MODULE 1 UNIT 1 Flashcards
study of fungi, termed specifically as
“Mycology”
Once in the scientific history, fungi were classified as “plants”. This was derived from the axiom attributed to Carl Linnaeus (father of modern taxonomy) “Plants grow and live, while animal grow, live and [?]”. This attribution begets misleading to the future discovery
and identification of plants.
feel
The term Mycology was derived from the Greek word [?] which means fungus (plural form fungi).
“mykes”
is the study of fungi encompassing environmental impact, genetic and biochemical properties
Mycology
is devoted to the study of fungi, its impact and relationship to human disease.
Medical Mycology
Approximately, [?] fungal pathogens out of more than 100,000 known fungal species are medically important, and about [?] of these species were often associated with mycosis.
- 200
- 50 to 60
Fungi play a very important role in various fields including but not limited to environmental, bioeconomy, etc.
is devoted primarily to the relationship of fungi to human.
Medical mycology
- Due to increasing number of [?] environmental molds.
ubiquitous
- Implicated as [?] pathogens, capable of producing serious or lethal disease in hosts that are immunocompromised (debilitated).
opportunistic
- One of the leading causes of [?] infection.
nosocomial
- Often mistaken as [?] infection that produces complicated and fatal consequences.
bacterial
- Increase [?] (travel to a geographical area where a fungus exists as part of the communal flora of the local population, or is endemic to the area).
morbidity
- [?] population.
Aging
an asexual form of a fungus
Anamorph
a cell that produces and extrudes conidia; the tip tapers lengthens and acquires a ring of cell wall material as each conidium is released; oil immersion magnification may be required to see the rings.
Annellide
ability of fungus to use a specific carbon or nitrogen source for growth; assimilation is read by the presence or absence of growth
Assimilation
enlarged, dome-shaped tip of a sporangiophore that extends into the sporangium.
Columella
the cell that produces conidia
Conidiogenous cell
a specialized hyphal structure that serves as a stalk on which conidia are formed. The shape and arrangement of the conidiophores and the conidia are generally characteristic of a genus. The suffix phore means “carrying” and is added to the word that denotes what it is carrying; e.g., conidiophores bear conidia and sporangiophores bear sporangia.
Conidiophore
An asexual propagule that forms on the side or the end of the hypha or conidiophore.
Conidium (plural conidia)
may consist of one or more cells, and the size, shape, and arrangement in groups are generally characteristic of the organism.
Conidium
Conidium is always borne [?], i.e., not enclosed within a saclike structure such as a sporangium.
externally
If a fungus produces two types of conidia, those that are small and usually single celled are referred to as
microconidia
whereas the larger [?] are usually segmented into two or more cells.
macroconidia
prefix meaning dark (brownish or blackish)
Pheo-
a subcutaneous or systemic disease caused by a variety of black fungi that develop in tissue or dark hyphae and(or) yeast-like cells.
Paeohyphomycosis
cut off sharply; ending abruptly with a flattened edge.
Truncate
fungi were used as an antiseptic and anesthesia due to the “magical and spiritual properties.
35,000 B.C.
people were convinced that association with fungi with entail the formation of disease(s).
Middle Ages
fungi were plants with no fruit nor seed
Renaissance period
Birth of the first mycological studies. Pier Anton Micheli (founder of modern mycological studies). It is in this period where mycology was separated from botany
18th Century
Fungi were recognized as a potential causative agents of diseases that are usually fatal in nature.
Mid-20th Century
TAXONOMY, (Gk.: [?]= arrangement; [?] = method).
- taxis
- anomia
Branch of science that deals with systematic biological classification of all living organisms.
TAXONOMY
Taxonomy is divided into three (3) disciplines viz.
Classification, Nomenclature, and Identification.
Synonym of “systematic” or biosynthetic
TAXONOMY
In 2011, a breakthrough in the field of fungal taxonomy occurred: the concept of “One Fungus = One Name” concept.
In 2011, a breakthrough in the field of fungal taxonomy occurred: the concept of [?] concept.
“One Fungus = One Name”
Throughout the past century, pleomorphic fungi had been defined with two different names based upon the phenotypic identification of the sexual stage (?) or the asexual form (?).
teleomorph
anamorph
For example, the anamorph form Blastomyces dermatitidis was known as [?] in the teleomorph form.
Ajellomyces dermatitidis
With the advent of numerous advances in molecular taxonomy, the teleomorphic and anamorphic stages of several medically important fungi were confirmed to be the
same organism
Recognizing the unique dilemma posed by the identification of pleomorphic fungi having two names, the [?] in Melbourne, Australia, in July 2011 recommended the discontinuation of the dual nomenclature system for fungi with anamorphic and teleomorphic forms
Nomenclature Section meeting of the International Botanical Congress
Known as the [?] this recommendation resulted in a historic revision of the International Code of Botanical Nomenclature.
Amsterdam Declaration on Fungal Nomenclature
Becoming effective on January 1, 2013, the principle of “One fungus = One name” assigned priority to the [?] independently of whether the organism was originally described as the anamorph or the teleomorph.
oldest genus (species) name
is the orderly arrangement of organisms into taxonomic groups on the basis of similarity.
Classification
is the orderly arrangement of organisms into taxonomic groups on the basis of similarity.
Classification
There are three criteria for taxonomical classification:
(i) Cell type
(ii) Level of cellular organization,
(iii) Nutritional status
this pertains whether a define organism is classified as Eukaryotic or Prokaryotic
(i) Cell type
discusses whether an organism is a unicellular or multicellular
(ii) Level of cellular organization
Nutritional status is divided further according to:
a. Food acquisition
b. Energy and carbon source
is the labelling of the units defined.
Nomenclature
is the process of determining whether an unknown belongs to one of the units defined in and labeled in.
Identification
Taxonomical classification of fungi: Domain
Eukarya
Taxonomical classification of fungi: Kingdom
Fungi
Taxonomical classification of fungi: Phylum
Zygomycota
Taxonomical classification of fungi: Class
NA
Taxonomical classification of fungi: Order
Mucorales
Taxonomical classification of fungi: Family
Mucoraceae
Taxonomical classification of fungi: Genus
Rhizopus
Taxonomical classification of fungi: Species
arrhizus
Taxonomical classification of fungi: Sub-species
NA
is one among the numerous causative agents of Mucormycosis, a fungal infection caused by ubiquitous environmental molds. This mold is one of the causative agents isolated from COVID-19-associated Mucormycosis epidemic in India started last May 2021.
Rhizopus arrhizus
The Cell wall structure is rigid contains primarily [?] of polysaccharide plus [?] protein and glycoproteins
90%
10%
is the major carbohydrate content consist of repeating monomers of N-acetyl-glucosamine (NAG) that provides shape and protection from external stress factors such as osmotic lysis (unaffected by antibiotics).
Chitin
Minor carbohydrates are also present viz.
Glucan, Mannan, Chitosan, and Galactosan.
There are various functional properties of the cell envelope. Simply provision of its shape and interference between the fungus and its external environment, acts as [?] for some enzymes, and processes [?] which allow interaction with other organisms.
- binding site
- antigenic properties
Phospholipid structure arranged in a two-layered fashion scattered randomly.
Cell Membrane
is the major type of sterol present that regulates solute intake and secretion (transport system) via selective permeability and target of some antibiotics such as Nystatin.
Ergosterol
Functions of CM include
facilitate biosynthesis of cell wall and capsular material
protects the cytoplasm
This is an external coating covering the cell wall, and few of the fungi exhibits this structure such as Cryptococcus neoformans
Capsule
an opportunistic fungi and the causative agent of Cryptococcosis among individuals who are immunosuppressed (eg, People living with HIV, PLHIV).
Cryptococcus neoformans
Considered as a virulence factor and compose of amorphous
polysaccharide and may influence the fungal growth by preventing
dissociation of buds and dispersion yeast.
Capsule
Bounded by a nuclear membrane and may vary in size, shape, and number. Contains usually 1 nucleolus of mostly RNA.
Nucleus
The [?] are linear in configuration, compared to bacterial chromosomes that are often circular.
chromosomes
are present at all times for protein synthesis.
Ribosomes
functions in energy generation.
Mitochondrion
associated cellular organelles of cytoplasm
(i) Nucleus
(ii) Ribosomes
(iii) Mitochondrion
(iv) Endoplasmic Reticulum
(v) Vacuoles
all fungi are non-motile, except those groups that belong to
Phylum Chytridiomycota (Chytrids)
The ecologically-derived modes of nutrition, fungi are considered as [?] (Gk., chemo = chemical; + hetero = an(other); troph = nourishment) and [?], utilize various organic compounds as sources of nutrients via absorption and capable of growing on dead and living organic matter respectively.
chemoheterotrophic
saprophytic
In terms of their atmospheric requirements, fungi are mostly [?] and to some extent few are [?].
aerobic
facultative anaerobic
Fungi produce [?], that is utilized for the synthesis of the amino acid lysine de novo through α-aminoadipate pathway and as a precursor for penicillin. This pathway is used as the target for the development of new antibiotics.
α-aminoadipate
plays a very important role in the identification of fungi for both macroscopic and microscopic methods.
Fungal morphology
Today with the luxury of molecular methods; with the appropriate selection of [?] plus the [?] of isolated fungi remains the key points in differentiating and in identification of fungi.
biochemical tests
traditional microscopic and macroscopic observation
Unicellular; Microscopic, characterized by spherical to ellipsoidal, varies in size, with a diameter of 3-5µ.
Yeasts
Mode of reproduction of yeast is through an asexual process known as the process of
budding
Budding comes in three sequential stages viz,
bud emergence, protuberance, and separation.
Yeasts are macroscopic, often observed in culture media (eg, Saboraud’s dextrose, Potato dextrose agar) and exhibits [?] colonies that measure 0.5 to 3.0mm in diameter. Optimum temperature for growth is 35-37ºC.
opaque, creamy to pasty
Microscopic, composed of a cylindrical structure called hypha (plural hyphae) which is the basic unit in a fungus.
Multicellular, 2-10µ in diameter; Microscopic, composed of a cylindrical structure called hypha (plural hyphae) which is the basic unit in a fungus.
Molds
The entire body of the hyphae referred to as [?] (colony surface) or the vegetative body of a fungus
thallus
the cross walls that cross sectionally divide a hypha is called
septum
is a mat of an intertwined hyphae that constitutes the colony surface of a mold.
Mycelium
Macroscopically, molds can be observe via
obverse (surface) or reverse (back).
creamy, bright or light gray to brown
obverse (surface)
non pigmented to yellow, orange, or red
reverse (back)
CLASSIFICATION OF HYPHAE
- Acc. to existence of septa
- Acc. shape and morphology
- Acc. to pigment production
- Acc. to hyphal growth
(fungus with cross walls), in most cases dichotomous occurs when a hyphae branches into two equal diameter to the hyphae from which they originated
Septate
(fungus without cross walls), Coenocytic hyphae results from nuclear division within a cell without division in the cytoplasm
Aseptate
the thin walls and lack of regular septation decreases the internal support of these broad hyphae and allows them to become characteristically twisted, collapsed, and folded in a ribbonlike fashion (Mucormycosis).
pauciseptate hyphae
characterized by multiple projections in a hypha resembling an old comb hyphae appearance (eg, Microsporum audouinii)
Pectinate
marked by hyphae with club-shaped cells, in which the layer end of one cell being attached to the smaller end of an adjacent (eg, Coccidioides sop.).
Racquet
the hypha forming a coiled or corkscrew like turns (eg, Trichophyton mentagrophytes)
Spiral
terminal hyphae branches that are irregular, broad and antler-like in appearance. (eg, Trichophyton schöenleinii).
Favic Chandelier
this is specified by a round knot-like structure formed by intertwined hyphae and seen among dermatophytes.
Nodular
produces root-like structure along the vegetative hyphae especially observed among Zygomycetes (eg, Rhizopous and Absidium)
Rhizoids
basically transparent, clear and colorless hyphae, as shown from Figure 1.6 among agents of mucormycosis (ie, Saksenaea vasiformis)
Hyaline
in comparison to hyaline, the structures of dematiaceous hyphae are brown to black in color, due to melanotic pigment in the cell walls.
Dematiaceous
forms filamentous (fuzzy) colonies
Apical elongation (macroscopic)
this is when mycelium buried down the culture medium for water exchange and nutrient absorption.
Vegetative
— projects on the surface of the medium, with reproductive structures known as spores which can be sexual or asexual.
Aerial
move towards the tip of the hyphae (towards the plasma
membrane) where they release various enzymes and other compounds.
Cell vesicles
Enzymes release by these particles involved in the
lysis and synthesis of the cell wall
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 [?]. Then disappearance of [?]
actin cap
Spitzenkörper
play an important role in organizing (regulating) hyphal growth. Found behind hyphal tip (apex)
Spitzenkörper
is triggered by stems for the sub apical accumulation of wall precursors (presumable vesicles) reaching a critical concentrations
Lateral elongation
is the ability of some fungi to grow into two (2) forms, specifically yeast and mold forms in which dependent on temperature.
FUNGAL DIMORPHISM
Yeast
Temperature:
Growth:
Form:
35-37ºC
In vivo
Pathogenic
Mold
Temperature:
Growth:
Form:
25-30ºC
In vitro
Infective
Some of the notable examples of fungi that exhibits dimorphism include:
- Sporothrix schenkii
- Blastomyces dermatitidis
- Histoplasma capsulatum
- Coccidioides immitis
- Paracoccidioides brasiliensis
- Penicilium notatum (P. chrysogenum)
- Talaromyces (Penicillium) marneffei
Requires the formation of special clusters for fertilization and nuclear fission
Sexual reproduction
— sex organ, gametes (sex cells) and either monoecious and dioecious. It involves mitosis and meiosis
Gametangium
are developed as a result of a primary nuclear fusion with reduction in chromosome number during their formation. Germinates and forms hyphae and mycelium
Sexual spores
•2-8 ascospores within an ascus (sac). the shape of each aids in
identification of fungus.
Ascospores (eg,, Saccharomyces spp., Ascomycetes)
•2-8 ascospores within an ascus (sac). the shape of each aids in
identification of fungus.
Ascospores (eg,, Saccharomyces spp., Ascomycetes)
2-4 basidiospores on the surface of a club-shaped called
basidium
Basidlospores (eg., Basidiomycetes)
When two sporangiophores sexually fuse to form a large thick-walled bodies
Zygospores (eg.. Mucor & Rhizopus)
contains zygospores along a non-septate hyphae
Zygosporangium
Produced from a fusion of 2 non-identical hyphae
Oospores (eg., Phythophthora. phythium, etc.)
are preferred terms used when there is a merging of nuclear material or genes combined.
Sporulation and spores
are formed by budding, no fusion of nuclei takes place in the formation of spore.
The asexual spores
Either unicellular (do not form mycelium often produced pasty type of colonies) or multicellular (form mycelium, produce
filamentous colony
Spores contained in sporangia or sacs are produced [?] (at the end)
terminally
specialized stalk which bears the sporangia
Sporangiophore
Sporangiophore are observed among
Zygomycetes, Rhizopus
Abundant club-shaped microconidia and oval-shaped macroconidia containing 5-8 cells.
Microsporum gypseum
Results from the transformation of a vegetative yeast or specialized hypha called [?].
conidiophores
Often referred to as macro- (?) and microcondidia (?)
multicellular
unicellular
Freed by abstraction at the point of attachment
Mitospores (Conidiospores)
Observed among Ascomycetes and Deuteromycetes
Mitospores (Conidiospores)
- Asexual spore formed from a budding process or blowing out along the mycelium or from another blastospore — blastogenesis
- Blastoconidia
Blastospores (Candida spp.)
•Formed by fragmentation (segmentation) or disarticulation of hyphae (mycelium) which results to cuffing of rectangular thick-walled spore
Arthrospores (eg., Dermatophytes, Coccidioides)
- Formed by budding from a pseudohypha
Chlamydospores (eg.. Candida spp.)
Chlamydospores are thick-walled resting spores either within (?) the segments: hyphal sides (?); or hyphal tip (?)
- intercallary
- sessile
- terminal
- Vesicle. enlarged swollen cell often at the end of a conidiophore or sporongiophore and ¡t bears conidia
- The conidiospores are formed usually at the sides (tips) of a hyphae and are usually ¡n chains
Phialospores (eg., Aspergillus spp.. Penicillium spp.)
Perfect fungi**
Ascomycetes
Basidiomycetes
Zygomycetes
Imperfect fungi
Deuteromycetes
Ascomycetes
Sexual spore:
Asexual spore:
Hyphae:
Ascospore
Any conidiospore
Septate
Basidiomycetes
Sexual spore:
Asexual spore:
Hyphae:
Basidiospore
Conidiospore
Sepate
Sexual spore:
Asexual spore:
Hyphae:
Zygospore
Conidiospore
Coenocytic
Deuteromycetes
Sexual spore:
Asexual spore:
Hyphae:
None
Conidia
Septate
are those capable of reproduction both sexual and asexual
Perfect fungi
reproduced via asexual type only (sporogenesis)
Imperfect fungi (fungi imperfecti)
CLASSIFICATION OF FUNGAL INFECTIONS
Superficial
Cutaneous
Subcutaneous
Systemic
Superficial
Tissue Involved (Site of Infection): Examples:
Outer skin layer, hair
Malassezia furfur
Cutaneous
Tissue Involved (Site of Infection): Examples:
Skin, hair, nails (keratinized area)
Trichophyton spp.
Subcutaneous
Tissue Involved (Site of Infection): Examples:
Connective tissue, muscles, bones
Sporothrix schenkii
Systemic
Tissue Involved (Site of Infection): Examples:
All body systems
Histoplasma capsulatum
Do not penetrate tissues
Superficial
Do not penetrate tissues but can cause inflammation
Cutaneous
Penetrate tissues; transmitted via traumatic inoculation
Subcutaneous
Thermal dimorphic
Systemic
Opportunistic
Tissue Involved (Site of Infection): Examples:
Any part (localized or systemic) Aspergillus, spp.,; Candida spp.
Infects immunosupressed hosts
Opportunistic
- [?] (ie, aseptic technique)
Proper collection
- Never use [?] for fungi
anaerobic transport media or anaerobic containers
- [?] is recommended for transport
Room temperature
- [?] delivery of the samples to the laboratory
Rapid
- [?] into proper and appropriate medium (eg, SDA, Cornmeal)
Inoculation
- [?] at a suitable temperature
Incubation
Optimum Temperature
Blood and CSF samples
Dermatological (ie, skin, hair, nails)
Others
30-37ºC
15-30ºC
4ºC
Pretreatment of Clinical Specimens prior to inoculation — importance
- Releases fungi [?] within cells
- [?] fungal material in the specimen
- Help reduce or eliminate bacteria present in contaminated specimens because of the action of mucolytic agents such as [?]
- enclosed
- Concentrate
- N-acetyl-L-cysteine, 5%\ oxalic acid, or dithiothreitol (sputolysin)
Biosafety Considerations:
- All work must be carried out in a certified [?] or higher, whenever is possible
- [?] are recommended for personnel working with clinical specimens that may contain dimorphic fungi as well as other potential pathogenic fungi.
- [?] must be worn for processing specimens and culture.
- type 2 laminar-airflow BSC
- BSL2 procedures
- Gloves
I. Skin and interspaces
(i) Wipe lesions and interspaces between toes with [?] to remove surface bacterial contaminants.
(ii) Using the edge of the glass slide or a blunt scalpel blade, firmly scrape the lesion, particularly at the [?]. If multiple lesions are present, choose the most recent for scrapings as old loose scale is often not satisfactory.
(iii) Place scrapings between [?] or place in a clean envelope labeled with the patient’s data.
70% alcohol
growing margin
two glass slides
II. Nail
(i) Clean nail with [?]
(ii) [?] Scrape outer surface and discard; scrape the deeper portion.
(iii) Remove a portion of debris from under the nail with a [?].
(iv) Collect [?] or nail clippings.
(v) Place all material in a [?] labeled with the patient’s data.
- 70% alcohol.
- Dorsal plate-
- scalpel
- whole nail
- clean envelope
III. Hair
(i) No cleaning of [?] is needed.
(ii) Select infected areas (with scaling or alopecia) and with forceps epilate at least [?]
(iii) For hairs broken off at the scalp level, use a [?]
(iv) Place hairs between [?] or in a clean envelope labeled with the patient’s data.
- scalp
- 10 hairs.
- scalpel or a blade knife.
- two clean glass slides
IV. Sputum [tracheal lavage, bronchial lavage, (endo)tracheal aspirate]
(i) Sputum should be [?] and collected in the early morning.
(ii) Have patient remove [?] and rinse vigorously with water.
(iii) Sputum should be the result of a [?] or induced by aqueous aerosol.
(iv) Collect [?] in a sterile container
- fresh
- dentures
- deep cough (not saliva)
- 5-10 ml
V. Urine
(i) The urine specimen most suitable for making a diagnosis of mycoses of the urinary tract is a [?] specimen. [?] should be collected when aspiration or cystoscopy cannot be done.
(ii) Early morning specimens are aseptically collected in . [?] have no value. If a delay in processing beyond [?] is anticipated the urine should be placed in a 4°C refrigerator for up to [?] to inhibit the overgrowth of rapidly growing bacteria.
- catheterized; clean-catch, midstream
- sterile containers; Twenty- four-hour collections; 2h; 12-14 hours
VI. Blood
(i) Blood is collected aseptically to avoid [?]. The collection site is cleaned with a disinfectant at the time of collection.
(ii) Uses sodium polyanethol sulfonate (SPS, Liquid) as an anticoagulant. Collect 8 ml blood in a yellow vacutainer tube (contains [?] SPS; final concentration with blood is 0.05%).
(iii) The [?] system is ideal for fungal blood culture. Ten ml of blood is directly collected in an isolator tube.
(iv) NOTE: [?] is the method of choice for patients with disseminated Histoplasmosis.
- microbial contamination
- 1.7 ml of 0.35%
- Lysis Centrifugation Isolator
- Blood culture
VII.Exudates
(i) The skin over [?] should be disinfected.
(ii) Using a sterile needle and syringe, aspirate material from [?]
(iii) Place the material in a sterile container. The syringe also serves as a transport container if the needle is [?]
- pustular lesions
- undrained abscesses
- capped
VIII.Vaginal material
(i) Using a [?], collect material from the vagina.
(ii) Insert swabs into a
- sterile swab
- sterile tube
IX. Tissue
(i) Tissue is aseptically collected from the [?] of the lesion.
(ii) Place between moist gauze squares, add a small amount of sterile water [?] to keep tissue from drying out and send immediately to the laboratory. Keep refrigerated not exceeding 8-10 hours at 4°C until processed.
- center and the edge
- 0.85% NaCl
X. Cerebrospinal Fluid
(i) As much spinal fluid as possible is collected and the placed in a [?]
(ii) Generally, a [?] is used.
(iii) If processing is to be delayed, samples should be stored at [?] and not be refrigerated is an adequate fluid culture in which fungal elements can survive until subcultured.
- sterile container
- number 3 tube
- 30-37 °C
XI. Bone Marrow
(i) Aspirate approximately [?] of bone marrow and place it in a sterile container.
(ii) [?] can be added as an anticoagulant.
(iii) The [?] may be used.
- 3-5mL
- SPS or heparin (sodium)
- pediatric isolated blood collection system
XII. Transudates (pleural fluid, synovial, peritoneal)
(i) Specimens are collected aseptically through [?]
(ii) Place in [?]
- thoracentesis, arthrocentesis, and paracentesis.
- sterile containers.
DIAGNOSIS OF HUMAN MYCOSIS: TWO APPROACHES
I. Clinical Diagnosis
II. Laboratory Diagnosis
based on demographic information, patient history (eg, travel, occupation, etc.), predisposing factors, and merely focused on manifestations (ie, signs and symptoms)
I. Clinical Diagnosis
usually microbiologic (ie, fungal CS), serologic, and molecular approach.
II. Laboratory Diagnosis
LABORATORY EVALUATION: TWO APPROACHES
Most organic substances might be confused with fungi are digested in the presence of moderately strong alkaline solutions.
Potassium hydroxide (KOH) preparation
Digestive capabilities can be enhanced with gentle heating or the addition of 40% dimethyl sulfoxide.
Potassium hydroxide (KOH) preparation
Digestive capabilities can be enhanced with gentle heating or the addition of 40% dimethyl sulfoxide.
Potassium hydroxide (KOH) preparation
Used to distinguish fungi in thick mucoid specimens or in specimens that contain keratinous material viz, skin, nails, and hairs.
Potassium hydroxide (KOH) preparation
10% concentration for skin and soft tissues, body fluids and 20% concentration for nails and hard tissues.
Potassium hydroxide (KOH) preparation
The polysaccharide capsule of organism is refractory to the particles of ink, and capsules appear as halo around the organism.
Colloidal carbon wet mounts (ie, India ink, Nigrosin)
Used for visualization of Cryptococcus spp.
Colloidal carbon wet mounts (ie, India ink, Nigrosin)
Nonspecific, non-immunological fluorochromes that bind to the β-(1⇥3) & β-(1⇥4) polysaccharides, specifically cellulose and chitin of fungal cell walls and fluoresces when exposed to long-wave UV light
Calcofluor white (CFW), syn. Blankofluor and Uvitex
• The fluorochrome can be mixed with KOH to clear the specimen for easier observation of fungal elements (eg., P. jirovecii) that appear bluish white agains dark background.
Calcofluor white (CFW), syn. Blankofluor and Uvitex
• Fluorescence microscope is required.
Calcofluor white (CFW), syn. Blankofluor and Uvitex
Basic mounting medium for fungi that consist of phenol, lactic acid, glycerol, and aniline (cotton) blue dye.
Basic mounting medium for fungi that consist of phenol, lactic acid, glycerol, and aniline (cotton) blue dye.
Lactophenol Cotton Blue (LCB)
• Used as both a mounting fluid and stain.
Lactophenol Cotton Blue (LCB)
Lactic acid acts as a clearing agent and aids in preserving the fungal structures, phenol acts as a killing agent, glycerol prevents drying, and cotton blue (Poirrier’s blue and aniline blue are analogous to cotton blue) gives color to the structures.
Lactophenol Cotton Blue (LCB)
Fungi (spores and hyphae) are often gram positive but some are stain poorly.
Gram stain (Hucker Modification)
Differentiates Nocardia form other aerobic actinomycetes.
Modified acid-fast stain
Fungus are usually seen as small, oval yeast cells is often contained with macrophages, stains blue and has a hyaline halo that represents poorly staining cell wall.
Wright’s (Giemsa) stain
• For the detection of H. capsulatum in bone marrow and Buffy coat specimens.
Wright’s (Giemsa) stain
• Also used to visualize trophozoite of P. jirovecii
Wright’s (Giemsa) stain
• Hydrolyzes the cell with aldehydes, which are often then able to combine with the modified Schiff reagent (Basic Fuchsin) coloring the cell wall carbohydrates a bright pink-magenta.
Periodic Acid-Schiff (PAS)
• Demonstrates the double-contoured refractile walls of Blastomyces dermatitidis that may not be
Periodic Acid-Schiff (PAS)
• Fungi, including P. jirovecii, are gray to black; background is green. It also stains Nocardia and other actinomycetes.
• Silver nitrate precipitates on the fungal cell wall
Gomori Methenamine Silver (GMS)
• Its drawback is that it often stains fungi too densely to observe structural details.
Gomori Methenamine Silver (GMS)
• Generally the preferred method for tissue sections
Gomori Methenamine Silver (GMS)
• Chromic acid oxidizes adjacent hydroxyl groups of cell-wall polysaccharides to aldehydes.
Gridley stain
• Cell walls purple to magenta, yeasts rose to purple, and capsules deep purple. Background yellow
Gridley stain
• Enhances, visualization of fungi and their morphology. Natural color of cell walls masked. Tissue response cannot be studied.
Gridley stain
Specific method of detecting fungi in body fluids.
Fluorescent antibody stain (immunofluorescence, IF)
• Direct technique: fluorescein-labeled Ab reacts with fungal antigen in cell wall.
Fluorescent antibody stain (immunofluorescence, IF)
• 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 (immunofluorescence, IF)
• Some use the immunoperoxidase technique. Specific and highly sensitive. Can be used to detect and measure antibodies.
Fluorescent antibody stain (immunofluorescence, IF)
The mucopolysaccharide in the capsular material of the fungus stains deep rose to red, whereas the other tissue elements stains yellow.
Mayer Mucicarmine stain (others: Southgate’s, Alcian blue)
• Useful for differentiating C. neoformans (gattii) from other fungi of similar size and shape when found in samples of tissue.
Mayer Mucicarmine stain (others: Southgate’s, Alcian blue)
• B. dermatitidis and Rhinosporidium seeberi may also react positive.
Mayer Mucicarmine stain (others: Southgate’s, Alcian blue)
• Good for differentiation of dimorphic fungi and works well on sputum smears
Papanicolaou (PAP) stain
Isolation of saprophytic & pathogenic fungi from sterile sites. Bacteria may grow in the medium.
Brain Heart Infusion Agar (BHIA)
(inhibits saprophytic molds),
Cycloheximide
(inhibits bacteria).
Chloramphenicol
Best for isolation of pathogenic fungi exclusive of dermatophytes.
BHIA + antimicrobials
Recovery of fungi from blood or bone marrow
BHI Biphasic Blood culture bottles
[?] comprises both liquid and solid medium.
Biphasic— culture system
Isolation of dermatophytes from keratinized material.
Dermatophyte test
medium (DTM)
produce alkaline metabolites that changes the pH indicator from yellow to red.
Dermatophytes
inhibit saprophytic fungi and bacteria.
Antibiotics
SDA + chloramphenicol, cycloheximide, and 1% glucose for the Isolation of dermatophytes.
Mycosel (Mycobiotic)
Primary recovery of saprobic and pathogenic fungi; also dermatophytes
Sabouraud Dextrose Agar (SDA)
SDA is consist of pancreatic
digest of casein, peptic digest of animal tissue, & dextrose at 4% conc’n & buffered at a pH of 5.6
Contain chloramphenicol; Similar to caffeic acid agar.
Breakdown of the medium by the phenol oxidase resulting to the production of melanin
Birdseed (Niger) Agar
For the isolation of C. neoformans that appear as dark brown to black colonies
Birdseed (Niger) Agar
Tween 80, a surfactant, isspecifically incorporated in lieu of dextrose for the demonstration of pseudohyphae, chlamydospores, and arthrospore formation
Cornmeal agar with Tween 80
Use for the cultivation and differentiation of Candida spp. on the basis of morphological characteristics. Used in micro (slide) culture
Cornmeal agar with Tween 80
Conversion of mold phase of B. dermatitidis to yeast phase
Cottonseed agar
Sucrose is the sole carbon source, with NaNO3 serving as the sole nitrogen source
Czapek-Dox Agar
Use for the differentiation of Aspergillus spp.
Czapek-Dox Agar
Stimulate production of spores and sporulating bodies
Potato Dextrose Agar (PDA)
Demonstrates pigment production of Trichophyton rubrum. Used in slide (micro) culture
Potato Dextrose Agar (PDA)
Identification of Microsporum audouinii
Rice medium
Nutritional requirement tests for the differentiation of Trichophyton spp. Contains either Casein agar base of NH4NO3 agar + inositol, thiamine, or nicotinic acid
Trichophyton Agars
Confirmation of nitrate reduction by C. neoformans
Nitrate reduction medium
Detection of urease production by C. neoformans and for differentiation of T. mentagrophytes from T. rubrum
Urea Agar
Detection of carbohydrate (eg., dextrose, maltose, lactose, etc.) assimilation through utilization of carbon or nitrogen of yeast in the presence of oxygen
Yeast Assimilation Media (CTA assimilaiton Sugars)
Identification of yeast by fermentation reactions with various carbohydrates
Yeast Fermentation Broth
Large (7,500mm2)
Petri DIsh
Small (1,500mm2)
Test Tube
Good Oxygen supply
Petri DIsh
Poor Oxygen supply
Test Tube
Relatively fast Rate of drying
Petri DIsh
Relatively slow Rate of drying
Test Tube
Poor (lid is easily displaced) Security of closure
Petri DIsh
Good Security of closure
Test Tube
Relatively large Probability of dissemination
Petri DIsh
Relatively small Probability of dissemination
Test Tube
Relatively easy Detection of mixed culture
Petri DIsh
Relatively difficult Detection of mixed culture
Test Tube
OTHER MATERIALS and CONSIDERATIONS for FUNGAL CULTURE
- Teasing needle (inoculating needle)
- Incinerator
- Sand overplayed with disinfectant
- Incubation of culture
Incubation of culture:
• [?], mold forms, most common temperature for incubation.
• [?], opportunistic and dimorphic organisms
• [?], conversion to yeast phase for dimorphic fungi.
25-30ºC
30ºC
25-37ºC
< 5 days
Saprobes, opportunistic fungi, yeast
Rapid growers
6 - 10 days
Subcutaneous & opportunistic fungi, dermatophytes
Intermediate growers
> 11 days
Systemic & subcutaneous fungi
Slow growers
observed at the entire verse or reverse side of the colony.
Pigment production
have different pigment on each side, both colors should be recorded.
Dermatophytes
Most are [?], having olive- green to dark brow —black color.
dematiaceous
Some are hyaline (?) pastel or colorless colonies.
moniliaceous
Appears leather-like, or waxy little mycelium, seems to merge with the agar
Glabrous
Resembles plush or suede, short aerial hyphae of equal length
Velvety
Resembles colonies of other Staphylococcus spp. (formerly CoNS), “bacteria like” but more dry and dull (waxy-pasty). No aerial mycelium, with a delicate fringe around the colonies in BAP
Yeast like
Wooly or “Floccose”, large quantities of long aerial hyphae that becomes entangled and may fill the entire petri dish
Cottony
Powdery due to heavy conidiation or sporulation, has even hyphae and abundant conidia
Granular
Presence of radial groves from the center of the culture toward the rim
Rugose
Random folds (long, short, parallel at right angles or combination)
Folded
Have central depression (concavity) surrounded by raised edges. Resembles S. pneumoniae colonies
Crateriform
Have many warts or rough knobs on the surface
Verrucose
Brain-like convolutions
Cerebriform
- Tease mount
(i) Place [?] of the mounting fluid (eg., LCB) on the slide.
(ii) Gently remove a [?] from the agar and place it on the slide
(iii) Using two [?], tease the strands apart.
(iv) Place a [?] and examine under a microscope
- 1-2 drops
- portion of the colony
- teasing needles
- coverslip
- Scotch tape method
(i) Wrap a piece of [?] around a tongue blade, with the sticky side out.
(ii) Touch the colony with the [?]
(iii) Lay the tape, sticky tape down on a [?] on a slide.
- clear cellophane tape
- sticky tape.
- drop of mounting fluid
Slide culture
(i) Cut a [?] form an agar (eg., SDA, Cornmeal, PDA), then transfer on a sterile glass slide.
(ii) Inoculate all sides of the agar block, then cover with a cover slip. (?)
(iii) Place the set-up in a [?] (wet house) and incubate until growth appears.
(iv) Prepare a sterile glass slide with [?] of mounting fluid (LCB).
(v) Carefully remove the coverslip from the block by [?]
(vi) [?] the coverslip carefully onto the mounting fluid.
(vii) [?] and examine under a microscope.
- square block (1cm2)
- 22x22mm
- moist chamber
- 1-2 drops
- lightly twisting.
- Lower
- Set aside
Conidia and spores form a fresh culture are washed off in sterile water and placed in labelled vials
Storage in water
Labelling glass tubes with a screw-capped and placed in a freezer (-70ºC)
Freezing
Layering an entire slant with mineral oil, capping the tube tightly and storing at room temperature
Mineral oil overlays
Freeze drying with the use of special equipment (lyophilizer)
Lyophilization
Antibody detection may be useful particularly when they occur in immunocompetent individuals.
Immunologic Methods
Several immunological techniques have been used to detect antibodies during fungal infections viz.:
(i) Immunodiffusion (ID)
(ii) Countercurrent immunoelectrophoresis (CIE)
(iii) Enzyme-linked immunosorbent assay (ELISA)
(iv) Complement-fixation test (CFTs)
(v) Fluorescent-enzyme immunoassay (FEIA)
Ab detection is helpful in diagnosing allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and Chronic cavitary aspergillosis
Aspergillus spp.
Limitation: lack of sensitivity due to cross-reactivity with dimorphic fungi
Blastomyces spp.
Sensitivity 62%
Specificity 53%
Candida spp.
Ab detection can be a marker for reactivation of disease in solid organ transplant recipient
Cryptococcus spp
Aspergillus spp.
Assay:
Antibod(ies)/Antigen(s) detected:
- CIE, FEIA, EIA
- Aspergillus IgG Aspergillus IgG
Blastomyces spp.
Assay:
Antibod(ies)/Antigen(s) detected:
- Commercial kits
- Antigens (eg., A Ag, WI1 Ag)
Candida spp.
Assay:
Antibod(ies)/Antigen(s) detected:
- EIA
- Mannan Ag Candida IgG, IgM, & IgA
Cryptococcus spp.
Assay:
Antibod(ies)/Antigen(s) detected:
- EIA
- Cryptococcus IgG, IgM, IgA; Ag: Glucuronoxylmannan
• A polysaccharide present in the cell wall of many fungi viz., Aspergillus, Candida, Fusarium with little or none on the following viz., Cryptococcus, Blastomyces, Mucoraceous molds.
1,3-β-D-glucan detection
• Found in the blood of patients with invasive fungal infections, and its ability to activate factor G of the horse-shoe crab coagulation pathways that allows it to be measured and quantified
1,3-β-D-glucan detection
Molecular Methods
Advantages:
(i) Has the ability to detect organisms that are present in [?] of that cannot be cultured.
(ii) Removal of the risks assoc. with culturing category 3 (BSL 3 fungi)— especially for
(iii) Decrease time for identification of the agent, & the potential to detect [?] in primary clinical specimens.
(iv) Offers the potential to identify pathogens in biopsy samples that have been [?] and even [?]
- small numbers
- H. capsulatum
- anti fungal resistance
- formalin-fixed; wax-embedded.
Molecular Methods
Advantages:
(i) Has the ability to detect organisms that are present in [?] of that cannot be cultured.
(ii) Removal of the risks assoc. with culturing category 3 (BSL 3 fungi)— especially for
(iii) Decrease time for identification of the agent, & the potential to detect [?] in primary clinical specimens.
(iv) Offers the potential to identify pathogens in biopsy samples that have been [?] and even [?]
- small numbers
- H. capsulatum
- anti fungal resistance
- formalin-fixed; wax-embedded.
