Lecture (2) mycology, Dr. kareema Amine Alkhafaji, Assistant professor.
Ascomycota and some mitosporic fungi:
Hyphae of fungi belonging to these groups (and the Basidiomycota) possess perforated septa at regular intervals along their length.
The septum consists of a simple plate with a relatively LARGE CENTRAL PORE (50-500 nm diameter) - this allows cytoplasmic streaming (the movement of organelles, incl. nuclei) between adjacent hyphal compartments. Cytoplasmic streaming enables sub-apical and intercalary (central) compartments of young hyphae to contribute towards growth of the hyphal tip - transporting nutrients and essential enzymes to the apex - so maximizing the capacity for somatic growth.
Associated with each septum are spherical, membrane-bound organelles called WORONIN BODIES that are composed of protein; remain close to the septal pore and tend not to be disturbed by the cytoplasmic streaming taking place; tend to be of the same or larger diameter than the septal pore and are, therefore, capable of blocking the pore; will block the septal pore if the adjacent hyphal compartment is damaged or ageing and becoming highly vacuolated.
Some other mitosporic fungi:
A number of mitosporic fungi possess septa with a single central pore, similar to that observed in the Ascomycota.
But other mitosporic fungi may possess MULTIPERFORATE SEPTA.
E.g. the septa of Geotrichum candidum (illustrated above) possess characteristic MICROPORES (approx. 9 nm diameter).
The number of pores in each septum can vary up to a maximum of approx. 50.
These micropores allow cytoplasmic continuity between adjacent hyphal compartments, but are too small to allow cytoplasmic streaming to occur to the extent observed in fungi possessing larger septal pores.
Basidiomycota:
The most complex type of septum is found in fungi belonging to the Basidiomycota.Each septum is characterized by a swelling around the central pore (DOLIPORE) and a hemispherical perforated cap (PARENTHOSOME) on either side of the pore - illustrated above. The perforated parenthosome allows cytoplasmic continuity but prevents the movement of major organelles. The plasma membrane lines both sides of the septum and the dolipore swelling, but the membrane of the parenthosome is derived from endoplasmic reticulum.
Functions of septa:1-Act as STRUCTURAL SUPPORTS: The addition of plate-like cross-walls to what is essentially a long tube-like structure (hypha) will help stabilize it. 2-Act as the FIRST LINE OF DEFENCE when part of a hypha is damaged ,large-pored septa that have Woronin bodies or large proteinaceous crystals associated with them have the advantage that cytoplasmic streaming can occur between adjacent compartments. But at the same time a mechanism exists for rapidly sealing the septal pore under conditions of stress (e.g. if the hypha is damaged) thereby helping protect the mycelium. 3 -Facilitate DIFFERENTIATION in fungi :Septa can isolate adjacent compartments from one another so that different biochemical and physiological processes can occur within them - these may result in differentiation of the hyphae into specialized structures, such as those associated with sporulation. It s unlikely to be coincidental that the most complex and highly differentiated sporulating structures we see are those produced by fungi possessing the most complex types of septa, i.e. fungi belonging to the Basidiomycota.
Growth kinetics
Definition of Growth in Fungi
Growth may be defined as an irreversible increase in the volume of an organism, usually accompanied by an increase in biomass.
Mycelial fungi exhibit extension growth of hyphae, accompanied by an increase in biomass.
Unicellular fungi (e.g. yeasts) may exhibit an increase in individual cell volume, accompanied by an increase in biomass. But collectively, the number of yeast cells within a culture (i.e. cell concentration) may also increase, resulting in an increase in biomass of the culture as a whole. Fungi may be cultured on SOLID or in LIQUID MEDIA:YEASTS are often cultured in LIQUID media .MYCELIAL SPECIES may be cultured in LIQUID or on SOLID growth media.
Growth Kinetics in Liquid Media: Batch Culture
If we wanted to estimate the growth of a MYCELIAL fungus growing in a LIQUID medium, we might first have to filter off the liquid medium and then determine the dry mass of the mycelium. To estimate growth of a YEAST species in liquid culture, we could:
EITHER filter the culture and determine the dry mass of all the yeast cells together (i.e. the biomass of the culture),
OR we could estimate the concentration of cells in the culture, using either a haemocytometer or optical density readings. If we plotted biomass or cell concentration
against time we might obtain the following characteristic S-shaped growth curve ........1. Characteristic S-shaped growth curve:
During an initial LAG phase the rate of growth or cell division is very slow.
Growth or cell division then starts to accelerate into the EXPONENTIAL phase - when, for example, with a unicellular organism (e.g. yeast species) any 1 cell produces 2 in a given period of time, those 2 produce 4, the 4 produce 8, 8 produce 16 and so on ........ This exponential phase (central red region in the graph opposite) represents the period when the fungus is growing or multiplying most rapidly. This phase will continue until one or more nutrients become limiting, oxygen becomes depleted and/or metabolic by-products accumulate to toxic levels, when ........ Growth will start to DECELERATE (DECLINE).This may be followed by a STATIONARY phase, during which there is no discernible change in cell concentration or biomass. Finally, we may observe a phase of CELL DEATH and LYSIS - which results in a decrease in cell number and/or biomass.
We are often most interested in determining the RATE OF GROWTH taking place during the EXPONENTIAL PHASE. But it would be difficult to determine the overall rate of growth during the exponential phase from the graph above, i.e. the red section of this graph, because the rate of growth (i.e. the slope of this region of the graph) changes with time. However,
Growth Kinetics in Liquid Media: Continuous Culture
An alternative to the liquid batch culture system is CONTINUOUS CULTURE in a liquid medium: This involves the CONTINUOUS ADDITION OF FRESH CULTURE MEDIUM to the vessel and the WITHDRAWAL (by means of an overflow devise) of a corresponding volume of OLD, SPENT MEDIUM, which will contain some of the microbial cells. The apparatus used is called a CHEMOSTAT.
Chemostat:
Many environmental factors (e.g. pH, oxygen levels, nutrients and temperature) can be controlled very precisely throughout the incubation period.
The culture is stirred continuously - ensuring nutrients and oxygen reach the cells and metabolic products are distributed away from them.
Chemostats may be used to culture yeasts or mycelial fungi. No additional nutrients are added to the culture vessel once it has been inoculated and incubation has commenced, and the only environmental factor controlled is the temperature of incubation - nutrients, oxygen levels and pH will change as incubation proceeds and the culture grows. Therefore, this is essentially a closed system. Batch cultures are used in some industries to obtain valuable microbial products (e.g. antibiotics, ethanol, organic acids) accumulate in the medium during the stationary phase of growth.
Growth Kinetics On Solid Media
Here we re concerned primarily with mycelial fungi.
While it s relatively easy to determine the biomass of a fungus growing in a liquid medium, it s more difficult to estimate biomass when a fungus is growing in all three dimensions over and through a solid medium.
For this reason we usually express the growth of a colony in terms of the RADIAL EXTENSION OF THE COLONY (i.e. we measure colony radius).
If we plotted colony radius v. time we might observe the following four stages ........
Radial extension v. time:
The LAG PHASE follows inoculation of the plate. During this phase spores are beginning to germinate (spore inoculum) or severed hyphae are beginning to branch (mycelial inoculum).
The EXPONENTIAL PHASE lasts until the colony radius is only approx. 100µm (0.1 mm). Because readings of colony radius are often made at daily intervals this phase is often missed.
During the LINEAR PHASE the rate of increase in colony radius is constant. The slope of this region represents the COLONY RADIAL GROWTH RATE = Kr.
A DECELERATION in colony extension occurs as the colony approaches the margin of the Petri dish and staling metabolites begin to accumulate within the growth medium. the colony margin.
Hyphal density: Although determining the rate of colony extension will provide us with a measure of growth, it will not necessarily be equivalent to the increase in biomass of the fungus because hyphae will be growing down and through the agar medium as well as across its surface. For this reason it s usually a good idea to estimate HYPHAL DENSITY as well, i.e. the number of hyphal branches produced per unit area.
Reproduction:
Two basic types of reproductive methods are found in the fungi:
(a) Sexual methods are produced by the fusion of two nuclei that then generally undergo meiosis. Sexual methods of reproduction involve plasmogamy (cytoplasmic fusion of two cells), karyogamy:(fusion of two nuclei), genetic recombination and meiosis. The resulting haploid spore is said to be a sexual spore, e.g. zygospores, ascospores and basidiospores. If a sexual spore is produced only by fusion of a nucleus of one mating type with a nucleus of another mating type (+ and - strains), the fungus is said to be heterothallic. In contrast, homothallic moulds produce sexual spores following the fusion of two nuclei from the same strain.
(b) Asexual methods are termed either spores or conidia depending on their mode of production, and which arise following mitosis of a parent nucleus. Conidia arise either by budding off conidiogenous hyphae or by differentiation of preformed hyphae. Asexual spores are commonly formed by consecutive cleavages of a sporangium. Asexual forms of reproduction represent the major method for the maintenance and dissemination of many fungi.
FUNGAL PATHOGENICITY
The ability of fungi to cause disease appears to be an accidental phenomenon. With the exception of a few dermatophytes, pathogenicity among the fungi is not necessary for the maintenance or dissemination of the species.
The two major physiologic barriers to fungal growth within the human body are temperature and redox potential. Most fungi are mesophilic and can not grow at 37oC. Similarly, most fungi are saprophytic and their enzymatic pathways function more efficiently at the redox potential of non-living substrates than at the relatively more reduced state of living metabolizing tissue. In addition, the body has a highly efficient set of cellular defences to combat fungal proliferation. Thus, the basic mechanism of fungal pathogenicity is its ability to adapt to the tissue environment and to withstand the lytic activity of the host s cellular defenses. In general, the development of human mycoses (fungal infections) is related primarily to the immunological status of the host and environmental exposure, rather than to the infecting organism. A small number of fungi have the ability to cause infections in normal healthy humans by (1) having a unique enzymatic capacity, (2) exhibiting thermal dimorphism and (3) by having an ability to block the cell-mediated immune defenses of the host. There are then many opportunistic" fungi which cause infections almost exclusively in debilitated patients whose normal defence mechanisms are impaired. The organisms involved are cosmopolitan fungi which have a very low inherent virulence. Currently, there has been a dramatic increase in fungal infections of this type, in particular candidiasis, cryptococcosis, aspergillosis, and zygomycosis. More recently described mycoses of this category include hyalohyphomycosis and phaeohyphomycosis. Altogether, some 200 "human pathogens" have been recognized from among an estimated 1.5 million species of fungi.
Clinical Techniques in Mycology
A. Visualization of fungi in tissue preparations
1. Treatment with 10% potassium hydroxide
2. Positive stain with
a. Lactophenol cotton blue
b. Grocott silver stain
c. Hematoxylin
d. Eosin
3. Negative stain with India ink
B. Fluorescence of fungi under ultraviolet light by using Wood s light examination
C. Culture of fungi on
1. Sabouraud s agar (favors fungal growth because of low pH)
2. Mycosel agar (selective for pathogenic fungi because of chloramphenicol and cycloheximide in medium)
D. Visualization of cultured fungi (25oC and 37oC)
1. Colonial morphology
2. Cellular morphology
a. Hyphal morphology
(1) Aseptate or coenocytic fungi (lack of septa) (2) Septate
(a) Regular connection (b) Clamp connection
b. Spore morphology
(1) Conidiospore (2) Sporangiospore
(3) Arthrospore (4) Chlamydospore
c. Yeast morphology
(1) Size (2) Thickness of walls (3) Capsule presence/absence
E. Identification of yeast by
1. Biochemical tests
2. Behavior in broth and serum (germ tube formation)
3. Behavior on cornmeal agar (pseudohyphae formation)
Metabolism
All fungi are free living, i.e., they are not obligate intracellular parasites. They do not contain chlorophyll and cannot synthesize macromolecules from carbon dioxide and energy derived from light rays. Therefore all fungi are heterotrophs, living on preformed organic matter. For medical purposes the important aspects of fungal metabolism are: 1- The synthesis of chitin, a polymer of N-acetyl glucosamine, and other compounds, for use in forming the cell wall. These induce immune hypersensitivity. 2. The synthesis of ergosterol for incorporation into the plasma membrane. This makes the plasma membrane sensitive to those
antimicrobial agents which either block the synthesis of ergosterol or prevent its incorporation into the membrane or bind to it, e.g. amphotericin B.
3. The synthesis of toxins such as
a. Ergot alkaloids- these are produced by Claviceps purpurea and cause an alpha adrenergic blockade
b. Psychotropic agents - these include psilocybin, psilocin and lysergic acid diethylamide (LSD)
c. Aflatoxins - these are carcinogens produced by Aspergillus flavus when growing on grain. When these grains are eaten by humans or when they are fed to dairy cattle and they get into the milk supply, they affect humans.
4. The synthesis of proteins on ribosomes that are different from those found in bacteria. This makes the fungi immune to those
antimicrobial agents that are directed against the bacterial ribosome, e.g., chloramphenicol.
5. The ability of certain metabolites to alter morphology of yeast and/or be assimilated by yeast with concomitant clinical identification affects
Antifungal Agents
Antifungal agents are classified according to their chemical structure as macrolides, azoles, allylamines, pyrimidine analogs and miscellaneous. . *The polyene antifungals are amphotericin B and nystatin which bind to ergosterol in the plasma membrane, thus disrupting it. . *The azole antifungals include fluconazole and ketoconazole plus numerous others. They all block ergosterol synthesis by binding to cytochrome P-450. *The allylamines include naftifine and terbinafine which inhibit squalene epoxidase, thus blocking ergosterol synthesis. * The pyrimidine analogs such as flucytosine incorporate into RNA and/or DNA thus blocking protein synthesis or DNA synthesis. *The miscellaneous antifungals include griseofulvin, haloprogin, ciclopirox olamine, tolnaftate and potassium iodide.
CLINICAL GROUPINGS FOR FUNGAL INFECTIONS
SKIN MYCOLOGY
Superficial Mycoses
Cutaneous Mycoses
Subcutaneous Mycoses
INFECTIOUS DISEASE MYCOLOGY
Dimorphic Systemic Mycoses
Opportunistic Systemic Mycoses
SKIN MYCOLOGY
The Superficial Mycoses :These are superficial cosmetic fungal infections of the skin or hair shaft. No living tissue is invaded and there is no cellular response from the host. Essentially no pathological changes are elicited. These infections are often so innocuous that patients are often unaware of their condition.
Pityriasis (tinea) versicolor A chronic, superficial fungal disease of the skin characterized by well-demarcated white, pink, fawn, or brownish lesions, often coalescing, and covered with thin fine scales. The colour varies according to the normal pigmentation of the patient, exposure of the area to sunlight, and the severity of the disease. Lesions occur on the trunk, shoulders and arms, rarely on the neck and face, and fluoresce a pale orange colour under Wood s ultra-violet light. Young adults are affected most often, but the disease may occur in childhood and old age.
Distribution: World-wide but more common in tropical than temperate climates.
Etiological Agent: Malassezia furfur a lipophilic yeast forming part of the normal flora of human skin.
The Cutaneous Mycoses
These are superficial fungal infections of the skin, hair or nails. No living tissue is invaded, however a variety of pathological changes occur in the host because of the presence of the infectious agent and its metabolic products.
Dermatophytosis - Ringworm or Tinea
Ringworm of scalp, glabrous skin, and nails caused by a closely related group of fungi known as dermatophytes which have the ability to utilize keratin as a nutrient source, i.e. they have a unique enzymatic capacity called keratinase.
The disease process in dermatophytosis is unique for two reasons:
(a) No living tissue is invaded the keratinized stratum corneum is simply colonized. However, the presence of the fungus and its metabolic products usually induces an allergic and inflammatory eczematous response in the host. The type and severity of the host response is often related to the species and strain of dermatophyte causing the infection.
(b) The dermatophytes are the only fungi that have evolved a dependency on human or animal infection for the survival and dissemination of their species.
Dermatophytes consists of three genera; epidermaphyon, trichophyton and microsporom. And can be divided into 3 broad epidemiological groups.
GEOPHILIC These dermatophytes normally inhabit the soil where they are believed to
decompose keratinaceous debris. Some species may cause infections in animals and man following contact with soil (e.g. Microsporum gypseum).
ZOOPHILIC These dermatophytes are primarily parasitic on animals. Infections may be transmitted to man following contact with animal host (e.g. Microsporum canis).
ANTHROPOPHILIC These dermatophytes are primarily parasitic on man and have only rarely been known to infect animals, presumably following contact with man (e.g.
Trichophyton rubrum).