Mycology Study Guide for Mid-Exam

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Mycology study guide for mid-exam

Differences between fungi, plant and bacteria
Different between yeast and molds
General features of fungi
Important terms (matching and MCQ)
Yeast-like fungi may be basidiomycetes, such as Cryptococcus neoformans or ascomycetes such as Candida albicans.
Moulds produce a great variety of conidia which are borne on specialized hyphae or conidiophores. Many moulds can be identified by the morphology of these spores and by their arrangement on the hyphae.
Fungal filaments are known as hyphae and a mass of hyphae collectively make up the mycelium. The terms "hyphae" and "mycelium" are used interchangeably.
Distinguish character for Zygomycetes, Basidiomycetes and Ascomycetes.
The main constituent of a fungal cell wall is chitin and not cellulose.
The fungal cell wall gives shape and form, protects against mechanical injury, prevents osmotic lysis, and provides passive protection against the ingress of potentially harmful macromolecules.
Difference between old part and newly growing fungi.
Sites of antifungal drug.
Periplasmic space in yeast.
NUCLEI: Nuclei are always present in living cells. The appearance is similar, though the amount of DNA is generally less, even allowing for the haploid state of most nuclei. Nuclei may move through the cytoplasm of most filamentous fungi, even through the dolipore septum of the Basidiomycota
Plasmids are also found in filamentous fungi, where some are associated with disease virulence
Tubules, Vesicles and Vacuoles
In older hyphae, the principle component of cellular compartments appears to be the vacuole. The vacuoles appear to have several functions. Recent evidence suggests that the vacuoles may have considerable activity and dynamism.
They may be involved with recycling, storage and transport. Vacuoles appear to store enzymes, other macromolecules, lipids, mineral nutrients such as polyphosphate, and toxins.
The control of pH and ion homeostasis in compartments appears to rest within the vacuole. Thus vacuoles play a critical and complex role in fungi.

•Filamentous forms always have an inclusion called the Woronin body that is associated with the simple pore of the septum. The contents are probably protein, surrounded by a membrane. While their function is unclear, Woronin bodies appear to block pores of the septum following damage to compartments.

some vesicles called lomasomes are located between the plasma membrane and the wall. The function of lomasomes is unclear. Because they are always located in zones of active hyphal elongation, a role in polymer synthesis and deposition has been suggested.
Each fungus can be found in a variety of nuclear states. That is, the number of nuclei (ploidy) and the degree of duplication (haploid/diploid) can vary throughout the life cycle of the fungus. Though most commonly haploid, each fungus may have significant periods in each part of the life cycle.
A haploid organism contains a single set of chromosomes in each of its nuclei.
Following plasmogamy (fusion of cytoplasm) in filamentous fungi, the number of nuclei doubles, though these need not necessarily go through karyogamy (fusion of the nuclei) immediately.
The state where two genetically distinct haploid nuclei coexist in one cell is called a dikaryon (adj. dikaryotic). When the nuclei fuse in karyogamy, the nucleus becomes diploid. In this cycle, fungi are similar to most organisms. However, diploid fungi almost always then pass immediately into meiosis, resulting in the formation of spores with a single haploid nucleus.
fungi with secondary homothallism (the fungus can mate with itself in the absence of a compatible mating type). The variation from common behavior is possible because nuclei may move from compartment to compartment, and the expression of mating type need not necessarily be found at one locus.
The filamentous fungi (molds) are so called because they form filamentous structures called hyphae (singular: hypha).
When hyphae grow together, they form a structure called a mycelium (plural: mycelia), which is generally white and can be detected by the naked eye.
As the mold matures, it develops spores (conidia) that are formed on the aerial branches of the hyphae. Conidia are asexual spores that are somewhat resistant to drying; their main function is to spread the fungus to new habitats, a fact that makes these types of organism’s common contaminants on most surfaces.
Conidia are also highly pigmented and create the spectrum of colors so typical of molds: black, blue-green, red, yellow, and brown.
Some molds also produce sexual spores that are more resistant to drying, heat, freezing, and even to some chemicals. Unlike bacterial spores, fungal spores are part of the normal life cycle of fungi, and, therefore, they are less resistant to chemicals and adverse environmental conditions
Yeasts are unicellular fungi typically classified with the Ascomycetes.
Yeast cells are generally round, oval, or cylindrical; under certain conditions,
yeasts can form filaments. One example of this growth pattern is seen with Candida albicans, which expresses pathogenicity only in the filamentous form.
Yeast colonies may appear similar to bacterial colonies on solid medium. However, under microscopic examination, yeast cells and can be easily distinguished from bacterial are much larger cells.
Yeast cells reproduce by budding or binary fission. During budding, a small outgrowth (daughter cell) is formed, and it grows until it separates from the mother cell to become a Some yeasts also undergo sexual separate cell. reproduction by a process called mating
Sexual reproduction occurs by the fusion of two haploid nuclei (karyogamy), followed by meiotic division of the diploid nucleus. The union of two hyphal protoplasts (plasmogamy) may be followed immediately by karyogamy, or it may be separated in time
This process involves release of specific proteolytic, glycolytic, or lipolytic enzymes from the hypha or yeast, extracellular breakdown of the substrate(s), and diffusion of the products of digestion through the fungal cell envelope. Fungal pathogens rely on these digestive enzymes to penetrate natural host barriers.
How bacteria-like fungi differ from fungi?
Factors that affect the growth of molds
Growth phases
On agar, initial hyphal growth results in undifferentiated mycelium. However, over time, this circular colony becomes differentiated because of differences in available food. The hyphae at the margin are exposed to abundant fresh organic nutrients, and the hyphae at the center to much less.
four zones can be differentiated, though note that they intergrade with one another. What are they?
As a consequence, the mycelium will become a complex of interlinked groupings of mycelia, with interlinking sections resulting in complex movements of cytoplasm around an ever-changing body. Hyphal anastomosis is an odd phenomenon given that negative autotrophic responses are commonly seen on agar. Hyphae normally avoid or do not react to adjacent hyphae. Yet, anastomosis is a regulated response to the presence of a vegetatively compatible hypha.
Hyphae grow above and below the surface of media. Penetration into media is a major differentiation between the fungi and other saprotrophic microbes.
Environmental Conditions
The balance between total carbon and nitrogen uptake is critical for fungal growth and development. Consideration of the carbon/nitrogen ratio is widespread in industry where specific physiological conditions need to be maintained for maximum rates of production. Relatively low nitrogen content will slow the rate of degradation, because of the demand for protein. Conversely, if the organic matter has a high protein content, then the protein may be used as a source of N and organic carbon. Once C and N requirements are met, the rate of growth is determined by availability of other minerals.
The hydrogen environment of fungi is difficult to study because fungi change the pH as they grow. Some species increase and others decrease pH of their medium.
pH of the medium is important for it influences mineral availability, enzyme activity and membrane function. Generally speaking, fungi can tolerate a wide range of pH, though most media used to culture fungi are acidic

•49. SporulationThe environment plays a major role in determining whether a fungus forms sexual or asexual spores. Spores are commonly formed as a fungus depletes its energy sources. A variety of environmental triggers may be involved in determining which type of spore is formed.

Taxonomy is defined as: the scientific classification and nomenclature of organisms
Fungi are classified based on their life cycle patterns, and over the years taxonomy of fungal isolates has been much of a challenge
Nomenclature of filamentous fungi is part of a classification system based on the organism’s appearance, especially their sexual stages, rather than on biochemical reactions and nutritional requirements that are key to classification of bacteria
Teliomorph and anamorph
the Deuteromycotina ( Imperfect Fungi)to accommodate of those fungi without a perfect state, but which may represent the asexual states (anamorphs) of either Basidiomycotina or Ascomycotina
Fungi that seldom or never reproduce sexually.

Asexual reproduction by vegetative growth and production of asexual spores common

Character of Zygomycetes
Epidemiology of Mycoses
Factors increase mycosis
Fungemia and independent risk factors
Modern molecular and classical epidemiology?

• RFLP (restriction fragment length polymorphisms) analysis

Electrophoretic karyotyping

Polymerase chain reaction-based methods of DNA fingerprinting

Protein-based methods include

Immunoblot fingerprinting

• Polyacrylamide gel electrophoresis

• Multilocus enzyme electrophoresis

Key question asked by epidemiology - are two or more isolates associated with an outbreak “the same” or “different”?
Endogenous and exogenous sources
Laboratory diagnosis:
• “Gold standard” is the recovery of the etiological agent from the clinical specimen
• Current methods include a mixture of the traditional, commercially-available kits, and new molecular approaches

65.Specimen selection

• Proper anatomical site

• Use of suitable container for transport

– Specimen collection

• Choose an active lesion

• Use aseptic technique

• Obtain an appropriate quantity

• Use sterile, appropriately labeled containers

Skin scrapping collection
CSF
• Examine microscopically as soon as possible
• Transport immediately using pertinent transport media if required
• Do not freeze specimens
• Do not refrigerate if not likely to contain contaminating microbes
• Do not desiccate
Microscopy:-

Microscopy is used to examine the clinical specimens for the presence of fungal elements, direct examination can be stained or unstained.

20% KOH mount (unstained):

Several specimens are subjected to KOH mount for direct examination.
The material is mixed with 20% KOH on a slide and a cover slip is placed, the slide is then gently heated by passing through the flame 2-3 times.
KOH serves to digest the protein debris and clears keratinized tissue to increases the visibility.
Addition of Dimethyl sulphoxide (DMSO) permits rapid clearing in the absence of heat.

Stain specimens:Calcofluor white:

This is a fluorescent dye, which binds selectively to chitin of the fungal cell wall.
The specimen then can be observed under fluorescent microscope.

C. India Ink:

This is negative staining technique, stain the back ground of the smear
Use for demonstration of Capsules
Example the Cryptococcus neoformans capsule

D. Periodic Acid-Schiff (PAS) stain:

On staining by this stain, fungal elements appear bright magenta color, while the background stains green.
It is useful in staining tissue specimens.

. Giemsa’s stain:

It is particularly useful in the detection of Histoplamsa capsulatum in the bone marrow smears.

F. Haematoxylin and Eosin (H&E) stain:

Useful for staining tissue sections
Candida and Aspergillus may be missed in H&E stained

G. Gram stain:

Candida is best demonstrated in clinical specimen by Gram stain, it is gram positive cell.

Direct microscopy is diagnostic in some fungal infection mainly Dermatophytes

Culture:

One of the most common media used to culture fungi in laboratory is Sabouraud’s Dextrose Agar (SDA).

It consists of peptone, dextrose and agar.

High concentration of sugar and a low pH (4.5-5.5) prevents growth of most bacteria and makes it selective for fungi.

Addition of antibiotics such as Chloramphenicol, or mixture of penicillin &Streptomycin to SDA serves to inhibit bacterial multiplication.

The saprophytic fungi can be inhibited by the addition of cycloheximide (actidione) to the SDA

An example of SDA with cycloheximide and Chloramphenicol is Mycosel agar.

Other basic media fungi growth include Potato Dextrose Agar, Malt Extract Agar etc…
For the fastidious fungi we use Brain Heart Infusion

Incubation methods:-

Most fungi are able to grow at room temperature 22oC, while few pathogenic fungi (e.g, Cryptococcus, dimorphic fungi) can grow at 37oC, So we need duplicate plate
Saprophytic fungi grow much quickly than pathogenic fungi
Since some fungi grow slowly cultures should not be discarded for 4-6 weeks& incubated aerobically.

Colonial morphology:-

Fungi are identified on the basis of colony morphology, and pigmentation(color of pigment, and if diffused or localized).

Needle mount:-
The first rule to remember in mould studies is to examine young, actively growing material. Older parts of colonies on natural material will often be partially decomposed or so covered with spores as to be unrecognizable. The best way to begin is to examine the growth from the margin of the colony.
The lactophenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare.

The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls.

under the microscope examine hyphae, conidia, spore or any other structure

6)Serology: -

Detection of anti-fungal antibody is helpful in diagnosis of sub-cutaneous and systemic mycoses& for the prognosis and response to anti-fungal drugs.
Different serologic techniques that are used include agglutination, immunodiffusion, counter-immunoelectrophoresis, complement fixation test, immuno fluorescence, RIA and ELISA.

7)Molecular techniques:-

Newer techniques such as DNA hybridization, PCR are useful in diagnosis of mycoses in a shorter period as well as detect those fungi that are difficult or dangerous to cultivate in vitro.

Diagnosis of fungal infection depend upon:
Histological stain ( application and limitions)