MLS 314 (MYCOLOGY AND VIROLOGY) – WEEK 1 & WEEK 2 Flashcards by Arriane Camacho

the asexual form of a fungus

Anamorph:

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a specialized conidiogenous cell from which a succession of spores is produced and which has a column of apical scars at its tip.

• Annellide

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: ability of a fungus to use a specific carbon or nitrogen source for growth.

•Assimilation

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: without cross-walls or septa.

•Aseptate

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: the process of conidium formation.

•Conidiogenesis

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[in which an existing hyphal cell is converted into one or more conidia]

thallic

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[in which conidia are produced as a result of some form of budding process]

blastic

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An asexual (mitosis only) propagule that forms on the side or the end of the hypha or conidiophore.

Conidium

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It may consist of one or more cells.

Conidium

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It is always borne externally, ie., not enclosed in a saclike structure such as sporangium.

Conidium

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If a fungus produces two types of conidia, those that are small and usually singled cell are referred to as (?)

microconidia

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are usually segmented into two or more cells

macroconidia

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: darkly pigmented.

•Dematiaceous

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: ability of a fungus to utilize a specific carbohydrate in the presence of other organic compounds, resulting in the production of gas.

•Fermentation

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(color change) may simply indicate that the carbohydrate has been assimilated.

Acid production

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All carbohydrates (?) by a fungus are also assimilated, but many compounds that are (?) are not necessarily fermented.

fermented
assimilated

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: colonies with a cotton-like texture.

•Floccose

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: term used to describe spores with a spindle-like shape.

•Fusiform

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: colonies with wax-like texture.

•Glabrous

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: a self-sterile fungus; sexual reproduction cannot take place unless two compatible mating strains are present.

•Heterothallic

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: a self-compatible fungus; sexual reproduction can take place within an individual strain.

•Homothallic

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: colorless, transparent, transluscent.

•Hyaline

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: one of the individual filaments that make up the mycelium of a fungus.

•Hypha (pl., hyphae)

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: a filamentous fungus.

•Mold

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: hyaline or lightly colored.

•Moniliaceous

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: a mass of branching filaments which make up the vegetative growth of a fungus.

•Mycelium

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: a cell with several nuclei.

•Oligokaryotic

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: having few septa.

•Pauciseptate

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: a chain of yeast cells which have arisen as a result of budding and have elongated without becoming detached from each other, forming a hypha-like filament. No marked constriction.

•Pseudohypha (pl., pseudohyphae)

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: a short branching hypha that resembles a root.

•Rhizoid

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: having cross-walls or septa.

•Septate

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: a monophyletic clade of species with equivalent clinical relevance.

•Species complex

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: a specialized hypha upon which a sporangium develops.

•Sporangiophore

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: term used to describe the development of a single conidium at successive sites along a lengthening conidiogenous cell.

•Sympodial

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: the sexual form of a fungus.

•Teleomorph

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: one of the two basic forms of conidiogenesis.

•Thallic

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: the vegetative growth of a fungus.

•Thallus

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: a unicellular, budding fungus.

•Yeast

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named species

undescribed species

new species described each year

135,000

1 million to more than 10 million

1,000 to 1,500

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human and zoonotic diseases

opportunistic infections

< 500
< 50

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Importance of
1.Increasing number of ubiquitous environmental molds implicated as (?) capable of producing serious diseases among debilitated or immunocompromised hosts.
2.One of the leading causes of (?)
3.Often mistaken for as (?) that produces fatal consequences.
4.Increase (?) (travel to an endemic area or a fungus that exists as part of indigenous flora of the local population)
5. (?) population.

opportunistic pathogens
nosocomial infections
bacterial infection
morbidity
Aging

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: Fungi were used as an antiseptic and anesthesia due to the “magical & spiritual” properties.

•35,000 BC

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: People were convinced that association with fungi will entail the formation of diseases. People believed that fungi were “the work of the devil”.

•Middle Ages

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: Fungi were plants with no fruit nor seed.

•Renaissance period

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: Birth of the 1st mycological studies. Pier Anton Micheli— founder of modern mycological studies. Mycology was separated from botany.

•18th Century

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: Fungi were recognized as a potential causative agents of diseases that are usually fatal in nature.

•Mid-20th Century

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: branch of science that deals with systemic biological classification of all living organisms and divided into three disciplines viz., classification, nomenclature, and identification.

•Taxonomy (Gk.: taxis = arrangement; anomia = method)

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•Taxonomy (Gk.: (?) = arrangement; (?) = method)

taxis
anomia

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•The scientific names of fungi are subject to the (?) that was adopted in 2011.

International Code of Nomenclature (ICN)

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•In 2011, a breakthrough in the development of (?) concept recommended the discontinuation of the dual nomenclature system for fungi with anamorphic and teleomorphic forms.

“One Fungus, One Name”

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Effective January 1, 2013 the system of permitting separate anamorph and teleomorph names ended; and assigned priority to the (?) independently of whether the organism was originally described as the anamorph or the teleomorph.

oldest genus (species) name

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•Eukaryotes are divided into five monophyletic lineages or supergroups as proposed by Adl et.al.1

1.SAR (one clade consists of three groups viz., Stramenopiles, Alveolata, and Rhizaria)
2.Archaeplastida
3.Excavata
4.Amoebozoa
5.Opisthokonta — True fungi

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— organisms that are not fungi sensu strictu (eg., Rhinosporidium seeberi) that share fungal-like morphological features with the true fungi.

Parafungi or pseudofungi

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Fungi Imperfecti also termed (?)— fungi that reproduced asexual type only (sporogenesis), while perfect fungi are capable of both sexual (teleomorph) and asexual (anamorph) type of reproduction.

“form-division Deuteromycota”

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Basic scheme of hierarchical organization of the Kingdom Fungi

Eukarya
Fungi (Mycetea)
- mycota (Basidiomycota), - mycotina
- mycetes (Agaricomycetes)
- ales (Agaricales)
- ace (Agaricaceae)
- Agaricus
- Agaricus bisporus

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Seven phyla that constitute the true fungi

1.Ascomycota
2.Basidiomycota
3.Blastocladiomycota
4.Chytridiomycota
5.Glomeromycota (formerly Zygomycota)
6.Microsporidia
7.Neocallimastigomycota

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•Consists of four subphyla incertae sedis.

Glomeromycota (formerly Zygomycota)

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accommodates Mucorales that includes the genera Lichtheimia (formerly Absidia), Mucor, Rhizomucor, and Rhizopus.

Subphylum Mucormycotina

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has been created for the Entomophthorales that includes the genera Basidiobolus and Conidiobolus — agents of subcutaneous infections

Subphylum Entomophthoromycotina

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Most members are septate, filamentous thallus, but some are atypical yeasts.

Phylum Basidiomycota

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produced via sexual reproduction of a generative cell (basidium).

Basidiospores (haploid)

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The most prominent are the basidiomycetous yeast with anamorphic stages belonging to the genera:

1.Cryptococcus
2.Malassezia
3.Trichosporon

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Polyphyletic, belong to subphylum Agaricomycotina, class Tremellomycetes, and order Tremellales and(or) Filobasidiales1

Cryptococcus

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Asexual reproduction is variable; do not produce spores like those of the Ascomytoca.

Phylum Basidiomycota

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Most filamentous basidiomycetes are wood-rotting fungi or obligate plant pathogens.

Phylum Basidiomycota

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Contains ~50% of all named fungal species and accounts for ~80% of fungi of medical importance.

Phylum Ascomycota

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Sexual reproduction leads to the development of haploid spores, termed (?) which are produced in a sac like structure called (?). (?) contains numerous asci.

ascospores
ascus
Ascocarps or ascomata

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Asexual reproduction leads to conidiation from a generative conidiogenous cell.

Phylum Ascomycota

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Three medically important subphylum:

1.Taphrinomycotina
2.Saccharomycotina
3.Pezizomycotina

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Taphrinomycotina contains the genus (?) formerly classified under kingdom Protozoa.

Pneumocystis

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Saccharomycotina contains the class (?) belongs to order Saccharomycetales

Saccharomycetes

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Pezizomycotina contains two classes: (1) and the (2).

Eurotiomycetes
Sordariomycetes

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Class Saccharomycetes

Phylum Ascomytoca

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Characterized by vegetative yeast cells which proliferate by budding or fission.

Class Saccharomycetes

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Do not produce ascomata, the ascus being formed by direct transformation of a budding vegetative cell, by “mother-bud” conjugation, or by conjugation between two independent single cells.

Class Saccharomycetes

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Members include the anamorphic stage belonging to genus Candida containing ~200 anamorphic species and has teleomorphs in more than 10 genera.

Class Saccharomycetes

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Belonging to class Eurotiomycetes or Sordariomycetes Teleomorphs of melanized fungi:

Capnodiales
Chaetothyriales
Microascales
Pleosporales
Ophiostomatales

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basic structural unit consists of a chain of multinucleate, tubular, filament-like cells.

Hypha

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Most multicellular fungi in their vegetative state, consists of a mass of branching hyphae called (?).

mycelium or thallus

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Each hypha has a rigid cell wall and increases in length as a result of apical growth with mitotic cell division.

Multicellular fungi

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In a more primitive fungi, the hyphae remain aseptate (coenocytic).

Multicellular fungi

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In a more advanced groups, the hyphae are divided into compartments or cells by the development of more or less frequent cross-walls, termed septa.— septate hyphae.

Multicellular fungi

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Fungi that exists in the form of microscopic multicellular mycelium are commonly referred to as (?).

molds

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Exists in the form of independent single cells propagate by budding out similar cells from their surface.

Unicellular fungi

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The bud may become detached from the parent cell or it may remain attached and itself produce another bud, leading to the production of a chain of cells or described as loosely arrangement of budding cells —

yeasts

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Under certain conditions, continued elongation of the parent cell before it buds out results in a chain of elongated cells, termed (?).

pseudohypha

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Compare to true hypha, the connection between adjacent they shows a marked constriction.

pseudohyphal cells

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Medically important fungi that change their growth form as a part of the process of tissue invasion.

Dimorphic fungi

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Dimorphic pathogens change from a multicellular mold form (25-30C) in the natural environment to a budding, single-celled yeasts form (35-37C) in tissue under the influence of temperature— (?).

thermal dimorphism

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Dimorphic fungi

1.Histoplasma capsulatum
2.Blastomyces dermatitidis
3.Coccidioides immitis
4.Paracoccidioides brasiliensis
5.Sporothrix schenckii

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Cell wall

Rigid, mostly composed of 90% polysaccharides eg., (?); 10% (?) in various combinations.

chitin, glucan, chitosan, galactosan, and mannan
proteins and glycoproteins

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the major carbohydrate consist of repeating monomers of N-acetyl-glucosamine (NAG) which provides shape and protection from osmotic lysis (unaffected by some antibiotics).

Chitin

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Barrier between fungal cell and its external environment.

Cell wall

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Binding site for some enzymes

Cell wall

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Possesses antigenic properties which allow interaction with other organisms

Cell wall

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Composed of structurally arranged phospholipids in two-layered configuration scattered randomly.

Cell membrane

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major component that regulates solute intake and secretion (transport system) through selective permeability and serves as the target of antifungal drugs like Nystatin.

Ergosterol

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Facilitates biosynthesis of cell wall and capsular material.

Cell membrane

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Protects the cytoplasm

Cell membrane

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External coating located outside or covering the cell wall and found only in certain fungi.

Capsule

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Composed of amorphous polysaccharide.

Capsule

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May influence growth of fungus by preventing dissociation of buds and dispersion of yeast.

Capsule

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Virulence factor

Capsule

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Contains the following organelles:
1.Nucleus
2.Chromosomes
3.Ribosomes
4.Mitochondrion
5.Endoplasmic reticulum
6.Vacuoles

Cytoplasm

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bounded by a nuclear membrane, may vary in size, shape, and number. Usually contains one nucleolus of mostly RNA.

Nucleus

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described as linear.

Chromosomes

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present at all times for protein synthesis.

Ribosomes

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generation of energy.

Mitochondrion

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Require preformed organic carbon compounds for nutrition.

Nutritional type is chemoheterotrophic (lacks chlorophyll)

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Obtain their nourishment by secreting enzymes into the external substrate and by absorbing the released nutrients through their cell wall.

Nutritional type is chemoheterotrophic (lacks chlorophyll)

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Capable of growing on dead (saprophytic) and living organic matter.

Nutritional type is chemoheterotrophic (lacks chlorophyll)

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Usually nonmotile except for the phylum Chytridiomycota and species of Rhizophidium.

Motility and atmospheric requirements

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Mostly aerobic, some are facultative anaerobic.

Motility and atmospheric requirements

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Lysine synthesis

Utilize -aminodiphate pathway.

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Lysine synthesis
Precursor for (?), also for the development of (?).

penicillin and lysine
antifungal drugs

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describes the propagules that result form an asexual process (mitosis only) and generally short-lived propagules.

Conidium (pl. conidia)

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Most fungi are capable of [?] reproduction (involving meiosis, preceded by fusion of the nuclei of two cells).

sexual

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Some species are [?] (self-fertile) and able to form sexual structures between different cells within an individual thallus.

homothallic

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Most are (?) that do not form their sexual structures unless two capable isolates come into contact

heterothallic

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— once two compatible nuclei have fused, meiosis can occur leading to the production of sexual (?).

Spores

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In some cases, spores are produced in millions in macroscopic “fruiting bodies” such as (?).

mushrooms

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Cell wall of Fungi

Polysaccharides (ie., Chitin, Glucan, Mannan, Galactosan, Chitosan)

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Cell wall of Bacteria

Polysaccharide
Peptidoglycan

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Cell membrane of Fungi

Ergosterol

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Cell membrane of Bacteria

No sterols, EXCEPT: Mycoplasma and Ureaplasma

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Nucleus of Fungi

Small, bound by nuclear membrane

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Nucleus of Bacteria

Nucleoid, no nuclear membrane

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Chromosome of Fungi

Linear

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Chromosome of Bacteria

Circular

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Ribosomes of Fungi

80s

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Ribosomes of Bacteria

70s

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Lysine synthesis of Fungi

-aminodiphate pathway

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Lysine synthesis of Bacteria

DAP (Diaminopimelate pathway)

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Identification of Yeasts

1.Morphological
2.Physiological, and
3.Biochemical characteristics

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Morphological includes:

a.color of colonies
b.size and shape of cells
c.presence of capsule around the cell
d.production of hyphae, pseudohyphae, arthroconidia, and(or) chlamydospores

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Biochemical characteristics

a.assimilation and fermentation of sugars
b.assimilation of nitrate

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•Most commercial tests (eg., ID YST, bioMérieux) are based on sugar assimilation of isolates.
•DNA sequencing
•Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)

Identification of Yeasts

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Identification of Molds

Macroscopic characteristics viz. colonial form, surface color (obverse) pigmentation, reverse pigmentation, and growth rate.

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Factors that influence the colonial form:

1.Culture medium
2.Incubation temperature
3.Age of culture
4.Amount of inoculum

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is important in speciation, hence it is important to select culture conditions which favor sporulation.

Sporulation

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promotes over production of mycelium which results in loss of sporulation, in such cases, subculture to a low-nutrient medium (stimulate sporulation)

Sabouraud’s dextrose agar (SDA)

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Compose of the basic unit of fungus (hyphae).

Mold cells

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The septum, represents the cross walls (divisions) of a hyphae.

Mold cells

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Mass of hyphae will produce mycelium.

Mold cells

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Cells are mostly spherical to ellipsoidal of 3 to 15 microns in size.

Yeast cells

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Multiply via asexual reproduction known as (?).

budding

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Stages of budding:

1.Bud emergence >
2.Protuberance
3.Elongation.

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Growth can be observe through obverse (surface) or reverse (back). Obverse observation can be white to cream, bright or light gray to brown, reverse may be non pigmented, to yellow, orange, or red.

Mold cells

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•As seen in culture, colonies may appear as white opaque, pasty to creamy to mucoid (encapsulated) with 0.5 to 3mm in diameter. •Colonies grow at 35-37C, optimum

Yeast cells

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are fungus with cross walls

Septate hyphae

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are nearly parallel to one another with dichotomous branching at acute angles — Aspergillus spp.

Septate hyphae

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are fungus without cross walls. Coenocytic hyphae results form nuclear division within a cell without division in the cytoplasm.

Aseptate

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Multiple projections in a hypha resembling an old comb hyphae appearance

Pectinate

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Hypha with club-shaped cells, the layer end of one cell being attached to the smaller end of an adjacent

Racquet

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•Hypha forming coiled or corkscrew like turns.
•Characteristics of Trichophyton mentagrophytes

Spiral

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Terminal hyphae branches that are irregular, broad, and antler-like in appearance. Especially characteristics of T. schöenleinii.

Favic chandelier

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Round knot-like structure formed by intertwined hyphae; seen especially among dermatophytes

Nodular

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•Root like structure along the vegetative hyphae
•Characteristics of certain Glomeromycota (formerly Zygomycota) (Rhizopus and Absidium)

Rhizoids

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Clear, transparent, and colorless hyphae

Hyaline

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Hyphae having structures that are brown to black, this is due to the melanotic pigment in the cell walls

Dematiaceous

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Triggered by stems from the sub apical accumulation of wall precursors (presumable vesicles) reaching a critical concentrations.

Lateral Elongation

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i.Cell vesicles move towards the tip of the hyphae (towards the plasma membrane) where they release various enzymes and other compounds.
ii.Enzymes release by these particles involved in the lysis and synthesis of the cell wall
iii.Following lysis of the cell wall, cell division and growth allows for the hyphae to be elongated at the tip.
iv.Once the new cell has formed, enzymes at the tip start synthesizing a new cell wall around the new cell to protect it. Followed by strengthening of the actin cap. Then disappearance of Spitzenkörper.
v.Spitzenkörper, play an important role in organizing (regulating) hyphal growth. Found behind hyphal tip (apex).

Process of apical elongation

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(?) move towards the tip of the hyphae (towards the plasma membrane) where they release various enzymes and other compounds.

Cell vesicles

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(?) release by these particles involved in the lysis and synthesis of the cell wall

Enzymes

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Following lysis of the (?), cell division and growth allows for the hyphae to be elongated at the tip.

cell wall

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Once the new cell has formed, enzymes at the tip start synthesizing a new (?) around the new cell to protect it. Followed by strengthening of the actin cap. Then disappearance of Spitzenkörper.

cell wall

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(?), play an important role in organizing (regulating) hyphal growth. Found behind hyphal tip (apex).

Spitzenkörper

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leads to the formation of filamentous (fuzzy) colonies — mycelium

Apical elongation

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Types of mycelium:

1.Vegetative
2.Aerial mycelium

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filamentous (fuzzy) colonies

mycelium

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buried down, for the water exchange and nutrient absorption.

Vegetative

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projects on the surface of the culture medium with reproductive structures known as spores which can be sexual or asexual.

Aerial mycelium

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Requires the formation of special clusters for fertilization and nuclear fission.

Sexual reproduction

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sex organ, gametes (sex cells) and either monoecious or dioecious

Gametangium

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Involves meiosis

Sexual reproduction

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are developed as a result of a primary nuclear fusion with reduction in chromosome number during their formation.

Sexual spores

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Germinates and forms hyphae and mycelium

Sexual reproduction

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2-8 ascospores within an ascus (sac), the shape of each aids in identification of fungus.

Ascospores

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Basidiospores

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When two sporangiophores sexually fuse to form a large thick-walled bodies

Zygospores

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contains zygospores along a nonseptate hyphae

Zygosporangium

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Produced from a fusion of 2 non-identical hyphae

Oospores

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Sporulation and spores are preferred terms used when there is a merging of nuclear material or genes combined.

Asexual reproduction

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spore formed by budding, no fusion of nuclei takes place in the formation of spore.

Asexual spores

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Do not form mycelium, produce pasty type of colony (yeast)

Unicellular

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Form mycelium, produce filamentous colony (mold)

Multicellular

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Spores contained in sporangia or sacs are produced terminally (at the end)

Sporangiospores

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specialized stalk which bears the sporangia

Sporangiophore

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Observed among, Zygomycetes, Rhizopus

Sporangiospores

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Results from the transformation of a vegetative yeast or specialized hypha called conidiophores. Often referred to as macro- (multicellular) and microconidia (unicellular)

Mitospores (Conidiospores)

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Freed by abstraction at the point of attachment

Mitospores (Conidiospores)

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Observed among Ascomycetes and Deuteromycetes

Mitospores (Conidiospores)

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produced on a thallus

Thallospores

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Formed by budding from a pseudohypha

Chlamydospores

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Thick-walled resting spores either within (intercallary) the segments; hyphal sides (sessile); or hyphal tip (terminal)

Chlamydospores

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highly developed conidiophores

Phialospores

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Vesicle, enlarged swollen cell often at the end of a conidiophore or sporangiophore and it bears conidia.

Phialospores

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The conidiospores are formed usually at the sides (tips) of a hyphae and are usually in chains.

Phialospores

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Outer skin layer (hair)

Do not penetrate tissues

Malassezia furfur

Superficial

Skin, hair, nails (keratinized area)

Do not penetrate tissues, but can cause inflammation

Trichophyton species
Microsporum species

Cutaneous

Connective tissues, Muscles, Bones

Penetrate tissues, transmitted via traumatic inoculation

Sporothrix schenkii
Rhinosporidium seeberi
Loboa loboi

Subcutaneous

All body system

Thermal (dimorphic)

Histoplasma capsulatum

Systemic

Any part

Infects immunocompromised host

Aspergillus fumigatus
Candida albicans

Opportunistic

Similar to bacteriology, the goal of mycology laboratory is to accurately (?) fungi suspected of causing infection.

isolate and identify

Important considerations on specimen collection, transport, and processing
1.Proper (?) (aseptic technique).
2.All specimens should be transported in (?) and processed ASAP.
3. (?) or anaerobic containers should never be used for fungi.
4. (?) is recommended for transport (some fungi can be affected by above 37C and below 10C— dermatophytes are sensitive to cold temperatures.
5.If delay is expected within 2h, specimen should be stored at (?).
6.Appropriate volume. Volume required for fungal culture is greater than in bacteriology, since fluids and respiratory secretions need to be (?) prior to plating.

collection
leakproof sterile containers
Anaerobic transport media
RT
4C
concentrated or pretreated

the appropriate physicians should be notified.

•For rejected or unacceptable specimen

requisition must accompany each specimen and must include the following demographics

1.Patient name
2.Age and sex
3.Location or address
4.Physician name
5.Specific culture site
6.Date and time of specimen collection
7.Clinical diagnosis
8.Any special culture request

Specimen must have a firmly attached label indicating the

patient name, specimen type, date and time of collection

Necessary to maximize recovery of fungi.

Pretreatment and optimum temperature for culture

Importance of Pretreatment and optimum temperature for culture

1.Release fungi enclosed within cells.
2.Concentrate fungal material in the specimen.
3.Help to reduce or eliminate bacteria present in contaminated specimens because of the action of mucolytic agents viz.

mucolytic agents

a.N-acetyl-L-cysteine
b.5% oxalic acid
c.Dithiothreitol (Sputolysin2)

Temperature requirements for culture:

1.Blood, CSF: 30-37C
2.Dermatological (ie., skin, hair, nails): 15-30C

Granules should be washed & crushed; other materials should be centrifuge at 2,000g for 10min.

Abscess, drainage, pus, granules

Lysis in Isolator tubes and then centrifugation for 30min. at 3,000g, in a 35 fixed-angle rotor or swing bucket.

Blood, bone marrow

Criteria for the detection of H. capsulatum and other dimorphic fungi.

Blood, bone marrow

Centrifugation at 2,000g for 10min. or membrane filtration.

Body fluids

Essential for best recovery with volumes 1mL; blood clots should be teased apart.

Body fluidsC

Mince; gently pushed pieces down into agar.

Nails

Essential for the recovery of dermatophytes.

Nails

Lysis with mucolytic agents, followed by centrifugation at 2,000g for 10min.

Respiratory secretions (BAL, sputum)

Critical for the recovery of Pneumocystis jirovecii as very difficult to isolate in culture; improves recovery of other mycoses.

Respiratory secretions (BAL, sputum)

Mince or grind in a mortar; push pieces down into agar

Tissue

Essential for best recovery; for zygomycetes and other molds, mincing is best; for H. capsulatum, grinding is best.

Tissue

Centrifugation at 2,000g for 10min.

Urine

Essential for best recovery, particularly deep mycoses.

Urine

Essential for best recovery.

Abscess, drainage, pus, granules

•All work in mycology should be carried out in a certified (?) especially when dealing with filamentous fungi.

type 2 laminar airflow BSC

can be handled on the bench in the same manner that bacterial cultures are routinely handled.

Yeasts cultures

are recommended for personnel working with clinical specimens that may contain dimorphic fungi.

•BSL 2 procedures

should be worn for processing specimens and culture.

•Appropriate PPE

of slanted media are safer to handle than petri plates.

•Screw-cap tubes

should NEVER be used if Coccidioides immitis is suspected.

•Petri plates

•Do not make slide cultures of isolates that may be:

1.Histoplasma capsulatum
2.Blastomyces dermatitidis
3.Coccidioides immitis
4.Coccidioides posadasii
5.Paracoccidioides brasiliensis
6.Talaromyces marneffei
7.Cladophialophora bantiana

Clean surface with 70% alcohol. Collect from active peripheral edge with sterile needle and syringe. If open, use swab system or aspirate.

Abscess (Drainage, exudate, pus, wound)

If thick, pretreatment similar to sputum specimen

Abscess (Drainage, exudate, pus, wound)

If 2h, RT

Abscess (Drainage, exudate, pus, wound)

Examine for grains or granules and note color if present

Abscess (Drainage, exudate, pus, wound)

Disinfect skin with iodine tincture or chlorhexidine prior to obtaining. Use max. volume of blood recommended for the system used.

Blood

Manual
Biphasic (Septi-Chek)
Automated (BacT/ALERT, bioMérieux)
Lysis-centrifugation

Blood

If 2h, RT; if longer, RT but process in 16h (lysis- centrifugation)

Blood

All systems for bacteria will recover all yeasts except for Malassezia spp. but will not recover molds.

Blood

Lysis centrifugation systems are good for recovery of molds, especially the causing endemic mycoses.

Gives high contamination and false positive rates.

Blood

Collect aseptically in a heparinized syringe or lysis-centrifugation tube.

Bone marrow

Clotted bone marrow is an unacceptable specimen.

Bone marrow

If 15min., RT; if longer RT

Bone marrow

Pediatric isolator tubes are best.

Bone marrow

Remove distal 3-5 cm of line tip and place in a sterile container.

Catheter tip (Intravascular)

Method of Maki et. al.2; used for catheter tips but not validated for fungi.

Catheter tip (Intravascular)

If 15min., RT; if longer,4C

Catheter tip (Intravascular)

Avoid media containing Cycloheximide.

Catheter tip (Intravascular)

Disinfect all types with 70% alcohol.

Cutaneous (Hair, skin, nails)

is most important, plucking is best; submit 10-12 hairs in sterile dry container or envelope.

Hair
hair root

scrape with dull edge of a scalpel or glass slide, or vigorously brush in a circular motion with soft-bristle brush.

skin

material under nail should also be scraped. submit in sterile container or envelope.

nails clip or scrape with scalpel

Only the leading edge of a lesion should be sampled, as centers are often nonviable.

Cutaneous (Hair, skin, nails)

All specimens should be pressed gently into the agar with a sterile swab, do not streak agar plates. If used, toothbrushes should be pressed gently into agar as well

Cutaneous (Hair, skin, nails)

If 72h., (very stable) Never refrigerate, as dermatophytes are sensitive to cold.

Cutaneous (Hair, skin, nails)

Select hairs with fluoresce under a Wood’s light.

Cutaneous (Hair, skin, nails)

can be collected with soft-bristle toothbrush.

Hair and skin

olive oil or a paper disc saturated with olive oil should be placed on the first quadrant of agar plate.

For pityriasis versicolor (M. furfur)

taken by physicians and media(slides) inoculated directly.

Corneal scraping

needle aspiration.

Vitreous humor

inoculate non-inhibitory media in X- or C-shape motion.

Corneal

concentrate by centrifugation, use sediment for media and smears.

Vitreous humor

If 15min., RT; if longer RT

Eye (corneal scraping, vitreous humor)

Very little material usually available. Avoid media with cycloheximide

Eye (corneal scraping, vitreous humor)

Collected surgically. Transport in sterile container.

Medical devices

Use sterile scalpel to collect (by scraping) biofilm or vegetative growth.

Medical devices

If 15min., RT; if longer, 4C

Medical devices

Avoid media containing cycloheximide. Device material recovered best by using liquid medium.

Medical devices

Have patient empty bladder and then massage prostate gland to yield fluid.

Prostate fluid

Inoculate media directly or transport in sterile wide mouth container.

Prostate fluid

If 15min., RT; if longer RT

Prostate fluid

Fluid should always be examined microscopically. The first urine following massage has a high yield. This fluid is excellent for detection of endemic mycoses.

Prostate fluid

Use first morning sputum collected after brushing teeth.

Collect brushing and BAL fluid surgically.

Place all samples in sterile containers.

Inoculate media containing antimicrobial agents with and without cycloheximide.

Respiratory tract, lower (sputum, bronchial aspirate, BAL fluid)

Viscous lower respiratory specimens should be pretreated and centrifuged to concentrate their contents.

Respiratory tract, lower (sputum, bronchial aspirate, BAL fluid)

If 2h., RT; if longer, 4C

Respiratory tract, lower (sputum, bronchial aspirate, BAL fluid)

Saliva and 24h sputum are unacceptable specimens. Methods for mycobacterial decontamination are not acceptable.

Respiratory tract, lower (sputum, bronchial aspirate, BAL fluid)

Swab oral lesions, avoiding tongue. Use thin wire or flexible swab or oropharynx. Collect sinus contents surgically.

Respiratory tract, upper (Oral, oropharyngeal, and sinus samples)

Use swab transport system for oral and oropharyngeal samples. Place sinus contents in sterile container

Respiratory tract, upper (Oral, oropharyngeal, and sinus samples)

Oral: if 2h, RT; if longer, RT
Sinus: if 15min. RT; if longer, RT

Respiratory tract, upper (Oral, oropharyngeal, and sinus samples)

Selective and chromogenic media are best for recovery of Candida spp.

Respiratory tract, upper (Oral, oropharyngeal, and sinus samples)

Collect as for bacteriology. Concentrate by centrifugation, and use sediment for inoculation. Clots should be grounded.

Sterile body fluids (CSF and pericardial, peritoneal, and synovial fluids)

Except CSF, put sterile body fluids in sterile Vacutainer tubes with heparin or lysis- centrifugation tube to prevent blood clotting.

Except for CSF, blood culture bottles can be used for recovery of yeast.

Sterile body fluids (CSF and pericardial, peritoneal, and synovial fluids)

If 15min., RT; if longer, RT; never refrigerate.

Sterile body fluids (CSF and pericardial, peritoneal, and synovial fluids)

should always be examined microscopically. With specimen volumes 2ml, fluid should be plated directly using as much fluid on each plate as possible.

Sterile fluid sediment

Specimen use should be discourage.

Stool

Collected surgically. A larger volume is needed than for Bacteriology.

Tissue

Use sterile container, keep moist (saline drops) to prevent drying.

Except with H. capsulatum, mincing (not grinding) is critical.

pieces should be pressed into the agar so they are partially embedded.

Grind tissue for recovery of H. capsulatum.

Tissue

If 15min., RT; if longer, RT

Tissue

recommended for invasive disease.

Examine subcutaneous portion for granules (see abscesses)

Tissue biopsy

First morning clean catch, suprapubic, or catheterized specimens; 24-h specimens are unacceptable.

Urine

Use a sterile container or urine transport system.

Concentrate specimens by centrifugation, and use sediment for inoculation.

Urine

If 2h, RT; if longer, 4C; urine transport systems can stay at RT for up to 72h.

Urine

Chromogenic media best for Candida. Use sediment for microscopic examination.

Urine

Collect as for bacteriology.

Vaginal

Swab transport system or sterile container for washings.

Vaginal

If 2h, RT; if longer, RT

Vaginal

Antibacterial media or chromogenic agars are best for recovery of Candida.

Vaginal

Common clinical sites for laboratory recovery of pathogenic fungi

General approaches for direct detection and identification of fungi

1.Culture based
2.Non-culture-based

2.Non-culture-based

a.Direct microscopic examination
b.Antibody and antigen detection
c.Detection of 1,3–D-glucan and other fungal metabolites
d.Mass spectrometry
e.Nucleic acid detection

Stains and methods used directly on unfixed tissue or samples

Potassium hydroxide (KOH) (5-30 min.)
Calcofluor white, CW (1-2 min.)
Rapid Giemsa-like (Diff-Quik) (2-3 min.)
Colloidal carbon wet mounts (India ink, nigrosin) (1 min.)
Lactophenol cotton blue (LPCB)

•Wet mount prepared in (?), used to distinguished fungi in thick mucoid specimens or in samples that contain keratinous material such as skin, hair, and nails.

10%

•Nails may require stronger concentration of up to (?) to digest the nail prior to microscopy.

25%

•The fungi remain unaffected while the (?) of the host cells are partially digested2.

proteinaceous components

•Solutions of NaOH (?) may be used as an alternative to KOH.

10% or 25% with added glycerin

•Visualization of fungal elements may be enhanced by the addition of (?) or glucan-binding fluorescent brighteners such as (?), which bind to chitin.

lactophenol cotton blue (LCB)
calcofluor white (CW)

KOH, basic procedure

Addition of (?)
Specimen covered in (?)
Wet house for (?)
Observe under the (?)

10% KOH
coverslips
5 to 30 minutes
microscope

•Nonspecific, nonimmunological fluorochromes (along with Uvitex 2B and Blankophor) that bind to β-1,3 and β-1,4 polysaccharides specifically cellulose and chitin of fungal cell walls and fluoresces when exposed to long-wave UV light

Calcofluor white, CW

•More rapid than auramine-rhodamine stain.

Calcofluor white, CW

•Fungal elements appear bluish white to green fluorescence against a dark background when excited with UV or blue-violet radiation

Calcofluor white, CW

are 5-8 m [], round, and uniform in size and exhibit a characteristic peripheral cyst wall staining with an intense “double parenthesis-like” structure in CW

P. jirovecii cyst

•Yeast cells are differentiated from P. jirovecii by

budding and intense staining

fluoresce strongly and must be differentiated from fungal hyphae.

•Cotton fibers

1.KOH (10%) is mixed in equal proportion with CW solution (?)

(0.1 g of CW M2R and 0.05 g of Evans blue in 100 ml H2O)

2.Specimen is covered with this mixture and a coverslip is applied for 5 to 10 min. (nail and tissue preparations require up to (?) incubation time).

During (?), clearing and interaction of stain with the fungal elements occur.

(?) of the slide may speed up the clearing process.

30 min.

incubation

Warming

CW, basic procedure

3.Examination of the preparation with a (?) containing appropriate (?) at x 100 to x 400 magnification

fluorescent microscopy
excitation and barrier filters

may stain poorly with CW due to pigmentation that masks the fluorescence.

Dematiaceous fungi

Cysts walls with internal thickenings (double comma).

P. jirovecii of BAL fluid

Dichotomous branching hyphae.

Aspergillus spp. of lung tissue

Chitinous walls of the fungus.

C. albicans from culture

•Also applied to other specimens, such as BAL fluid or CSF.

Rapid Giemsa-like (Diff-Quik)

•Yeasts and trophozoites appear as blue purple

Rapid Giemsa-like (Diff-Quik)

(?)

•Differentiate form Leishmania;

Leishmania has a (?) and Histoplasma does not.

Rapid Giemsa-like (Diff-Quik)

Kinetoplast

A.Thick cluster of mostly trophic forms (2-3m) with small reddish purple nuclei and light blue to red violet cytoplasm.

Rapid Giemsa-like (Diff-Quik)

B.CSF with C. neoformans.

Rapid Giemsa-like (Diff-Quik)

•Detection of encapsulated microorganisms of C. neoformans in CSF.

Colloidal carbon wet mounts (India ink, nigrosin)

•The organism is refractory to the particles of ink, and capsules appear as clear halos around the organism against a black background.

C. neoformans in Colloidal carbon wet mounts

•Artifacts viz. erythrocytes, leukocytes, and talc particles from gloves or bubbles following a myelogram may

displace the colloidal suspension and mimic yeast (false positive)

Determine the test

•When positive in CSF, diagnostic of meningitis. Negative in many cases of meningitis; not reliable. But the sensitivity is (?) followed by lateral flow assay (?) and CALAS (?)

Colloidal carbon wet mounts

97%
94%
74%

India ink, basic procedure (1 min.)

Addition of 1 drop (?) on a slide
Addition of 1 drop
Add (?)
Observe under the

CSF
India ink
cover slip
microscope 400x

Determine the test

•Basic mounting medium for fungi consisting of

Lactophenol cotton blue (LPCB)

phenol, lactic acid, glycerol, and aniline (cotton) blue dye.

•Commonly used for the microscopic examination of fungal cultures by tease or tape preparation.

Lactophenol cotton blue (LPCB)

makes an excellent permanent stain or fixative for mounting slide culture preparations2.

•LPCB with 10% polyvinyl alcohol (LPCP-PVA)

acts as a clearing agent and aids in preserving the fungal structures.

Lactic acid

acts as a killing agent

Phenol

prevents drying

Glycerol

provides color to the structures.

Cotton blue

are analogous to cotton blue

Poirrier’s blue and aniline blue

LPCB, basic procedure

A drop of (?) is added to a glass slide, and a (?) is prepared.
Add a (?) and examine at (?)

LPCB solution; tease or tape mount
coverslip; x 100 to x400 magnification

Note the crescent spindle-shaped conidia. x 400

Fusarium spp. in LPCB

Cells appear as dark-walled and some budding cells x 400.

C. neoformans in LPCP isolated form CSF

•Primarily used for the examination of bone marrow, buffy coat, and peripheral blood smears.

Giemsa stain

•For the detection of intracellular yeast forms of H. capsulatum and fission yeast cells of Talaromyces marneffei.

Giemsa stain

•The fungus is seen as small oval yeast cells, often contained within macrophages, and stains blue; a hyaline halo represents poorly staining cell wall.

H. capsulatum in Giemsa stain

•The stain can also be used to visualize trophozoite of P. jirovecii.

Giemsa stain

Giemsa stain, basic procedure (15 min.)

PBS is prepared on a glass slide and placed in (?).
Slide is flooded with freshly prepared Giemsa stain (?)
After 5 min. Rinse slide with (?) and air dried. Examine at x 100 to x 400 magnification

100% methanol for 1 min
stock Giemsa stain diluted 1:10 with phosphate buffered H2O
d’H2O;

Yeast appears as small, oval cells that stain light to dark blue with a hyaline halo due to the unstained cell wall.

Macrophages from a lymph node filled with H. capsulatum.

BAL fluid showing characteristic fission yeast cells of

T. marneffei

Detection of bacteria and fungi.

Gram stain

Gram positive
Gram negative

yeasts and pseudohyphae
hyphae (septate and coenocytic)

may decolorize and appear either Gram negative or stippled

Cryptococcus

stain weakly in some instances and exhibit only stippling

Often have orange amorphous material around yeasts

Cryptococcus spp

Not all fungi are detected.

Gram stain

Filaments of Nocardia stain at least partially acid-fast (pink); Actinomyces and other actinomycetes are negative.

Modified acid-fast

Detection of P. jirovecii in respiratory specimens viz. lung biopsy specimen imprints and BAL specimens

Toluidine blue O

Stains cyst walls of P. jirovecii reddish blue or dark purple against a light blue background.

Toluidine blue O

Cysts are often clumped and may be punched in, appearing crescent shaped.

Toluidine blue O

Trophozoites are not discernible.

Toluidine blue O

1.Place the air-dried slide in the sulfation reagent (?) for 10 min.
2.Rinse in cold H2O for 5 min., drain, and place in toluidine blue O (?) for 3 min.
3.Rinse in (?), followed by absolute ethanol and then xylene.
4.Examine at (?) magnification.

45 ml of glacial acetic acid mixed with 15 ml of conc’d H2SO4
0.3 g of dye in 60 ml of H2O
95% ethanol
x 100 to x 1000

Cytologic stain used primarily to detect malignant cells. But it works well in sputum smears and good for differentiation of dimorphic fungi.

Papanicolaou

Depending on the cell type detected, background stains in subtle range of green blue, orange to pink hues.

Papanicolaou

Some species of Candida stains gold, while other fungi may not stain at all.

Papanicolaou

Mucopolysaccharide stain, not commonly used (more commonly used is mucicarmine stain); like India ink, does not detect all cases.

Alcian blue

Useful in visualizing polysaccharide capsule of Cryptococcus that stains blue against a pink background in histological sections of tissue.

Alcian blue

The polysaccharide capsule of Cryptococcus neoformans stains turquoise blue; the finer details of the capsule may be better preserved in some specimens when compared with mucicarmine

Alcian blue

General-purpose histologic stain. Stains some fungal elements violet to bluish purple in mild to moderate contrast to the lighter background tissue.

Hematoxylin and eosin (H&E)

Has advantage of allowing observation of natural pigments of fungi.

Hematoxylin and eosin (H&E)

The best stain to demonstrate host tissue reaction and pigment of dematiaceous fungi.

Hematoxylin and eosin (H&E)

Fungal cytoplasm is pink and the nuclei are blue.

Hematoxylin and eosin (H&E)

Aspergillus spp. and mucoraceous moulds stain well.

Hematoxylin and eosin (H&E)

Splendore-Hoeppli phenomenon may be seen with some fungi, including Basidiobolus and Sporothrix.

Hematoxylin and eosin (H&E)

Fungi stain pink to red purple, nuclei may be blue depending on the counterstain used.

Periodic acid Schiff (PAS)

PAS stains glycogen, so other tissue structures can have a similar appearance to yeast cells.

Periodic acid Schiff (PAS)

Demonstrates the double-contoured refractile walls of Blastomyces dermatitidis that may not be visible with GMS stain.

Periodic acid Schiff (PAS)

Fungal stain of choice of many dermatopathologists.

Periodic acid Schiff (PAS)

Fungal elements including Pneumocystis jirovecii stain gray to black. Background is green.

Gomori methenamine silver (GMS)

It also stains Nocardia and other actinomycetes.

Gomori methenamine silver (GMS)

Often stains fungi too densely to observe structural details.

Gomori methenamine silver (GMS)

Yeast cells and cysts of Pneumocystis may appear similar in size and shape.

Gomori methenamine silver (GMS)

Stains mucin

Mucicarmine

Stains the capsule of Cryptococcus that appears pinkish red and may also stain the cell walls of Blastomyces dermatitidis and Rhinosporidium seeberi.

Mucicarmine

Useful for differentiating C. neoformans (gattii) from other fungi of similar size and shape when found in samples of tissue

Mucicarmine

Detection of melanin of dematiaceous fungi and C. neoformans.

Fontana-Mason (FM)

Cell walls appear as brown to black with pale pink background.

Fontana-Mason (FM)

It was originally thought to stain only dematiaceous fungi and C. neoformans (organisms known to contain melanin or melanin precursors), but it has now been shown to stain variably, but less intensely, A. fumigatus, A. flavus, Trichosporon spp., Fusarium chlamydosporum, and some Mucorales.

Fontana-Mason (FM)

Gram stain for bacteria; demonstrates the bacterial filaments of the actinomycetes, eg., Nocardia, Actinomadura.

Brown and Brenn (B&B)

Specific method of detecting fungi in body fluids

Fluorescent antibody stain

Direct technique: fluorescein-labeled Ab reacts with fungal antigen in cell wall.

Fluorescent antibody stain

Indirect technique: unlabeled Ab complexes with fungal antigens. Fluorescein-labeled conjugate reacts with globulins attached to fungal antigens. Cell walls yellow-green.

Fluorescent antibody stain

Some use the immunoperoxidase technique. Specific and highly sensitive. Can be used to detect and measure antibodies.

Fluorescent antibody stain

Used for the cultivation of ascosporogenous yeasts such as Saccharomyces cerevisiae.

Acetate ascospore agar

The potassium acetate formulation has been shown to be a better sporulation medium than sodium acetate.

Acetate ascospore agar

Ascospores produced in this medium are visible microscopically after staining Kinyoun carbol-fuchsin acid-fast stain.

Acetate ascospore agar

Selective and differential medium used for the isolation of Cryptococcus spp. esp. C. neoformans and C. gattii which is unique in that they produce the enzyme phenol oxidase.

Birdseed agar

The breakdown of substrate (Guizotia abyssinica seeds or niger seeds) produces melanin, which is absorbed into the yeast wall and imparts a tan to brown pigmentation of the colonies.

Birdseed agar

Colonies of other yeasts are beige or cream in color.

Birdseed agar

Chloramphenicol is selective agent that inhibits bacteria and some fungi.

Birdseed agar

Creatinine enhances melanization of some strains of C. neoformans.

Birdseed agar

Selective and differential medium used for the isolation and differentiation of Candida spp.