PCCSOM 2026 Microbiology F.01 Introduction to Mycology PDF

PCC SOM 2026 MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY MICROBIOLOGY LECTURE LECTURER: DR. ARLENE QUITASOL, FPSP DATE: APRIL 29, 2024 TOPIC OUTLINE I. Medical Mycology II. Morphologic Forms of Fungi III. Variant Forms IV. Structure of Yeast and Hyphae V. Fungal Pigment VI. Reproductive Structures VI...

PCC SOM 2026 MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY MICROBIOLOGY LECTURE LECTURER: DR. ARLENE QUITASOL, FPSP DATE: APRIL 29, 2024 TOPIC OUTLINE I. Medical Mycology II. Morphologic Forms of Fungi III. Variant Forms IV. Structure of Yeast and Hyphae V. Fungal Pigment VI. Reproductive Structures VII. Coidiogenesis VIII. Dimorphism IX. Diagnostic Techniques I. 2. MOLDS Multicellular Filamentous fungi Colonies: o woolly, fluffy, or velvety appearance, sometimes punctuated with a granular or powdery aspect o produced by the formation of asexual reproductive structures o At other times, the colony may have a glabrous (smooth) appearance MEDICAL MYCOLOGY Study of fungi and their relationship to human disease Encompasses 1. Single-celled yeasts 2. Filamentous molds Disease ranging from superficial skin infection (cutaneous mycosis) to disseminated deep seated visceral disease (systemic mycosis) Fungal agents include the historically pathogenic fungi (true pathogens), as well as the saprophytic fungal agents elevated to the status of pathogen by modern therapies and diseases (opportunistic pathogens) II. MORPHOLOGIC FORMS OF FUNGI 1. YEASTS Unicellular organisms Blastoconidia, aka “Budding” o Asexual reproduction to produce a daughter cell Colonial mass of yeast o collection of distinct individual organisms that macroscopically may resemble bacterial colonies on the agar surface Yeast colonies o smooth with a regular edge, some species may be rough with irregular edges o Heaped and dull colonies, like staphylococci o Produces catalase Identification of colony as yeast o Microscopic presence of “budding yeast” with or without pseudohyphae Pseudohyphae of yeast o often visible macroscopically as filamentous extensions from the edges of the colony, known colloquially as “FEET” NOTE TAKER: BALDOS, PADAYAO, FERRER III. 1. VARIANT FORMS DIMORPHIC FUNGI In agar media and environment: o behave as molds at 25-30 ᵒC (Room Tᵒ) o behave as tissue forms at 37 ᵒC a. YEAST Blastomyces Histoplasma Sporothrix b. SPHERULE Coccidioides immitis Coccidioides posadasii SCLEROTIC BODIES o Nonhyphal cells produced by black molds such as Fonsecaea pedrosoi and Phialophora verrucosa o appear as molds when grown on solid media at room temperature. Page 1 | 8 PCC SOM 2026 2. MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY REVERSE DIMORPHISM produces both yeast cells and hyphae in cutaneous lesions seen in pityriasis versicolor caused by Malassezia furfur IV. STRUCTURE OF YEASTS AND HYPHAE A. MOLD 1. 2. 3. 4. 5. 6. Hyphae (singular, hypha) filamentous structure of a mold Mycelium mass of hyphae Vegetative mycelium mycelium growing on the surface of or within the agar Aerial mycelium filamentous extensions above the colony Conidiophores aseptate or septate specialized hyphae that bear asexual reproductive structures. True hyphae may have cross walls that contain pores for communication through the hyphae or cross walls that are complete, dividing the hyphae into multiple cells. a. b. c. SEPTATE: with cross walls ASEPTATE: without cross walls SPARSELY SEPTATE: nonseptate fungi that have occasional cross walls B. YEAST “Budding” asexual reproduction by the formation of BLASTOCONIDIA, where the daughter cell usually appears at one end of a yeast cell and eventually enlarges to form a new yeast cell a. Pseudohypha (plural, pseudohyphae) Produced if a series of daughter cells do not detach fully from the originating cells distinguished from true hyphae by the presence of a constriction at the junction of adjacent cells and by the restriction in size of the daughter cell to that of the parent True hyphae Walls are parallel without constrictions b. Cryptococcus spp. produces only budding yeast and rarely rudimentary or primitive pseudohyphae, but true hyphae are not produced. The width of the hyphae and the angle of branching are important clues to fungal identity. Mucormycetes also called zygomycetes and members of the order Mucorales, such as Rhizopus spp. broad, ribbonlike hyphae that have haphazard branching Aspergillus spp. narrow hyphae that branch at acute angles (dichotomous branching) V. FUNGAL PIGMENT - 1. NOTE TAKER: BALDOS, PADAYAO, FERRER Some mold hyphae and yeast can be characterized by the production of a dark pigment MELANIN brown coloration (microscopic preparations) colonies of the fungi to appear dark green, brown, or black. Melanin containing fungi are interchangeably called o “Dematiaceous” o “Melanized” o “Dark” o “Phaeoid” Page 2 | 8 PCC SOM 2026 2. MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY HYALINE fungi that lack dark hyphal pigmentation clear or colorless True appearance of these molds usually can be discerned by observing the back of the colony o Hyaline: maintains a light coloration. o Dematiaceous: demonstrate dark pigment both the front (obverse) and the back (reverse) VI. REPRODUCTIVE STRUCTURES Primary means for identification of mold fungi is by characterization of asexual reproductive structures. In yeast, phenotypic studies are the mainstay in identification, with asexual reproductive structures serving as ancillary clues in the identification process. Two principal asexual structures 1. SPORES (which may also be present as sexual structures) ASEXUAL SPORES “SPORANGIOSPORES” produced by cleavage within an encompassing structure called a SPORANGIUM 2. CONIDIA (singular, conidium) much more diverse and form by differentiation from the tip or side of a fertile hypha, such as a conidiophore, or by hyphal differentiation. “BUDDING” Principal means of asexual reproduction in yeast is by the formation of blastoconidia Bud starts as a softening of the cell wall of the mother cell, followed by expansion of the cell wall (blown out) and migration of nucleus and cytoplasm to the swollen area. Septum seals the boundary between daughter and parent cells If separation does not occur, a pseudohypha results. STRUCTURES FOR REPRODUCTION 1. CONIDIOGENOUS CELLS o portions of the vegetative mycelium o differentiate into conidia 2. CONIDIOPHORES o specialized hyphae that support the conidia o may be the conidiogenous cell itself arising from the vegetative mycelium or may be a supporting hypha **In Aspergillus spp. conidiophore, which is aseptate, enlarges at the tip to form a swollen vesicle Conidiogenous cells, now termed PHIALIDES, arise from the vesicle to support chains of conidia Some species of Aspergillus produce a row of phialides, which occur on a row of sterile cells called METULAE, with the conidia arising from the distal phialides. NOTE TAKER: BALDOS, PADAYAO, FERRER Page 3 | 8 PCC SOM 2026 VII. 1. MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY SPORANGIOPHORES produced by Mucormycetes, which support the sporangium enclosed by sporangiospores COLUMELLA an extension of the apex of the sporangiophore into the sporangium 2. BLASTIC CONIDIOGENESIS Protoplasm of the conidiogenous cell is blown out or blasted to form the conidium Simplest form of blastic conidiogenesis is the budding process by which many types of yeast, ie. Candida spp. Blastoconidia are divided into two types: a. one where the cell wall is involved in the process b. the other where cell wall is not involved o Further division of blastic conidiogenesis is made among those species in which the outer cell wall does not participate in the process (called enteroblastic conidiogenesis) 3. ENTEROBLASTIC CONIDIOGENESIS Phialidic and annellidic conidiogenous cells recognized i. PHIALIDES o Conidiogenous cells that often have a collarette at the apices, produced when the tip releases the first conidium. ▪ Collarette May be a conspicuous flask shaped structure, as observed in Phialophora spp., or an inconspicuous structure, as seen in Aspergillus spp. ii. ANNELLIDES o Conidiogenous cells that rupture to leave a distinct ring of cellular material at the base of the conidium when it separates from the annellide. o Formation of sequential conidia at the base pushes the oldest cell to the tip of the chain and leaves a series of rings or annellations at the apex of the annellide, providing a record of past events, like rings on a tree CONIDIOGENESIS THALLIC CONIDIOGENESIS Process in which the conidium does not develop until a septum is formed between the conidium and the parent cell. Conidium originates from the whole of the parent cell Most important human pathogens that exhibit thallic conidiogenesis are: o dermatophytes o dimorphic fungi in the genus Coccidioides Arthroconidia: Produced as conidiogenesis progresses, barrel-shaped conidia these fragment easily and are disseminated with little difficulty, resulting in a high degree of infectivity demonstrated by these important human pathogens Thallic conidia of the dermatophytes are separated by size into two types: a. Macroconidia: large septate b. Microconidia: small, one celled, simpler NOTE TAKER: BALDOS, PADAYAO, FERRER Page 4 | 8 PCC SOM 2026 MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY o ✓ In general, Enriched media: favors vegetative mycelium (sexual) Basal (starving) media: favors asexual reproductive structures MEDIA Subculture onto water agar or potato flake agar Czapek Dox agar Potato dextrose or potato flake agar or on cornmeal agar with 1% glucose. VIII. FINDINGS General technique for encouraging the development of conidial structures. Where colors of Aspergillus colonies are best studied on Where Trichophyton rubrum produce red pigment DIMORPHISM Demonstration of dimorphism is the traditional approach to definitive identification of endemic fungal pathogens. In most laboratories, the initial isolate is the mold phase because culture plates usually are incubated at 25 to 30 ᵒC. Conversion to the tissue phase (yeast or yeastlike structure) is accomplished by incubating a subculture at 37 ᵒC. A rich medium, such as brain heart infusion agar with a blood supplement, should be included with the primary isolation medium, particularly when H. capsulatum is suspected. ✓ The introduction of molecular probes has considerably simplified the task of definitive identification for these endemic fungi and has eliminated the need to demonstrate both phases of the dimorphic fungi in the laboratory. Although demonstration of the characteristic yeast or yeastlike phase in tissue strongly supports the diagnosis of a dimorphic fungus, identification of the pathogen in culture is still necessary for disease confirmation. ✓ IX. o B. SPECIMEN COLLECTION AND TRANSPORT DIAGNOSTIC TECHNIQUES With the increased number of patients undergoing transplantation procedures or receiving aggressive immunosuppression and chemotherapy, the growing population of immunosuppressed hosts has dramatically increased, leading to a subsequent rise in the prevalence of invasive fungal infection Early recognition of a fungal infection to optimize patient management is becoming more realistic with advances in diagnostic applications for the identification of fungal etiologic agents. A. LABORATORY SAFETY o Biosafety considerations from specimen collection through culture conformation in the mycology laboratory are critical Inoculation of specimens and manipulation of mold colonies should always be performed in a biologic safety cabinet to prevent dissemination of the highly mobile fungal conidia. The greatest hazard for laboratory personnel comes from handling mold cultures of the dimorphic pathogens, Coccidioides immitis posadasii and H.capsulatum. NOTE TAKER: BALDOS, PADAYAO, FERRER Classified as risk group 3 (RG3) pathogens, and biosafety level 3 practices, containment equipment, and facilities are highly recommended for propagating and manipulating cultures, as well as for processing soil or other environmental materials known or likely to contain infectious conidia from these agents Screw capped tubes of culture media should be used for culture if these pathogens are suspected. Unfortunately, the diagnosis may not be appreciated before the pathogen is isolated in some cases. All work with molds, however, should be performed in a certified biologic safety cabinet. The correct specimen for submission to the mycology laboratory depends on the clinical presentation and the organ system affected Swabs o Discouraged o inferior for collecting specimens in most areas of the infectious process o Swab fibers may be mistaken for hyphal elements upon direct examination specimens Best specimens for mycologic diagnosis are: o Scrapings o Curettings o Aspirates o Lesion biopsies Hairs infected with a dermatophytic fungus (e.g., Microsporum canis) fluoresce under a long wavelength ultraviolet light (Wood’s lamp) and can be specifically selected for examination. Cutaneous lesions of dermatophytosis (ringworm) are characterized by an active advancing edge with central healing, so that scrapings can be collected from the edge of the process. Hair, nails, scalp, and skin scrapings should be sent to the laboratory in a clean, dry container. Tissue samples should be sent in a sterile container with a small amount of sterile, preservative free saline or a transport medium to prevent drying of the sample. C. DIRECT EXAMINATION Multiple manipulations of specimen material in the laboratory may render the material not acceptable for culture, so in cases in which culture is warranted, specimens must be separated before this type of examination is performed. 1. Wet Preparation Simplest method for direct examination of a specimen o Observe a suspension placed on a slide and coverslip under reduced light. Dry or viscous specimens o Addition of a wetting agent such as saline is necessary Specimens containing distracting tissue debris and cells, such as vaginal secretions, nails, and skin scrapings Page 5 | 8 PCC SOM 2026 MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY POTASSIUM HYDROXIDE (KOH) o may be used to dissolve the tissue material, so that fungal elements are more visible DIMETHYL SULFOXIDE (DMSO) o Facilitates rapid breakdown of keratinized tissue CHLORAZOLE o lipophilic dye that is attracted to and stains fungal elements an iridescent green-black 2. Gram Stain Useful for the detection of yeast Most yeasts stain partially or completely gram positive and are generally differentiated from bacteria by their larger size and by the presence of budding cells Hyphae of molds may appear gram positive or gram negative by this stain Molds may stain less reliably than yeast cells and are easily missed in clinical specimens using this method. 3. Giemsa or Wright Stain Useful if histoplasmosis is suspected 4. India Ink Preparation 5. Histopathologic Stains a. b. c. Periodic Acid-Schiff (PAS) method o useful for demonstrating internal details. Gomori’s Methenamine Silver (GMS) o considered one of the better stains o demonstrating fungi because it provides high contrast with minimal background staining, thus allowing for the demonstration of sparsely present fungal elements in the sample Hematoxylin and Eosin (H&E) stain o best used for studying the host reaction and for determining whether a fungus is hyaline (colorless) or dematiaceous (naturally pigmented). 6. Calcofluor White Stain Fluorochrome compound Whitener used in the textile and paper industries Binds to the chitin in the walls of fungal cells and fluoresces white or apple green (depending on the filter combination used) when exposed to short-wavelength ultraviolet light from a fluorescence Calcofluor white can also be mixed with potassium hydroxide to clear the specimen for easier observation of fungi Polysaccharide capsule of species within the Cryptococcus neoformans/C. gattii species complex can be demonstrated by negative staining using India ink particles; it is especially evident when infected cerebrospinal fluid (CSF) is examined When examined with the light microscope, the capsule stands out as a clear space around the fungal cell, with ink particles bouncing off the edge as Brownian motion occurs India ink technique is still useful when the antigen test is not immediately available and for examination of capsules in cells from isolated colonies that suggest C. neoformans/C. gattii species complex 7. Other Specialized Stains a. MAYER’S MUCICARMINE demonstration of the mucoid capsule of C. neoformans b. FONTANA-MASSON STAIN demonstration of melanin or melanin like substances in the lightly pigmented agents of phaeohyphomycosis as well as the staining of minute amounts of melanin pigment in the cell wall of C. neoformans/C. gattii species complex. D. ISOLATION IN CULTURE 1. Unfortunately, some C. neoformans strains are poorly encapsulated; thus the sensitivity of the India ink test is generally less than 50%, and the organism is especially evident in patients who are not infected with the human immunodeficiency virus (HIV) Cryptococcal Antigen Latex Agglutination System (CALAS) direct detection of antigen in CSF or serum using a latex agglutination or enzyme immunoassay technique has increased the sensitivity to close to 100%. NOTE TAKER: BALDOS, PADAYAO, FERRER Selection of Media: All specimens should be inoculated onto a general-purpose fungal growth medium a. SABOURAUD DEXTROSE AGAR pH of 5.5 to 5.6 designed for the isolation of dermatophytic fungi b. EMMONS MODIFICATION OF SABOURAUD DEXTROSE AGAR less glucose with a pH of 6.8 to 7.0 more widely used in the clinical mycology laboratory as a general growth medium Page 6 | 8 PCC SOM 2026 c. MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY INHIBITORY MOLD AGAR and POTATO DEXTROSE AGAR or POTATO FLAKE AGAR utilized as general isolation media in the laboratory spread from person to person via respiratory droplets ✓ For specimens from nonsterile sites or from sites that are likely to be contaminated with other microbial flora, inoculation of a nonselective agar and a selective agar in tandem is recommended. 3. Blood Cultures Two systems commonly used: 1. BACTEC System 2. BacTAlert System Yeasts are aerobic organisms, aerobic culture bottles are used for detection. E. ✓ The most commonly used selective agar 1. Cycloheximide: 400 to 500 mg/L inhibit saprobic fungi 2. Chloramphenicol: 50 mg/L 3. Gentamicin: 50 mg/L , to inhibit bacteria ✓ For tissue specimens, especially when dimorphic fungi might be the etiologic agent, an enriched agar: o Brain Heart Infusion agar + 5% to 10% sheep blood and o Supplemented with chloramphenicol and gentamicin or o SABHI agar with antibiotic d. 2. CHROMagar Candida selective and differential medium used for the isolation and differentiation of major clinically significant Candida spp. uses a chromogenic mixture to differentiate major Candida spp. by colony color 1. Candida albicans, green 2. Candida krusei, pink 3. Candida tropicalis, metallic blue 4. other species: white to mauve Inoculation and Incubation Processing of clinical specimens for fungal detection requires careful handling because some processes commonly used to prepare specimens for bacterial culture may be too disruptive for the fungal organism (e.g., grinding in a homogenizer). Inoculation of scrapings or curettings onto and into the agar at multiple points on the agar surface is a reliable technique for culture. Tissue should be teased or minced, after which the fragments are similarly inoculated. Plates or tubes are incubated in ambient air at 25-30 ᵒC. Most fungi grow within a 2-week incubation period, and among commonly isolated fungi, only the dimorphic fungi such as H. capsulatum and B. dermatitidis are frequently detected after 14 days of incubation. NOTE TAKER: BALDOS, PADAYAO, FERRER GROWTH TEMPERATURE Most molds grow best at 25 to 30 ᵒC Most yeasts grow well at 35 to 37 ᵒC and are often recovered first on enriched blood agar plates in the bacteriology laboratory. F. MORPHOLOGIC IDENTIFICATION CORNMEAL AGAR SUPPLEMENTED WITH TWEEN 80 (CM T80) visualization of yeast morphology (+) pseudohyphae with chlamydoconidia is confirmatory for i. Candida albicans ii. Candida dubliniensis 1. YEAST MORPHOLOGY a. GERM TUBE TEST Important initial step in the identification of yeast isolates. Germ tubes: Elongated, fingerlike extensions from a yeast cell, represent the beginnings of a true hypha Structure can be differentiated from pseudohyphae by the lack of a constriction at the junction of germ tube and yeast cell and by the parallel cell walls in the germ tube. True germ tubes are formed after growth in serum at 37 ᵒC for up to 4 hours 1. C. albicans 2. C. dubliniensis 2. MOLD MORPHOLOGY a. Cellophane tape mount A simple method for examination of molds using clear tape and staining with LACTOPHENOL COTTON (ANILINE) BLUE (LPCB) STAIN. b. The established method used in observing mold morphology tease the mycelium apart with inoculating needles and examine the teased hyphae with LPCB stain. Page 7 | 8 PCC SOM 2026 ✓ MICROBIOLOGY F.01 INTRODUCTION TO MYCOLOGY Occasionally, a slide culture may be necessary to preserve easily disrupted conidial structures in their original relationships. 1. The classic approach involves cutting a square of an appropriate agar medium (usually Sabouraud dextrose, potato dextrose, or potato flake agar), which is suspended on a glass slide and overlaid with a coverslip. 2. The slide is supported by glass rods in a Petri dish, to which sterile water is added for maintenance of humidity. 3. The coverslip subsequently can be removed after a few days of incubation, placed in a drop of LPCB, and observed for undisturbed reproductive structures. Urease production o tested by inoculation of the yeast isolate onto a slant of Christensen urea agar or into urea broth o incubated at 25-30 ᵒC for at least 3 days. o useful to differentiate the dermatophytes ▪ Trichophyton mentagrophytes: urease positive ▪ T. rubrum: urease negative Alkalinization of the medium o after production of NH3 by urea-splitting organisms o detected by a pH indicator Urease may be produced by other nonpathogenic species of Cryptococcus, by Rhodotorula spp., and by some isolates of Trichosporon spp. and C. krusei. H. SEROLOGIC IDENTIFICATION ✓ When a mold isolate is suspected of being a dimorphic fungus (e.g., growth on cycloheximide containing medium), o a slide culture should not be performed o a cellophane tape test or teased preparation should be examined only after the preparation has been sealed in a biosafety cabinet certified for use. o Lactophenol cotton blue is fungicidal but sealing the coverslip with nail polish before observation provides additional protection. o Alternatively, the culture may be flooded with 10% formalin (4% formaldehyde solution) and incubated at room temperature overnight before the mold is manipulated G. BIOCHEMICAL IDENTIFICATION Biochemical tests are at the core of identification schemes for yeast and occasionally are useful for identification of molds. Biochemical characterization of yeasts may be accomplished by study of fermentation or assimilation patterns. Assimilation test o assesses the ability of an isolate to use (for growth) i. carbohydrate as the sole source of carbon ii. nitrate as the sole source of nitrogen Urease production in cryptococci o differentiate these species from Candida spp., particularly in respiratory specimens i. C. neoformans: pulmonary and systemic pathogen ii. Candida spp. frequent inhabitants of the upper airways but uncommon causes of primary pneumonia. 3. 4. 5. 6. 7. 8. 9. 10. 1. 2. False, Pseudohyphae Conidiogenous cells, Conidiophores Phialides Thallic Condogenesis B. C. tropicalis Assimilation test T. metagrophytes True C. ` NOTE TAKER: BALDOS, PADAYAO, FERRER CALAS (Crytocococcal Antigen Latex Agglutination System) Multiple antigen detection methods are used for the specific diagnosis of a variety of diseases, including: o Aspergillosis o Histoplasmosis o Blastomycosis o Paracoccidioidomycosis o Talaromycosis (formerly penicilliosis) ▪ caused by Talaromyces marneffei (formerly Penicillium marneffei) I. MOLECULAR IDENTIFICATION A. Probe-based assays commercially available commonly used for the identification of Histoplasma, Blastomyces, and Coccidioides spp. from culture B. Peptide nucleic acid fluorescence insitu hybridization (PNA -FISH) assay another probe-based method commercially available for the qualitative identification of certain Candida spp. in blood culture bottles CHECKPOINT ! 1. T/F: A true hyphae is characterized by a presence of constriction at the junction of the adjacent cells. 2. What are the two structures for reproduction? 3. This structure arises from the vesicle to support chains of conidia. a. Conidiophores b. Columella c. Sporangium d. Phialides 4. Type of conidiogenesis in which the conidium does not develop until a septum is formed between the conidium and the parent cell. 5. This stain is recommended if Histoplasmosis is suspected. a. India Ink b. Wright Stain c. PAS Stain 6. Using CHROM agar, a metallic blue colony is a characteristic of what Candida spp? 7. This test assesses the ability of an isolate to use CHO as sole source of carbon. 8. A trichophyton spp that tests positive in Urease Production Test. 9. T/F: “C” in CALAS means “Cryptococcal”. 10. The following are important clues for fungal identity, except a. Width of the hyphae b. Angle of branching c. Length of hyphae Page 8 | 8