Micro Prax 1- Mycology

Thanks to Renn, Lilia, Kate and Iami for the pictures Some pictures are from past prax reviewer (batch 2013)Thank you! Some I got from the internet thank you google!!! Go Batch 2014!!!!

Exercise 10- KOH Mount

Potassium hydroxide (KOH) mount a rapid method for the demonstration of fungal forms in clinical material facilitates the clearing of specimens for enhanced microscopic observation without altering the fungal elements partially digests proteinaceous components (e.g. host epithelial cells) that are collected along with the fungi leaves the polysaccharide-containing cell walls intact ***rapid because the specimen placed in a drop of KOH will dissolve at a faster rate than the fungi ***proteinaceous components are partially digested by the alkali, leaving the cell walls intact ***the chitinous cell walls of fungi protects fungal elements from disintegration 10% KOH- for skin scrapings 20% KOH- for nail scrapings

KOH Mount :Skin Scrapings

Possible Agent: Malassezia furfur Spaghetti and meatballs appearance

Hyphal filaments

hyphae

Upon potassium hydroxide (KOH) examination, hyphae are visible and grow into strands within clumps of keratinocytes. Thick-walled spores frequently occur in grapelike clumps. Individual spores and short stubby hyphae float in the clear areas between clumps of keratinocytes. Many of the short hyphae are dystrophic.

spores

KOH Mount Skin Scrapings

Hyphal filaments

Spores

Ptyriasis Versicolor
Basidiomycota Infectious forms: short hyphae and yeast like cells Can be visualized by woods lamp Culture not done Scaly patches of variable color Mostly endogenous Lipophilic Also called as tinea versicolor, tinea alba and tinea flava Spread from person to person

Exercise 11- Microscopic Morphology of Fungal Culture

Accurate identification of filamentous fungi is based on the microscopic examination of sporulating parts of a colony, since each species has a characteristic morphology and arrangement of its spores and fruiting bodies. Tease Mount Preparation - traditional procedure used by most laboratories Principle: To demonstrate conidia or other reproductive structures or morphological forms which might give information toward the identification of the organism. Slide culture or van Tieghem cell - best method for preserving and observing the actual structure of a fungus Principle: In an undisturbed state, important microscopic structures and morphologic details are demonstrated Lactophenol Cotton Blue (LPCB) - for very quick evaluation of fungal structures Principles: It has 3 components: lactic acid which preserves fungal structures, cotton blue (an acid dye) which stains the chitin present in the cell walls, and phenol which kills any live organisms suspended in the stain

Candida albicans

Tease Preparation
Candida albicans
Ovoid yeast cells

Note: budding yeast cells (arrow)

Below: Candida albicans in its hyphal form

Budding yeast (arrows)

Phylum: Ascomycota Representative Genus:

Candida albicans

Brief Description:

Opaque colored colonies with pasty consistency Appear as budding yeast cells, psudohyphae, or a combination of both Asexual spores chlamydiospores pseudohyphae occurring in elongated chains

Reproduction: no sexual
Hyphae:

Candidiasis
Most common opportunistic fungal infection worldwide C. albicans most common and most pathogenic GIT- major colonizing habitat Reproduce by budding Infectious forms: yeast cells and pseudohyphae KOH: small budding yeast cells and pseudohyphae Culture: pseudohyphae with clusters of round blastoconidia

Epidermophyton floccosum

Tease Preparation
Epidermophyton floccosum
Smooth, thin-walled macroconidia which are often produced in clusters growing directly from the hyphae

Macroconidia

Numerous chlamydoconidia are formed in older cultures. No microconidia are formed.

Chlamydoconidia Hyphae

Phylum: Deuteromycota Representative Genus: Epidermophyton floccosum Brief Description:

Colonies are olive green or khaki-colored with powdery surface that become folded with floccose patches as culture ages. Reverse is orange to brownish at times with yellow border
no sexual reproduction

Reproduction: Hyphae: septate Macroconidia:

best seen in young culture- smooth, club-shaped with rounded ends, contain 2-6 cells, found singly or in characteristic clusters; transform to chlamydiospores

Microconidia: absent

Trichophyton mentagrophyte

Microconidia

Trichophyton mentagrophyte
Brief Description:
Colonies vary greatly. Surface may be buff and powdery or white and downy; powdery form exhibit concentric and radiate margin no sexual reproduction, only in anamorph stage

Reproduction:

Hyphae

Phylum: Deuteromycota

Representative Genus/Species:
Trichophyton mentagrophytes

Hyphae:
coiled spiral hyphae maybe present

Macroconidia: Usually rare, not always present, cigar shaped thin-walled, narrowly attached to the hyphae and contain 1-6 cells
Microconidia: numeous, borne in clusters along the hyphae

Microsporum gypseum

Macroconidia

Microsporum gypseum
Brief Description:
Colonies re flat and spreading with powdery to granular surface, irregularly bifringed border; buff at first, then becomes tan to cinnamon brown No sexual reproduction

Reproduction:

Hyphae

Phylum: Deuteromycota Representative Genus: Microsporum gypseum Hyphae:

With septate

Macroconidia: numerous, symmetric with rounded ends and having no more than 6 cells
Microconidia: club-shaped and usually seen among hyphae

Aspergillus sp.

Slide Culture Preparation

Vesicle

Aspergillus sp.
Conidiophore

Hyphae

Swollen Vesicle

Conidiophore

Septated Hyphae

Phylum: Ascomycota Representative Genus: Aspergillus sp. Brief Description: Smokey green color colonies, Reproduction: No sexual reproduction

Hyphae: Wide, septated, dichotomously branching with conidial heads Classification Accg. To Habitat: Ubiquitous (soil, plants, organic debris)

Aspergillosis
Infectious form: Conidia Tissue form: Hyphae Most common species: A. Fumigatus Natural reservoir: Soil and air Occurs as:
Allergic form Colonizing form Disseminated Mycotoxicosis

In culture: Conidiophore with swollen vesicle, rows of phialides bearing radial chains

Penicillium sp.

Slide Culture Preparation

Penicillium sp.

Conidia Hyphae Conidia Metullae Metullae

Conidiophore

Conidiophore

Metullae

Conidia

Hyphae

Phylum: Deuteromycota Representative Genus: Penicillium marneffei Brief Description: For P. marneffei- the only species that is dimorphic Flat, powdery to velvety, and tan to reddish yellow colonies Reproduction: no sexual reproduction Hyphae: septate

Penicillium
Produce red pigments Reproduction: transverse fission/ schizogony Unknown habitat Infectious form: Conidia KOH: small ovoid yeast cells with transverse septum Culture RT: conidiophores (flask shape) branch into metullae with phialides Culture at 37C- Round yeast cells with cross walls

PKMVY