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hi MT 6322: MYCOLOGY AND VIROLOGY LABORATORY Course Instructors: Jerry F. Ching Jr. and Martin M. Cruz...

hi MT 6322: MYCOLOGY AND VIROLOGY LABORATORY Course Instructors: Jerry F. Ching Jr. and Martin M. Cruz A.Y. 2024-2025, Term 1 SUPPLEMENTARY MATERIALS ESSENTIAL COMPONENTS Peptone ○ source of amino acids and nitrogenous compounds Dextrose (glucose) ○ source of carbon energy pH ○ 5.6 at 25 C ○ inhibit bacterial growth LEGEND BOOK / RESOURCES OTHER FACTS & DETAILS TO NOTE ✄ ✎ SABOURAUD DEXTROSE AGAR (SDA) POTATO DEXTROSE AGAR General-purpose medium for yeasts and molds Frequently used microbial growth media for cultivation of molds, yeasts and other fungi Potato Infusion and Dextrose: ○ Carbohydrate source support the luxuriant growth of fungi and bacteria ○ Encourage mold sporulation and pigment first formulated by a French Physician Dr. Raymond production in certain dermatophytes Jacques Adrien Sabouraud Supplemented with selective agents such as acids Selective media for fungal culture and primarily (eg. tartaric acid) or antibiotics (e.g. used for the isolation of dermatophytes, yeasts and chloramphenicol, or chlortetracycline) to inhibit various other pathogenic and non-pathogenic fungi the growth of bacteria 1  Microsporum spp. GENERAL COLONIAL CHARACTERISTICS ON CULTURE MEDIA General Appearance: ○ Granular to wooly/cottony Color: ○ Varies from yellow, white and brown ○ Certain strains can exude a cream color BRAIN HEART INFUSION AGAR (BHI) General purpose medium Isolation, cultivation, and maintenance of a variety of fastidious and nonfastidious microorganisms GENERAL MICROSCOPIC CHARACTERISTICS Highly nutritious base that meets the growth Macroconidia requirements of many types of microorganisms ○ Describes as hyaline, multi-septate, with including bacteria, yeast and molds. thick rough cell walls, and are clavate, Selective formulation containing chloramphenicol fusiform or spindle-shaped and cycloheximide is also available that will allow Microconidia the recovery of pathogenic fungi while inhibiting a ○ Single-cell, hyaline, smooth-walled, and are wide range of bacteria and saprophytic fungi. predominantly clavate in shape Microsporum audouinii Microscopic features: ○ Macroconidia and microconidia are absent or are rarely found ○ Most cultures are sterile or produce only the thick-walled chlamydospores ○ If they are present, they have longer appearance than Microsporum canis ○ Microconidia are pyriform to clavate in shape. These features are not specific for this species. WATER AGAR Recommended for enumeration, cultivation, and observation of sporulation of some fungi 1.5% (Water Agar) contains agar as the solidifying agent and purified deionized water Chlamydoconidia Confirmatory Tests: ○ No growth on rice grains ○ Reverse Pigmentation on Potato Dextrose Agar (PDA) - Salmon Pink to Pinkish Brown 2  ○ BCP Milk Solids Glucose Agar - Older cultures may develop white Demonstrates rapid pH change to alkaline pleomorphic tufts of mycelium. A deep (purple coloration) yellowish-brown reverse pigment is usually ○ Hair Perforation Test Negative present. Microsporum canis Microscopic Features: ○ Macroconidia: Spindle-shaped (5-15 cells), verrucose, thick-walled and have terminal knobs Microscopic Features: ○ Smooth, thin-walled macroconidia, which are often produced in clusters growing directly from the hyphae. Numerous chlamydospores are formed in older cultures. ○ Microconidia are absent ○ Microconidia: Pyriform to clavate Confirmatory Tests: ○ Good growth on rice grain ○ Reverse Pigmentation on PDA - Bright Yellow ○ Positive Hair Perforation Test Microsporum ferrugineum Microscopic Features: ○ No macroconidia and microconidia. Macroconidium and So-called "bamboo" hyphae are a Chlamydoconidium (E. flocossum) distinctive feature of this species ○ Visualization of these bamboo hyphae Trichosporon spp. confirms this species and the other specific Trichosporon asahii colonial features of the Microsporum genus. Colony characteristics: ○ Colonies are white to cream-colored, powdery, suede-like to farinose with radial furrows and irregular folds Bamboo hyphae Epidermophyton Microscopic Morphology: It has two species: Epidermophyton floccosum and ○ Budding cells and lateral conidia are absent Epidermophyton stockdaleae ○ Arthroconidia are barrel-shaped Epidermophyton floccosum - it is the clinically significant species affecting humans Colonial Characteristics on Culture Medium: ○ Greenish-brown with a suede-like surface, raised and folded in the center, with a flat periphery and submerged fringe of growth. 3  Microscopic Morphology: Laboratory Diagnosis: ○ Budding cells and lateral conidia are absent. ○ This species assimilates L-arabinose but Arthroconidia are long cylindrical not melibiose. Growth at 37C. Most common species, especially from invasive infections. Physiological Tests: + Positive - Negative v Variable Laboratory Diagnosis: ○ This species assimilates myo-inositol but w Weak not melibiose and is tolerant to 0.01% (variable tolerance to 0.1%) cycloheximide. s Slow Growth at 37C. Usually associated with nd No Data white piedra on pubic hairs. Physiological Tests: Glucose + Melibiose + L-Rhamnose - D-Glucitol + Glucose + Melibiose - L-Rhamnose + D-Glucitol v Galactose v Raffinose + D- v a-M-D- + Galactose + Raffinose - D- + a-M-D- + Glucosamime Glucoside Glucosamime Glucoside L-Sorbose v Melezitose + N-A-D- + D-Gluconate + L-Sorbose v Melezitose v N-A-D- + D-Gluconate + glucosamine glucosamine Sucrose + Soluble + Glycerol v DL-Lactate + Sucrose v Soluble v Glycerol v DL-Lactate v Starch Starch Maltose + D-Xylose + Erythritol + myo-Inositol + Maltose + D-Xylose v Erythritol + myo-Inositol v Cellobiose + L-Arabinose + Ribitol - Nitrate - Cellobiose + L-Arabinose + Ribitol v Nitrate - Trehalose + D- + Galactitol - 2-K-D- + Trehalose + D- + Galactitol - 2-K-D- + Arabinose Gluconate Arabinose Gluconate Lactose + D-Ribose + D-Mannitol v D-Glucuronate + Lactose + D-Ribose + D-Mannitol v D-Glucuronate + Malassezia furfur Trichosporon inkin Other associated terms: Tinea versicolor, Pityriasis Colony characteristics: versicolor, Dermatomycosis furfuracea, tinea flava, ○ Colonies are restricted, white, finely chromophytosis cerebriform with a granular covering, Laboratory Diagnosis without marginal zone, often cracking the ○ KOH/PAS preparation of skin scrapings media. ○ Wood's lamp - golden yellow to light green fluorescence in lesions/hair 4  Microscopic examination: ○ Colonies: shiny, moist, yeast-like, dirty ○ KOH test: Round budding yeast cells with white-brown coloration. colonies eventually hyphae (Fig. A) turn to OLIVE to GREENISH BLACK ○ PAS: "Spaghetti and meatball appearance" ○ Microscopic examination: (Fig B.) Young cultures: Budding blastoconidia (Fig. A) Old cultures: Cluster of blastoconidia (Fig B.) Older hyphal colonies: Annelloconidia (Fig. C) Culture: ○ Saboraud's Dextrose Agar (SDA) - must be overlaid with Olive, Peanut, or Vegetable oil due to their affinity to lipids (lipophilic). MACROSCOPIC MICROSCOPIC Phialophora spp. Phialophora verrucosa Type of Mycoses: Subcutaneous Causative Agent of chromoblastomycosis, characterized as verrucous nodules that often become ulcerated and crusted. Diagnosis: Presence of characteristic lesions accompanied by microscopic sclerotic bodies, often Dry, smooth or lightly Presence of collarettes referred to as copper pennies because of their shape wrinkled, glistening or during conidiogenesis. and staining properties in tissue sections. dull, white to creamy Laboratory Diagnosis Methods: 1. Growth - rapid growing producing olive-gray to black cottony or wooly colonies. Hortaea werneckii Other associated terms: Tinea nigra palmaris, Exophiala werneckii, Phaeoannellomyces werneckii, Cladosporium werneckii Laboratory Diagnosis ○ Specimen: Skin scrapings from darkly pigmented lesions Direct Microscopic Examination: ○ 10-20% KOH test: Septate hyphal elements and budding cells Culture: Phialophora verrucosa (CBS 140325). ○ SDA with antibiotics (e.g. a. Colonies grown on MEA (Malt Extract Agar) for 3 wk; Chloramphenicol/Gentamicin) at 37°C b. colonies grown on OA (Oatmeal Agar) for 3 wk; 5 c-k. micromorphology showing phialides and conidia. — Scale bar = 10 pm. 2. Microscopy - Conidiogenous cells, phaeoid, flask-shaped phialides, with collarettes Conidia of Phialophora verrucosa at the tips of phialides with collarettes (Nomarski optics, X 1000.) Fusarium spp. Type of Mycoses: Cutaneous (Associated with mycotic keratitis) Diagnosis: Implicated in a number of infectious diseases, including keratomycosis, burn wounds, and invasive disease in immunocompromised patients. Also, considered the second most common pathogenic molds causing invasive disease Laboratory Diagnosis Methods: 1. Isolation - via Blood culture and may appear yeast-like on initial recovery 2. Growth - rapidly growing hyaline fungus producing a fluffy aerial mycelium that can develop various colors with age, ranging from rose to mauve to purple to yellow as well as pink, lavender or salmon color. Diffusible pigments may be seen in the surrounding agar. 3. Microscopy - abundant macroconidia with fewer microconidia are produced on vegetative hyphae. Macroconidia are banana- or canoe-shaped and are formed singly, in small clusters, or clustered together in mats termed sporodochia. Macroconidia typically are multicelled. 6  ASPERGILLUS VS. PENICILLIUM Aspergillus Penicillium Generalities The conidiophore is aseptate and Conidiophore is long slender septate unbranched. and branched. Conidiophore arises from a Conidiophore arises from any specialized T shaped thick walled vegetative cell of the mycelium. foot cell. There are no foot cells. Conidiophore enlarges into a vesicle The conidiophore ends in a whorl of at its tip bearing the finger link branches which gives a broom like sterigmata. The structure forms a appearance spherical head. Appearance Definition Aspergillus is a genus of ascomycetes fungi Penicillium is a genus of ascomycetes that that have an unbranched, non-septate stalk have a brush link conidiophore which is like conidiophores. branched and septate Color Mostly green and black. There are yellow to Mostly blue brown color species Conidiophore Septum tissue is correctly formed; Septum tissues are not correctly formed; Aseptate and unbranched septate and branched Causing diseases Disease causing ability is high Disease causing ability is low Antibiotic Aspergillus species are not popular in Penicillium is popular in antibiotic production antibiotic production production 7  RHIZOPUS (BREAD MOLD) Specimen for Sputum, Urine, Scrapings from Diagnosis lesions, Bone Marrow biopsy, Skin, Lymph nodes Histoplasma capsulatum Morphology Microscopic: small, ovoid cells intracellularly in histologic section stained with Gomori methanamine silver or Giemsa stain Macroconidia Thick-walled spherical structures 8-15 μm diameter with surface projections Microconidia Smooth spherical structures 2-4 μm in diameter Disease HISTOPLASMOSIS Miscellaneous Serological tests: immunodiffusion, Tests latex agglutination test Mode of Inhalation of Conidia Transmission Treatment Amphotericin B Pathogenesis An intracellular mycosis of the reticuloendothelial Coccidioides immitis system Infection is self-limited S/S: fever, chills, myalgias, headaches, non-productive cough Severe disseminated histoplasmosis → RES involved, with lymphadenopathy, enlarged spleen, liver, high fever, anemia Colony Cultivation in SDA: white to tan Characteristics cottony colonies; pale yellow brown on reverse Disease COCCIDIOIDOMYCOSIS Mode of Inhalation of airborne arthroconidia Transmission Pathogenesis Infection is self-limited Dissemination is rare but may be fatal 8  Blastomyces dermatitidis Patients with primary infection are asymptomatic S/S: fever, malaise, cough, headache Late-stage patient forms a hypersensitivity reaction in the form of erythema nodosum or erythema multiforme (rashes) Colony Cultivation in SDA: white to tan Characteristics colony Specimen for Sputum, Pus, CSF, Tissue Biopsy Diagnosis Material Morphology Microscopic: spherule with thick, doubly refractile wall and endospores form within the spherule and fill it Macroconidia Globose macroconidia 8-15 μm With distinctive tuberculate or finger-like cell wall Macroscopic Slow to moderate growth ornamentation Morphology White to dark tan young (at 22C): colonies, tenacious Microconidia Ovoid microconidia Old colonies glabrous to wooly 2-4 μm Which appear smooth or Microscopic Oval, pyriform to globose finely roughened Morphology smooth conidia borne on (at 22C): short, lateral hypha like Miscellaneous Serological tests: immunodiffusion, conidiophores Tests latex agglutination test Microscopic Large yeast (8-12 μm) Morphology Blastoconidia attached by (BAP at 37C): broad base, small, oval yeast (2-5 μm) 9  Paracoccidioides brasiliensis Macroscopic Slow growth Morphology White to beige colony (at 22C): Glabrous, leathery, flat to wrinkled, folded or velvety Microscopic Colonies frequently only Morphology produce sterile hyphae (at 22C): Fresh isolates may produce conidia similar to those of B. dermatitidis Microscopic Multiple blastoconidia Morphology budding from singe, large (BAP at 37C): yeast (15-30 μm) 10