PRELIM: INTRODUCTION TO MYCOLOGY Flashcards
Mykos
fungus
Logus
study
Study of fungi
Mycology
Branch of biology concerned with the study of fungi, including their biochemical, physical and microscopic properties, their use to humans and as well as the human diseases they can cause, such as toxin production or infection
Mycology
is the study of fungi that produce pathology or disease in humans and animals including their ecology and epidemiology
MEDICAL MYCOLOGY
is the study of the general characteristics, pathology & pathogenesis, and laboratory tests & methods of medically important fungi that aids in their identification and treatment
DIAGNOSTIC MYCOLOGY
Fungi – exist in two forms:
- Molds
- Yeasts
Fungi can exist as in mold form only or yeast
form only or having both forms. T OR F
T
Eukaryotic
Fungi
With membrane-bound nucleus, organelles, 60s-40s ribosomal subunits, etc.
Fungi
Fungi
Contains chitin in their cell walls:
- Peptidoglycan for bacteria
- Cellulose for plants
Contains chitin in their cell walls
Fungi
Can be single-celled (yeast) or multicellular (mold )
Fungi
Can reproduce sexually or asexually (produce spores)
Fungi
the characteristic of fungi that differ it from plants (autotrophs); heterotrophs depend on other organisms as their source of nutrients.
Heterotrophs
HETEROTROPHIC ORGANISM CAN BE:
- Saprophytic
- Symbiotic
- Parasitic
feeds on dead organic material (ex: mushrooms growing on dead trees)
Saprophytic
fungi living in another organism and causes harm to the latter.
Parasitic
Fungi that are responsible for breaking down and recycling dead plant and animal materials
SAPHROPHYTIC FUNGI
fungi is living together with other organisms and establishes mutualism; will not particularly cause disease or any harm
Symbiotic
Example: Mushroom growing on dead trees/animal bodies
SAPHROPHYTIC FUNGI
SYMBIOTIC FUNGI:
MYCORRHIZA
Mycorrhizae fungi germinates in soil.
SYMBIOTIC FUNGI: MYCORRHIZA
Its hyphae penetrate the roots of the plant.
SYMBIOTIC FUNGI: MYCORRHIZA
The plant roots provide essential nutrients for the growth of the fungi.
SYMBIOTIC FUNGI: MYCORRHIZA
In return, the large mass of fungal hyphae acts as a virtual root system for the plants,
ncreasing the amount of water and nutrients that the plant may obtain from the surrounding soil.
SYMBIOTIC FUNGI: MYCORRHIZA
- depends on environment
- can be yeast or mold
DIMORPHIC FUNGI
*
only ____ spp. generally recognized as causes of disease in humans
100-150 spp.
accidental hosts by spores inhalation or introduction via trauma
humans
healthy humans are relatively resistant to infection of fungi except for dimorphic fungi. T OR F
T
no cellulose (has hypae/roots)
fungi
has cellulose
plants
*
Fungi that attack living organisms,penetrate their outer defenses, invade them. And obtain nourishment from living cytoplasm, thereby
causing disease and so metimes death of the host.
PARASITIC FUNGI
________infiltrating their bodies and minds Cordyceps spore can manipulate the Bullet ant then erupts from the ant’s head
Bullet Ants with spores from (parasitic fungus)
*
2 FORMS OF FUNGi:
- yeast
- mold
UNICELLULAR Grow at body temperature (37 C)
YEAST
Moist, creamy, opaque, pasty colonies
YEAST
MULTICELLULAR Grow best at room temperature (22-25 C)
MOLD
Fluffy, cotonny, woolly, powdery
colonies
MOLD
Unicellular
YEAST
Oval, Spherical
YEAST
Oval, Spherical
YEAST
Parasitic
YEAST
Facultative anaerobe
YEAST
Multicellular
MOLD
Filamentous
MOLD
Room Temp
MOLD
Saphrophytic
MOLD
Aerobic
MOLD
multicellular with tubualr, filamentous hyphae (branches)
MOLD
any organic environment having a moist/humid atmosphere and not exposed to harsh weather conditions
MOLD
production of sexual or axesual, airborne spores
MOLD
threadlike, come in a wide variety of colors and hues
MOLD
mostly unicellular and existing individually or with buds growing on them
YEAST
mostly occur naturally in oceans
YEAST
budding or binary fission
YEAST
round or oval-shaped, dull colored and mostly monochromatic
YEAST
Unicellular circular, spherical or oval-shaped forms of fungi
YEAST
*
Colonies are moist, pasty, creamy and opaque
YEAST
Yeasts are less colorful compared to molds, some yeast colonies can be pigmented
YEAST
May be mistaken as bacterial colonies
YEAST
Yeast colonies should be biochemically
differentiated from bacterial colonies
YEAST
YEAST
Optimum temperature:
near body temperature
(37 C)
Parasitic
YEAST
form of fungi that can develop
disease in human
Parasitic
Appears to be “multicellular” but just undergoing process of Budding.
BUDDING
Most of the clinical specimen processed in
the laboratory are in YEAST form. T OR F
T
Multicellular
MOLDS
Mold Colonies appearing to be Filamentous or Cotton
MOLDS
Branching form
MOLDS
“Hyphae”
Branches
MOLDS
Optimum temperature for growth:
Room temperature (35- 37 C)
Saprophytic
MOLDS
called such because they can survive outside the human body; can rely on dead plants/animals as source of nutrients
Saprophytic
Ability that fungi can exist in the form of both mold and yeast.
DIMORPHISM
This is usually brought about by change in temperature
DIMORPHISM
EXAMPLE OF DIMORPHISM
Histoplasma
Blastomyces
Coccidioides (not thermally dimorphic)
Paracoccidiodes
Sporothrix (Subcutaneous)
Penicillium
SPOROTHRIX SCHENCKI
Acquired via: accidental pricking of the skin by the rose thorn
SPOROTHRIX SCHENCKI
Skin nodule ( subcutaneous tissue) bipsy will demonstrate yeast cell
SPOROTHRIX SCHENCKI
optimum temperature of growth for saprophytes (mold) is ____ and _______ for parasite fungi (yeast)
20-30 C; 30-37 C
The standard temperature for incubation of fungi is ____ and cultures should be incubated in a humidified environment for ____. While Bacterial cultures require incubation temperature of _____
30ºC
21 days
37 degree Celsius
grow best at pH of 4-6; average of 5.0
Fungi
are more resistant to osmotic pressure than bacteria
Fungi
requires less nitrogen and moisture than bacteria
Fungi
can be aerobic or anaerobic
Fungi
are capable of metabolizing complex carbohydrates such as lignin in wood.
Fungi
*
what percent is lignin in woods?
15-25%
are aerobic
Molds
are facultative anaerobes
Yeasts
*
HARMFUL EFFECTS OF FUNGI
- ALLERGY
- MYCOSES
- MYCOTOXICOSES
Spores are potent triggers of allergic reactions
ALLERGY
Mold spores get into your nose and can cause allergy symptoms.
ALLERGY
Mold form of a fungi with asexual spores at the tip released on the air new fungi.
ALLERGY
- Some spores of molds can trigger allergic reactions (Allergic rhinitis or asthma attacks)
ALLERGY
a direct fungal infection of animals, including humans
MYCOSES
Fungi causing infection in any parts of the body.
MYCOSES
Fungi directing one specific tissue, location or organ in the body.
MYCOSES
Disease cause by the natural toxin produced by fungi
MYCOTOXICOSES
*
No presence of fungi in the affected tissue or organ. What causes the disease is the toxin
produced by the fungi.
MYCOTOXICOSES
EXAMPLE OF MYCOTOXICOSES
Aspergillus flavus
can produce aflatoxin that can cause hepatocellular carcinoma (seen in both children and adults)
Aspergillus flavus
Aflatoxin, once ingested in large amount, causes in children:
o Neurological problem
o Stunted growth
*
BENEFICAIL EFFECTS OF FUNGI
- PREPARATION OF BREAD
- ALCOHOLIC FERMENTATION
- PREPARATION OF VACCINE
- SOURCES OF DRUGS
- FOOD
- RECYCLING OF ORGANISC MATTER
- MAINTAIN ECOLOGICAL BALANCE
Yeast cells are used in preparation of bread
PREPARATION OF BREAD
Bread = sugar + gluten + yeast cells + water
PREPARATION OF BREAD
*
Gluten forms the framework of bread
PREPARATION OF BREAD
Yeast converts the sugar into carbon dioxide
PREPARATION OF BREAD
Yeast cells are used in induce alcoholic fermentation in grape juice/ barley grain to produce wine and beer; respectively
ALCOHOLIC FERMENTATION
“Say YEAST to beer and wine”
very true
Human hepatitis B virus vaccine is prepared using antigen produced by recombinant technology in yeast (Saccharomyces)
PREPARATION OF VACCINE
Get the DNA that encodes for the surface antigen incorporate the viral DNA to the yeast cells Yeast cell will now have the capacity to produce the surface antigen. Yeast cell produce ßßby budding Extract as Vaccine
RECOMBINANT TECHNOLOGY
Penicillin sources:
Penicillium notatum
*
used against gram+ bacteria. clinically it aginst pneumonia, rheumatic fever, tonsillites, tetanus, diptheria and many other diseases
penicillin
griseofulvin sources:
Penicillium griseofulvum
used against mycosis
griseofulvin
cephalosporin sources:
Cephalosporium acremonium (a marine fungus)
used against gram+ and gram- bacteria; typhoid
cephalosporin
who discoverd Penicillin?
Alexander Flemming
Higher fungi may be eaten directly as mushrooms
FOOD
Saprophytic fungi in soil produce degradative
enzymes essential for the biologic recycling of organic matter
RECYCLING OF ORGANISC MATTER
Parasitic fungi can also help regulate the number of other species to maintain ecological balance
MAINTAIN ECOLOGICAL BALANCE
Primarily a saphrophytic fungi but if the environment where it is living is low in
NITROGEN = transform into parasitic fungi
Arthrobotrys conoides
using hyphae to form loops that are motion-sensitive. Whenever a nematode passes thru the loops will constrict trapping the nematode and then produce enzymes that will eventually invade the surface of the nematode and kill and digest it.
Arthrobotrys conoides
Unicellular form of fungi
YEAST
Spherical or elliptical in shape
YEAST
YEAST Reproduces by:
- Budding – most common
- Binary fission
- Formation of pseudohyphae
Parent yeast cell dividing equally.
BINARY FISSION
-Daughter cell (Blastosphore or Blastoconidia) is smaller than parent yeast cell.
BUDDING
-Most common way of reproduction among yeast cell.
BUDDING
Asymmetric division of yeast cells
BUDDING
A new yeast cell (blastospore) is formed through mitotic cell division and remains attached as a bud on the old cell until it splits and becomes independent.
BUDDING
*
A yeast cell duplicates its genetic material, or deoxyribonucleic acid (DNA), and then divides into two parts (cytokinesis), with each new organism receiving one copy of DNA.
BINARY FISSION
“ FALSE HYPHAE “
PSEUDOHYPAHE
Buds fail to detach producing elongated yeast cells
PSEUDOHYPAHE
- found yeast
- always arise from a constricted area
pseudohyphae
- root-like structures found in molds
- elongated structure arising form a non-constricted area
hyphae
Consist of branching cylindrical tubules with diameter called hyphae
MOLD
Hyphae grow to form a filamentous mass of
intertwining strands called a mycelium
MOLD
MOLD COLONIES 2 PORTIONS:
- reproductive/aerial hyphae
- vegetative portion
grow on the top of the agar’s surface of the culture media
reproductive/aerial hyphae
contains the reproductive structures such as spores
reproductive/aerial hyphae
portion of the mycelium that anchors the mold and absorbs nutrients
vegetative portion
grows in or on a substrate and absorbs water
vegetative portion
another name of vegetative portion
thallus
1.
can be septate or nonseptate.
HYPHAE
have dividers between the cells, called septa (singular septum).
SEPTATE
*
The septa have openings called pores between the cells, to allow the flow of cytoplasm and nutrients throughout the mycelium.
SEPTATE
Its septate and not septated.
SEPTATE
Nonseptate or Coenocytic hyphae
NONSEPTATE
- lack septum and cell membranes between the cells.
NONSEPTATE
Septa can be seen in branching points
NONSEPTATE
NONSEPTATE EXAMPLE
Zygomycetes:
Mucor, Rhizopus, Absidia
Nonseptated Hyphae:
Mucor, Rhizopus, Absidia
no rhizoids
Mucor
Nodal rhizoids
Rhizopus
Internodal
Absidia
root-like structures attached to the
nonseptated hyphae.
Rhizoids
Long stalk arising from nonseptated hyphae
Sporangiophore
dilated part of sporangiophore; serve as source of nutrients of the spores
Columella
sack that contains the spores
Sporangium
spores produced within a sack
Sporangiospore
Hyaline if fungal structures are colorless
HYPHAE
Highly refractile
HYPHAE
are group of fungi that produce melanin in their cell walls, giving them a characteristic brown colour when grown on agar
Dematiaceous
Agents causing Chromoblastomycosis
DEMATIACEOUS FUNGI
Group of fungi with dark colonies and pigmented fungal elements
DEMATIACEOUS FUNGI
DEMATIACEOUS FUNGI
Curvularia
Curvularia
Alternaria
Phialophora
Exophiala
*
An extracellular layer which lies outside the cell wall and it is usually composed of polysaccharides.
CAPSULE
It protects the cell from different environmental dangers such as phagocytosis, desiccation and harmful chemicals
CAPSULE
Capsule of ________ has antiphagocytic properties and is associated with virulence.
Cryptococcus neoformans
Cryptococcus neoformans can cause ________ among immunocompromised patients
Fungal meningitis
Boundary of the cell (innermost outermost layer):
cell membrane > cell wall > Capsule
Most common cause of Fungal meningitis:
Cryptococcus neoformans
Abundant in: eucalyptus tree and pigeon droppings
Cryptococcus neoformans
Immunocompromised patient not be able to eliminate the spores
Cryptococcus neoformans
Used to identify the capsule of Cryptococcus neoformans
INDIA INK STAINING / INDIA INK WET MOUNT
is directly examined by adding one drop of India ink.
CSF
Capsule appear as a _______ against a dark background (negative staining). in india ink
clear halo
CSF centrifuged to let capsule to form sediment to be stained:
sediment
to stain everything in the slide except the capsule of Cryptococcus neoformans (+:white halo against black background)
India ink
White Capsule against a black background
Negative Staining
Used for: isolation of Cryptococcus neoformans (brown or dark brown color)
BIRD SEED AGAR
Candida albicans color:
colorless
The cell wall is a characteristic structure of fungi and is composed mainly of glucans, chitin and mannan.
CELL WALL
As the components of the fungal cell wall are not present in humans, this structure is an excellent target for antifungal therapy
CELL WALL
CELL WALL COMPOSED OF:
- Chitin
- Glucans
- Mannan
forms the cross-linking
Glucans
topmost
Mannan
MOLDS FUNGAL CELL WALL MAJOR COMPONENT:
Chitin
(polymers of N- acetylglucosamine)
YEAST FUNGAL CELL WALL MAJOR COMPONENT
Glucan (polymers of glucose)
Human Cell Membrane: main sterol is
Cholesterol
provides stability and flexibility to cell membrane
Fungi: Ergosterol
*
Bilayered membrane composed of several phospholipids
CELL MEMBRANE
Contain sterols which are essential for the viability of fungi
CELL MEMBRANE
Nuclei, mitochondria, ER, storage vacuoles containing hydrolytic enzymes, ions, metabolites such as amino acids
CELL MEMBRANE
Principal fungal sterol is
ergosterol
fungal cell wall is poorly stained with routine hematoxylin and eosin. T OR F?
T
LABORATORY IDENTIFICATION OF FUNGI
Microscopic Methods (recommended)
Cultivation (recommended)
Biochemical tests
Serologic Test
quick & simple method to visualize budding yeasts, hyphae, & pseudohyphae. Lack of contrast is a major disadvantage making it difficult to appreciate fungal elements microscopically.
Saline Mount
MICROSCOPIC METHODS WET MOUNT:
- saline mount
- potassium hydroxide (KOH) mount
- calcoflour white stain
- lactophenol cotton blue (LPCB)
- giemsa or wright stain
- periodic acid-schiff (PAS)
- methenamine-silver nitrate stain
rapid & simple technique
Potassium Hydroxide (KOH) Mount
KOH acts as clearing agent:
o Dissolves keratin
o Eliminates debris o Dissolves fat droplets (may appear as yeast cells)
is used for nails (highly keratinized tissue)
20% KOH
*
Although ca also result to poor contract like saline mount. Used to visualize budding yeasts, hyphae, & spherules.
Potassium Hydroxide (KOH) Mount
Can also be used to examine hair to determine whether hair is infected within the shaft (ENDOTHRIX INFECTION) or hair is infected outside the hair shaft (ECTOTHRIX INFECTION)
Potassium Hydroxide (KOH) Mount
used as a brightening agent since calcoflour white BINDS to CHITIN in the fungal cell wall which provides excellent contrast over a dark background when visualized using a
fluorescent microscope.
Calcoflour White Stain
used to vIsualize fungal structure by staining chitin in the fungal cell wall color BLUE.
Lactophenol Cotton Blue (LPCB)
Also useful in staining tease preparation (wet mount) or slide cultures
Lactophenol Cotton Blue (LPCB)
*
kills other organisms (bacteria)
Phenol
preserves fungal elements
Lactic Acid
stains the chitin in the fungal cell wall blue.
Cotton Blue
Advantages:
- Structures are readily seen microscopically
- Has GLYCERINE component – slows down The drying of the LPCB
Lactophenol Cotton Blue (LPCB)
used to visualize intracellular Histoplasma
capsulatum in blood smears, lymph nodes, lung, liver, or bone marrow. The organism appears as small, oval yeast cell staining light to dark blue.
Giemsa or Wright Stain
also stains well using this method (GIEMSA STAIN) result to “ SUNBURST APPEARANCE”
Cryptococcus neoformans
Giemsa or Wright Stain:
demonstrates budding while bacteria equal circle
Candida albicans
used to stain the hypha of moulds and some yeasts. Periodic Acid oxidizes the OHin the cell wall CHO to form aldehydes which reacts with the basic fuchsin dye to form a pink-purple complex. A counterstain (fast green) can be used to provide Contrast
Periodic Acid-Schiff (PAS)
useful for screening of clinical specimens for presence of fungal elements which stains black. Fungi appear outlined in black against a pale-black ground.
Methenamine-Silver Nitrate Stain
is a modification of this method used for histological examination of specimens
GOMORI METHENAMINE-SILVER NITRATE STAIN
● Wood’s lamp (UV light)
● Emits wavelength 320–450 nm (peak 365 nm)
● The light is held over an area of skin in
darkened room.
● Infected hair and skin will fluoresce when examined in the dark
FLUORESCENCE TEST
Culture media must include sources of:
- Nitrogen
- Nitrate
- Amino acids
- Carbon
- Vitamins and minerals
Has a low pH which can inhibit growth of bacteria
SABOURAUD DEXTROSE AGAR
PRIMARY ISOLATION MEDIA:
- sabouraud dextrose agar
- brain heart infusion media
- mycosel - SDA-CC
Used for isolation of most fungi
SABOURAUD DEXTROSE AGAR
*
Disadvantages
~ May not allow yeast phase of the fungi to grow
~ Too many fungi (including saprophytes) can grow on the agar
~ Histopasma capsulatum fails to grow in this medium
SABOURAUD DEXTROSE AGAR
Recommended for the culture of yeast phase of dimorphic fungi at 35-37 degree Celsius
BRAIN HEART INFUSION MEDIA
Made from pig heart and calf brain
BRAIN HEART INFUSION MEDIA
Antimicrobial supplements
MYCOSEL - SDA-CC
inhibit contaminating saprophytic fungi
Cycloheximide
inhibit gram bacterial growth
Chloramphenicol
Chloramphenicol can be substituted with Gentamicin or Tetracycline
MYCOSEL - SDA-CC
DIFFERENTIAL/ SPECIAL ISOLATION MEDIA:
- potato dextrose agar (PDA)
- corn meal tween 80 agar (CMT 80)
- dermatophyte test medium (DTM)
- christensen urea slant
used as a subculture medium rather than a primary isolation medium. This medium enhances the sporulation & pigmentation of fungi
Potato Dextrose Agar (PDA)
used for the demonstration of blastoconidia, pseudohyphae, arthroconidia, and chlamydospores in Candida spp. and some other yeasts
Corn Meal Tween 80 Agar (CMT 80)
used for the selective growth of dermatophytes. This medium will turn from yellow to red within 14 days incubation at room temperature if dermatphytes are growing. It should be checked once every week
Dermatophyte Test Medium (DTM)
used to detect urease production by turning the slant to pinkish purple color after 48 hours
incubation.
Christensen UREA Slant
Christensen UREA Slant
UREASE POSITIVE (+):
Trichosporon, Rhodotorula, & Cryptococcus
Christensen UREA Slant
UREASE NRGATIVE (-):
Geotrichum, Saccharomyces, & most Candida spp
IMPORTANT BIOCHEMICAL TEST FOR THE IDENTIFICATION OF YEAST & YEAST LIKE ORGANISM
- Carbohydrate (CHO) Fermentation
- Carbohydrate (CHO) Assimilation
- Nitrogen Assimilation
- Germ Tube Test (Reynold’s Brande Phenomena)
Principle: growth and utilization of CHO
under anaerobic conditions as determined by
acid & gas production
Carbohydrate (CHO) Fermentation
Carbohydrate (CHO) Fermentation
Indicator:
Bromcresol Purple (BCP)
Carbohydrate (CHO) Fermentation
Positive Reaction:
Yellow Color for Acid production and bubbles trapped in the fermentation tube for gas production
NOTE: Observe every 48 hours for 14 days
Carbohydrate (CHO) Fermentation
Principle: the yeast ‘s ability to utilize a particular CHO is determined by using a CHO-free (nitrogen-based) agar and filter paper disks that are impregnated with various CHO. Growth around the disk indicates the yeast can utilize the CHO
Carbohydrate (CHO) Assimilation
Carbohydrate (CHO) Assimilation
Positive Reaction:
growth around the disk indicates the CHO has been assimilated by the yeast
NOTE: Incubate plate for 24 hours at 300C. Plates should be re-incubated for another 24 hours and read again if the growth is insufficient.
Carbohydrate (CHO) Assimilation
Principle: Nitrate assimilation is defined simple as the utilization of a nitrogen source by a microorganism in the presence of oxygen. A positive reaction is indicated by the presence of growth or the use of a pH indicator in medium. The indicator method is a modification of the Wickerham method that was devised by Adams and Cooper
Nitrogen Assimilation
Nitrogen Assimilation
Indicator:
Bromthymol Blue (BTB)
Nitrogen Assimilation
Positive Reaction:
Green to Blue Slant due to alkaline pH when nitrate is assimilated.
NOTE: Incubate aerobically with loosen caps at 35-370C for 24-72 hours.
Nitrogen Assimilation
a screening procedure which is used for the identification and differentiation of Candida albicans from other yeasts.
Germ Tube Test (Reynold’s Brande Phenomena)
Principle: This test allows the detection of the Germ Tubes which are the initial stage of hyphae formation. These are the short, non septate germinating hyphae which are one half the width and three to four times the length of the yeast from which they originate. Approximately 95-97% of Candida albicans isolate develop germ tube when incubated in a proteinaceous media at 35C for 2.5-3 hour
Germ Tube Test (Reynold’s Brande Phenomena)
