rhizopus between → Absidia sporangiophore Rhizoids not immediately beneath the sporangiophore Source: Dr. Salaver’s PPT E. HYALINE and DEMATIACEOUS HYPHAE HYALINE HYPHAE If fungal structures are colorless Figure 27. Nonseptate Hyphae Highly refractile Source: Dr. Salaver’s PPT 6 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 ○ Causative agent of fungal meningitis among DEMATIACEOUS FUNGI immunocompromised patients Group of fungi that produce melanin in their cell walls, ○ Found in pigeon droppings giving them a characteristic brown color when grown on agar Group of fungi with dark colonies and pigmented fungal elements Figure 31. Transmission of Cryptococcus neoformans Source: Dr. Salaver’s PPT Figure 29. Dematiaceous Fungi Source: Dr. Salaver’s PPT India Ink Wet Mount Used to identify the capsule of Cryptococcus Dr. Salaver: neoformans CSF is directly examined by adding one drop of India ink If the hyphae is brown in color even without the stain, Capsule appear as a clear halo against a dark you call the fungi DEMATIACEOUS background (negative staining – white capsule against What do you call the group of fungi that produce a black background) melanin like pigments? – DEMATIACEOUS Figure 30. Lesion infected by a dematiaceous fungus (appears pigmented) Figure 32. India Ink Staining Source: Dr. Salaver’s PPT Source: Dr. Salaver’s PPT Dr. Salaver: Dr. Salaver: (Refer to Figure above) If they are brown, most likely the lesions that they will What do you call the white halo around each yeast cell? produce are also brown in color – CAPSULE What stain was used? – INDIA INK What structure was demonstrated? – CAPSULE (BROWN CAPPE) Name the organism – CRYPTOCOCCUS ○ Curvularia – cause plant diseases but can cause NEOFORMANS infection among immunocompromised individuals Name the specimen – CSF ○ Alternaria – cause plant diseases but can cause Temperature - ROOM TEMPERATURE infection among immunocompromised individuals Unicellular or Multicellular - UNICELLULAR ○ Phialophora ○ Piedraia Staining Procedure (Dr. Salaver: “Wag niyo na to memorize’in ha. Wag niyo ○ Exophiala na pahirapan ang life niyo.”) 1. Place a loopful of India ink on the side of a clean REMEMBER: slide EX ni PIED si PHIA, naghanap siya ng ALTERnative na 2. Mix a loopful of liquid culture with the ink may CURVes 3. Place a clean cover slip over the preparation avoiding air bubble 4. Examine microscopically F. FUNGAL STRUCTURES Bird Seed Agar 1. 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 Cryptococcus neoformans ○ Capsule has antiphagocytic properties and is Figure 33. Bird Seed Agar associated with virulence Source: Dr. Salaver’s PPT 7 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 The extract of Guizotia abyssinica seeds contains ○ The nuclei inside the fungal hyphae are caffeic acid which serves as a substrate for the HAPLOID, unlike the diploid cells of most detection of phenoloxidase, an enzyme produced by C. plants and animals neoformans. The action of phenoloxidase on caffeic acid results in the production of melanin which is absorbed by the yeast cell wall forming a tan to reddish-brown pigmentation. Dr. Salaver: Appearance of colonies in bird seed agar: ○ Cryptococcus - brown ○ Candida - whitish Figure 35. Sexual and Asexual Reproduction of Molds Table 4. Human vs Bacteria vs Fungi Structures Source: Dr. Salaver’s PPT Cell Membrane Cell Wall MOLDS: Spores Formation Human Present Absent Bacteria Present Present Sexual (peptidoglycan) → Production of sexual spores Fungi Present Present Asexual (chitin, glucans, → Fragmentation of hyphae mannan) ▪ Each fragment develops into new vegetative cells Source: Dr. Salaver’s PPT → Production of asexual spores B. ASEXUAL REPRODUCTION 2. CELL WALL (FRAGMENTATION OF HYPHAE) 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. 3. CELL MEMBRANE Bilayered membrane composed of several phospholipids Contain sterols which are essential for the viability of Figure 36. Fragmentation of Hyphae fungi Source: Dr. Salaver’s PPT ○ Principal fungal sterols are: Ergosterol (provides Dr. Salaver: stability and flexibility to the cell membrane) and Asexual spores - created by 1 fungus Zymosterol Fragmentation of hyphae - “for example, I am a fungus, I am a mold, I have a lot of hyphae, if gusto ko mag asexual (cause walay partner), i-cut nako ang hand nako and throw it didto, naa na another Salaver” Most popular organism multiplying by this mechanism - MUSHROOMS ○ as the hyphae are crawling underneath the ground, they are undergoing fragmentation Figure 34. Fungal Cell Membrane Source: Dr. Salaver’s PPT ASEXUAL REPRODUCTION: ASEXUAL SPORES 1. Chlamydospores Dr. Salaver: 2. Arthrospores The sterol found in the fungal cell membrane is called 3. Macroconidia and microconidia ERGOSTEROL (but if not found in the choices, go for 4. Conidiospores/Phialospores ZYMOSTEROL) 5. Sporangiospore 1. CHLAMYDOSPORES IV. MOLD REPRODUCTION A. REPRODUCTION (Molds) Yeast cells multiply by ○ binary fission ○ budding ○ pseudohyphae formation Molds multiply by sexual and asexual means – production of spores (main reproductive material of fungi) Figure 37. Chlamydospores Source: Dr. Salaver’s PPT 8 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 Box-like in shape Thick-walled asexual spores formed by rounding up and enlargement of hyphal segments Coccidioides immitis ○ Terminal ○ Intercalary Candida albicans MOLDS Hyphae suddenly become rounded CANDIDA ALBICANS Dimorphic fungus - grows as yeast form in a carbohydrate Figure 39. Arthrospores medium Source: Dr. Salaver’s PPT - forms molds (hyphae) when the medium is Dr. Salaver: low in nutrients. If the hyphae will become box-like - Arthrosphores The yeast-to-mold transition (filamentation) is a process induced by environmental factors. Yeast cells 3. MACROCONIDIA / MICROCONIDIA - Suited for the dissemination in the bloodstream while; Macroconidia - Hyphal cells have been proposed as a ○ Exospore virulence factor. ○ Large, septated could be spindle-shaped Hyphal cells or club-shaped - Invasive and speculated to be important Microconidia for the penetration, colonization of organs ○ Smaller than macroconidia and surviving plus escaping macrophages ○ Round, elliptical. pyriform, tear-shaped ○ Born singly or in grape like clusters along the hyphae Figure 40. Macroconidium and Microconidium When C. albicans cells are grown in a medium that Source: Dr. Salaver’s PPT mimics the physiological environment of a human host, they grow as filamentous cells (both true Dr. Salaver: hyphae and pseudohyphae). If you see tear drop shaped structure - either Macroconidia or microconidia Dr. Salaver: ○ Macroconidia - bigger, with septations If you see a hyphae was suddenly become ○ Microconidia - smaller, no septations rounded - Chlamydospore DERMATOPHYTES Dermatophytes are fungi that require keratin for growth. These fungi can cause superficial infections of the skin, hair, and nails. → Secrete enzymes keratinase, which digest keratin (infect only superficial keratinized structures.) → Unable to grow in 37 degree Celsius thus no deep infection THREE IMPORTANT DERMATOPHYTE GENERA Microsporum Trichophyton Epidermophyton → The genus Trichophyton is capable of invading the hair, skin and nails. (All three are TRI). → The genus Epidermophyton involves the skin and nails only (Not Hair). → The genus Microsporum involves only the hair and the skin (Not Nail). Table 5. DERMATOPHYTES Figure 38. Asexual Spores Organism Shape Skin Hair Nails Source: Dr. Salaver’s PPT Trichophyton Pencil- ✓ ✓ ✓ Shaped 2. ARTHROSPORES Microsporum Spindle- ✓ ✓ ✘ Shaped Produced by the fragmentation of hyphae into Epidermophyton Club- ✓ ✘ ✓ compartments separated by septa Shape 9 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 MICROSPORUM Large multicellular spindle shaped macroconidia with few microconidia Cannot infect the nails Dr. Salaver: Trichophyton – called as such because it can infect TRI (three) – hair, skin, nails). Predominantly MICRO, looks like a Christmas light. Epidermophyton – can’t infect the eeeeehair, therefore only skin and nails. Microsporum – can’t infect the next letter to letter “M” which is N – nails. Predominantly MACRO, little Figure 41. Microsporum micro. It is spindle shape – “Chinese eyes” Source: Dr. Salaver’s PPT Treatment for Ringworm: ○ Anti-fungal Medication ○ Lifestyle modification TRICHOPHYTON Example: Tinea Pedis – wear Predominant forms are microconidia with few or no socks first before pants. Prevent macroconidia travel of fungus through the If present, macroconidia is cylindrical in shape pants. Tinea capitis cannot be caused by Epidermophyton Tinea unguium cannot be caused by Microsporum Where to collect the sample? At the periphery/raised portion. Dermatophytosis Also called tinea or ringworm Disease of the nails, hair and skin caused by filamentous Figure 42. Trichophyton fungi called dermatophytes Source: Dr. Salaver’s PPT Etiological agents: → Epidermophyton floccosum EPIDERMOPHYTON → Microsporum spp. Club-shaped Macroconidia only; microconidia are not → Trichophyton spp. produced/present. Cannot infect the hair Table 6. DERMATOPHYTES Genus Macroconidia Microconidia Epidermophyton Present None Microsporum spp. Many Rare Trichophyton spp. Rare Many 4. CONIDIOSPORES Conidiospores – spores produced in a chain at the tip of Figure 43. Epidermophyton a conidiophore Phialides -an elongated and flask shaped projection rising Source: Dr. Salaver’s PPT rom the vesicle or conidiophore DERMATOPHYTES CLINICAL CLASSIFICATION → Also known as Sterigma or Sterigmata Infection is named according to the anatomic locations involved. → Tinea faciei (face excluding beard and mustache) → Tinea barbae (beard and mustache) → Tinea corporis (trunk and extremities) → Tinea capitis (scalp) → Tinea cruris (Jock itch/groin) → Tinea pedis (Athlete’s foot/ soles or toes) → Tinea manuum (hand, palm) Figure 44. Conidiospores → Tinea unguium (nail) Source: Dr. Salaver’s PPT 10 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 Dr. Salaver: FOUR IMPORTANT CONIDIOSPORE GENERA Exophiala - NO PHIALIDES Aspergillus Penicillium Phialophora E. Alternaria Exophiala Chain of club-shaped spores with septations A. Aspergillus Septate hyphae which bears terminal conidiophore F. Curvularia Conidiophore expands to large inverted flask shaped Curved-shaped vesicle” Vesicle is covered with phialides Phialides bear the chain of conidiospores 5. SPORANGIOSPORES Aspergillus flavus- no black pigment (green spores) Figure 49. Sporangiospores Figure 45. Aspergillus Source: Dr. Salaver’s PPT Source: Dr. Salaver’s PPT Asexual spores contained in a sac-like or B. Penicillium sporangium Unique among fungi with nonseptated or Septate Hyphae with conidiophore that exhibit branching coenocytic hyphae to form metulae Ex. Rhizopus, Mucor, Absidia Brush-like conidiophore (metulae) give rise to phialides with conidiospores A. Rhizopus Nonseptated hyphae With Rhizoids With Sporangiophores With Sporangium Figure 46. Penicillium Source: Dr. Salaver’s PPT Dr. Salaver: Spores are arranged in chains. Use Conidiospores. C. Phialophora Septate hyphae with flask-shaped phialides with cup-shaped collarettes with conidiospores Figure 50. Rhizopus Source: Dr. Salaver’s PPT B. Mucor Nonseptated hyphae with no rhizoids The sporangiophores arise singly with sac called sporangium Sporangium contains sporangiospores Figure 47. Phialophora Source: Dr. Salaver’s PPT Dr. Salaver: Phialophora - NO CONIDIOPHORE For moving exam- Septate hyphae with phialides with collarettes with conidiospores D. Exophiala Septate hyphae with with “loooong” conidiophore that has conidiospores at its tapering end Figure 51. Mucor Source: Dr. Salaver’s PPT C. Absidia Has internodal rhizoids Figure 48. Exophiala Source: Dr. Salaver’s PPT 11 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 C. SEXUAL REPRODUCTION 3 phases: 1. Plasmogamy – fusion of haploid fungi resulting to one organism with two nuclei (dikaryotic fungi) 2. Karyogamy – fusion of nuclei to form diploid organism 3. Meiosis – diploid nucleus become haploid Figure 53. Ascospores Table 7. ASCOMYCETES REPRODUCTION SEXUAL SPORE ASEXUAL SPORE Ascospores Conidiospore Microconidia and Macroconidia Source: Dr. Salaver’s PPT Figure 52. Phases of sexual reproduction Source: Dr. Salaver’s PPT Dr. Salaver: Fusion of the cell membranes (haploid) is called Plasmogamy. Figure 54. Life Cycle of Ascomycetes ○ Haploid + haploid= Dikaryotic fungi Source: Dr. Salaver’s PPT ○ Nuclei is not yet fused Dr. Salaver: Plasmogamy is followed by Karyogamy ○ Nuclei is fused = Diploid fungi Why are fungi called Ascomycete? Meiosis convert fungi from diploid to HAPLOID ○ Because they form ascospores (sexually) ○ Fungi normally exist as haploid ○ Ascospores are released as haploid ○ Haploid spores will mate with another haploid organism and the cycle will D. SEXUAL SPORES continue Mnemonic : ZAB What if they can’t find a partner? ○ Asexual reproduction 1. Z- Zygospores (Phycomycetes) ○ Ascomycete can reproduce by producing 2. A- Ascospores (aspergillus) ASCOSPORE= sexually 3. B- Basidiospores (Cryptococcus) CONIDIOSPORE= asexually Microconidia and Dr. Salaver: Macroconidia= asexually; also Sexual spores are named after wherever they are belong to Ascomycete group contained. Ex. ○ Spores contained in a sac called Ascus= ZYGOSPORES ASCOSPORES Fusion of two identical cells arising from fused hyphae ASCOSPORES Thick-walled spore of fungi that is formed by union Fungal spore formed within an ascus of of two similar sexual cells ascomycetes Kind of spore is specific to fungi classified as ascomycetes (Ascomycota) Clusters of four or eight spores within a sac called the ascus Sac fungi Figure 55. Zygospores Source: Dr. Salaver’s PPT ZYGOMYCOTA Nonseptate hyphae Asexual spores: sporangiospores Sexual spores: zygospores Clinically important members: ○ Rhizopus ○ Mucor ○ Absidia 12 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 Table 8. ZYGOMYCOTA REPRODUCTION Gentle heating may hasten the action of the KOH Fungal elements appear as hyaline structures. SEXUAL SPORE ASEXUAL SPORE Some may be slightly pigmented. Zygospores Sporangiospores Disadvantage: poor contrast BASIDIOSPORES Contained in a club-shaped basidium Hyphal Fragmentation Edible mushrooms Figure 57. Hyaline structures (Macroconidia & Microconidia) ○ Club fungi Source: Dr. Salaver’s PPT Table 9. BASIDIOMYCETES REPRODUCTION Lactophenol Cotton Blue (Aman’s medium) SEXUAL SPORE ASEXUAL Basidiospore Hyphal Lactic acid- preserve the fungal structures fragmentation Phenol- killing agent Cotton blue- imparts blue color to the fungal structures Replaces KOH Advantages- structures are readily seen microscopically Figure 58. LPCB Source: Dr. Salaver’s PPT 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 Figure 56. Basidiospores room. Source: Dr. Salaver’s PPT Infected hair and skin will fluoresce when examined in the dark IMPERFECT FUNGI (DEUTEROMYCETES) Those fungi that do not show any sexual stage Ex. Penicillium V. LABORATORY METHODS Microscopic Methods Cultivation Biochemical Tests Figure 59. Tinea manuum in Wood’s lamp Serologic Tests Source: Dr. Salaver’s PPT A. MICROSCOPIC METHODS Wet Mounts – placing fluid into the slide and Condition Color suspending the specimen into the fluid ○ KOH Acne Orange-red ○ Lactophenol cotton blue (LPCB) Stained smears Burns infection Green ○ India ink ○ Special stains for fungal cell walls Erythrasma Coral pink 10% Potassium hydroxide (KOH) Folliculitis Bluish-white KOH acts as clearing agent ○ Dissolves keratin Hereditary punctate White ○ Eliminates debris and cells keratoderma 20% KOH is used for nails (highly keratinized tissue) Pityriasis versicolor Yellowish-brown 1-2 drops of KOH is added to specimen on a slide Cover with a coverslip Porphyria Red-pink Allow the specimen to clear for 20 minutes (10-30 minutes). Tinea capitis Blue-green 13 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 2. Staphylococcus spp: of equal size FUNGAL STRUCTURES: CELL WALL Fungal cell wall is poorly stained with routine Hematoxylin and Eosin Fungal cell wall stains → Periodic acid Schiff → Gomori’s Methenamine silver stain → Calcofluor white - a fluorescent stain Figure 64. Gram stain: Cryptococcus neoformans → Gram stain is useful for Candida and Cryptococcus “Starburst / Burning star appearance” Source: Dr. Salaver’s PPT FUNGAL CELL WALL STAINS: Dr. Salaver: 1. PERIODIC ACID SCHIFF Sample case: A immunocompromised patient with poor compliance to antiretroviral medicines went to Stains magenta red the ER. Patient has nuchal rigidity, headache and fever. What sample should be sent to the laboratory for testing? ANSWER: CSF (for meningitis) Follow up question: If India ink is not available for this specimen, what should be used? ANSWER: 1) Bird Seed Agar Figure 60. Periodic Acid Schiff 2) Gram stain- look for starburst appearance Source: Dr. Salaver’s PPT SPECIAL STAINS 2. GOMORI’S METHENAMINE SILVER STAIN Stains black A. INDIA INK STAINING Negative staining used for detection of organism’s capsule Figure 65. India ink Staining Figure 61. Gomori's Methenamine Silver Stain Source: Dr. Salaver’s PPT Source: Dr. Salaver’s PPT 3. CALCOFLUOR WHITE B. GIEMSA OR WRIGHT STAIN Fluorescent stain For blood and bone marrow smears Fluoresces into apple green or bluish white color when exposed to ultraviolet light from the fluorescence Use this if the organisms already reached the blood microscope Added with potassium hydroxide Figure 66. Giemsa or Wright Stain Source: Dr. Salaver’s PPT B. CULTIVATION Figure 62. Calcofluor White SIMPLE STREAKING Source: Dr. Salavers ppt For bacteria: 4 quadrant streaking method to dilute the number of organisms in order to see isolated colonies 4. GRAM STAIN For fungi: simple streaking method Usually used to demonstrate bacteria HOW: Useful for Candida and Cryptococcus 1. Get a swab and moisten it 2. Wipe the swab on the surface of your sample 3. Simple streaking on the agar plate 4. Incubate the sample: Room Temperature: grow as MOLD Body Temperature: grow as YEAST DON’ T FORGET TO TURN THE AGAR PLATE UPSIDE Candida spp. Staphylococcus spp. DOWN TO AVOID CONDENSATION! Figure 63. Gram Stain Source: Dr. Salaver’s PPT Dr. Salaver: To know the difference between the two under the microscope: Figure 67. Simple Streaking 1. Candida spp: with budding yeast Source: Dr. Salaver’s PPT 14 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 MACROSCOPIC EXAMINATION OF FUNGAL CULTURES → Septated vs Nonseptated hyphae → Types of spores present DISADVANTAGE: Structures may be destroyed by the RATE OF GROWTH teasing procedure Should not be done if dealing with Rapid grower - growth within 1 to 2 days systemic fungi Slow grower- growth may take weeks ADVANTAGE: can use sample for both aerial and vegetative spores sampling TOPOGRAPHY No longer practiced in the laboratory Only applicable when dealing with yeast Flat- uniformly elevated; elevation from surface of agar Raised- elevated colony above the agar surface Convex- colony is broader at the center than the edges; center is raised while the edges are flat Umbonate- knob in the center of the colony Crateriform- saucer or goblet-shaped; looks like the mouth of a volcano Rugose- wrinkled; looks like the rugae of a stomach Figure 69. Tease Method Source: Dr. Salaver’s PPT CELLOPHANE TAPE PREPARATION A piece of scotch tape is laid gently over the portion of the Figure 68. Topography of Fungal Cultures fungal colonies and slowly lifted and place on a slide with Source: Dr. Salaver’s PPT KOH or LPCB Good for sampling sporulating fungi SURFACE TEXTURE OF COLONY Hyphae and rhizoids may not be seen Cottony or Woolly Should be done in processing systemic fungi Velvety or Silky- mostly appears wrinkled ADVANTAGE: will not destroy the morphology of the fungi Granular or Powdery DISADVANTAGE: only limited to sampling reproductive Moist spores and not the vegetative spores PIGMENTATION Best way to observe the color is to look at the underside of the medium Can be observed on the surface or reverse side of the culture medium Can be used to identify fungi in the laboratory HAIR BAITING TECHNIQUE Put soil on the petri dish Get hair strands sample Figure 70. Cellophane Tape Preparation Soak the hair strands on acetone to remove the sebum on Source: Dr. Salaver’s PPT it in order to expose the keratin since some fungi feed on it RIDDELL’S SLIDE CULTURE TECHNIQUE Create a hole at the center of the soil Put the hair strands at the hole at the center so that the Used to study undisturbed morphological details of fungi fungi from the soil will crawl their way to the hair strand Reproductive hyphae are preserved Under the microscope you can see the fungi crawling on Prepare petri dish with moisten tissue paper the hair Place sterile slide on bent glass/ applicator stick in the Spray water petri dish Incubate the sample Cut 1x1cm SDA agar and place it on top of the slide Inoculate fungi at the four sides of the agar block QUESTION!!! Cover the SDA block with cover slip and incubate at room What is the purpose of soaking hair in acetone? temperature The purpose of soaking hair in acetone is to Always moisten the base of the petri dish remove sebum and other oils, exposing the keratin. This creates an environment that promotes the growth of keratinophilic fungi, which feed on keratin. What is the source of the fungi? Soil FUNGAL CULTURES: MICROSCOPIC EVALUATION TEASE METHOD Fragments of fungal culture is teased using needles and place on a glass slide with KOH and LPCB Study the hyphae and spores 15 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 Figure 73. Brain Heart Infusion Agar Source: Dr. Salaver’s PPT GERM TUBE TEST Germ tubes are short outgrowth, non-septate germinating hyphae They are ½ the width and 3-4 times the length of the cell from which they arise When cells of Candida are incubated in rabbit serum at 37°C for 2-4 hours, Candida albicans produce short, slender, tube like structures called germ tubes INTERPRETATION: → GERM TUBE POSITIVE: Figure 71. Riddell’s Slide Culture Technique 1. Candida albicans Source: Dr. Salaver’s PPT 2. Candida dubliniensis -Culture with Corneal agar with Tween 80 to differentiate these two species SABOURAUD DEXTROSE AGAR → GERM TUBE NEGATIVE: 1. Other Candida species Most commonly used for isolation of most fungi in the laboratory Has low pH which inhibits growth of bacteria since fungi grows at this pH Good for isolating dermatophytes Also a good culture media for ring worms Dextrose is used as an energy source of the organisms DISADVANTAGE: May not allow yeast phase of the pathogenic/ dimorphic fungi to grow May not support growth of fastidious fungi Saprophytes can grow on the agar Figure 74. Germ Tube Test Source: Dr. Salaver’s PPT CORNMEAL AGAR WITH TWEEN 80 Corn Meal Agar allows Candida albicans and Candida dubliniensis to produce Chlamydospores. The addition of the Tween 80 solution enhances the formation of the chlamydospores Incubate at room temperature for 48 hours Figure 72. Dr. Salavers ppt Dr. Salaver: Sabouraud Dextrose Agar To differentiate the two germ tube positive Candida SDA-CC species, observe the following: ○ Candida albicans: lesser chlamydospores Modified SDA ○ Candida dublinensis: more With antimicrobial supplements: chlamydospores → CYCLOHEXIMIDE: inhibit contaminating saprophytic fungi → CHLORAMPHENICOL: inhibit gram bacterial growth ▪ If chloramphenicol is not available, it can be substituted with GENTAMICIN BRAIN HEART INFUSION MEDIA Recommended for the culture of molds, heast and fastidious microorganisms Good for culturing dimorphic fungi Made from pig heart and calf brain and peptone (animal source): source of carbon, nitrogen, vitamins Figure 75. Corn Meal Agar with Tween 80 Source: Dr. Salaver’s PPT Dextrose 16 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 Identify CHROMAGAR Also used in differentiating different Candida species based on their pigmentation on the culture media growth 5 What chemical was added to LPCB to slow down the drying out of prepared specimens? a. Lactic acid b. Phenol 6 c. Cotton blue d. Glycerine e. Castor oil Risk factor for this systemic mycoses is exposure to bird and bat droppings: a. Blastomycosis 7 b. Paracoccidiodomycosis Figure 76. ChromAgar c. Coccidioidomycosis Source: Dr. Salaver’s PPT d. Histoplasmosis CARBOHYDRATE ASSIMILATION TEST # ANSWER Determines the ability of a yeast isolate to use a particular 1 A carbohydrate substrate as its sole carbon in a medium 2 C 3 B Yeasts were cultures in Yeast Extract Peptone Dextrose 4 Cryptococcus neoformans medium for 24 hours at 30°C 5 Arthrospore Place sugar molecule in each well 6 D. Glycerine Check which sugar was used by the organism in its 7 D.Histoplasmosis growth by looking at the halo around the well X. FREEDOM WALL Figure 77. Carbohydrate Assimilation Test Source: Dr. Salaver’s PPT VII. APA REFERENCES [Salaver, F.]. (2023). Mycology [Powerpoint Slides]. College of Medicine, Davao Medical School Foundation, Inc. Jawetz, Melnick and Adelberg’s Medical Microbiology (28th ed.). McGrawHill IX. REVIEW QUESTIONS # QUESTION Saccharomyces is used to produce vaccines against? A. Hepatitis B 1 B. COVID C. DPT D. Tetanus toxoid Which of the following is NOT TRUE about fungi A. Unicellular or multicellular 2 B. has membrane-bound nucleus C. can produce food on their own D. reproduce sexually and asexually A mycelium is A. fungi that is incapable of sexual reproduction 3 B. group of hyphae C. fungi that grow on top of agar Identify 4 17 of 18  Lesson 8: Basic Mycology 1-3 NMD2027 INDEX: APPENDIX Dermatophytes Clinical Classifications Clinical Lesions Site of Lesions Clinical Appearance Organisms Most Frequently Isolated Tinea Capitis, epidemic Scalp Circular patches with short hair stubs of T tonsurans, M audouini broken hair within hair follicles (US), T violaceum, M ferrugineum (outside U.S.) Tinea capitis, nonepidemic Scalp M canis, T verrucosum, M gypseum (rare) Tinea favosa (favus) Scalp, torso T schoenleinii, T violaceum Tinea barbae Beard Edematous, erythematous lesion T rubrum, T verrucosum Tinea corporis Arms, Legs, Torso T rubrum, M canis, T mentagrophytes Tinea cruris Genitocrural folds T rubrum, T mentagrophytes, Epidermophy Tinea pedis and manus Feet, Hands T rubrum, T mentagrophytes Tinea unguium Nails Thickened or crumbling distally; discolored; T rubrum, T lusterless mentagrophtes, E. loccosum 18 of 18