A Brief History of Microbiology

Development of microscopy:

Aristotle (384-322) and others believed that living organisms could develop from non-living materials. 1590: Hans and Zacharias Janssen (Dutch lens grinders) mounted two lenses in a tube to produce the first compound microscope. 1660: Robert Hooke (1635-1703) published "Micrographia", containing drawings and detailed observations of biological materials made with the best compound microscope and illumination system of the time. 1676: Anton van Leeuwenhoek (1632-1723) was the first person to observe microorganisms. 1883: Carl Zeiss and Ernst Abbe pioneered developments in microscopy (such as immersion lenses and apochromatic lenses which reduce chromatic aberration) which perist until the present day. 1931: Ernst Ruska constructed the first electron microscope.

Spontaneous generation controversy:

1688: Francesco Redi (1626-1678) was an Italian physician who refuted the idea of spontaneous generation by showing that rotting meat carefully kept from flies will not spontaneously produce maggots. 1836: Theodor Schwann (1810-1882) helped develop the cell theory of living organisms, namely that that all living organisms are composed of one or more cells and that the cell is the basic functional unit of living organisms.

1861: Louis Pasteur's (1822-1895) famous experiments with swannecked flasks finally proved that microorganisms do not arise by spontaneous generation. Development of sterilization Development of aseptic technique

This eventually led to:

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Proof that microbes cause disease:

1546: Hieronymus Fracastorius (Girolamo Fracastoro) wrote "On Contagion" ("De contagione et contagiosis morbis et curatione"), the the first known discussion of the phenomenon of contagious infection. 1835 Agostino Bassi de Lodi showed that a disease affecting silkworms was caused by a fungus - the first microorganism to be recognized as a contagious agent of animal disease.

1847: Ignaz Semmelweiss (1818-1865), a Hungarian physician who decided that doctors in Vienna hospitals were spreading childbed fever while delivering babies. He started forcing doctors under his supervision to wash their hands before touching patients. 1857: Louis Pasteur proposed the "germ theory" of disease. 1867: Joseph Lister (1827-1912) introduced antiseptics in surgery. By spraying carbolic acid on surgical instruments, wounds and dressings, he reduced surgical mortality due to bacterial infection considerably. 1876: Robert Koch (1843-1910). German bacteriologist was the first to cultivate anthrax bacteria outside the body using blood serum at body temperature. Building on pasteur's "germ theory", he subsequently published "Koch's postulates" (1884), the critical test for the involvement of a microorganism in a disease: 1. The agent must be present in every case of the disease. 2. The agent must be isolated and cultured in vitro. 3. The disease must be reproduced when a pure culture of the agent is inoculated into a susceptible host. 4. The agent must be recoverable from the experimentally-infected host. This eventually led to:

Development of pure culture techniques Stains, agar, culture media, petri dishes Haeckel 1865: Protista. Cohn 1980 --> Development of microscopy, structure of nucleus. Chatton 1930 --> Stanier & Van Niel 1962. Whittaker 5 Kingdom classification

Microbes create problems for conventional taxonomies:

The Eukaryote/Prokaryote Divide

Main differences between prokaryotes and eukaryotes: 1. Nature and arrangement of genetic material. 2. Membrane-bounded organelles.
3. Protein synthesis machinery, 70S vs. 80S ribosomes.

4. The chemistry of the cell wall (where present).

Other differences:

Endospores in some prokaryotes. Gas vesicles in some prokaryotes. Flagellum structure/function. Microtubules in eukaryotes. Size ??? Online Experiment: How Big is a Bug?
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The Chemistry of the Prokaryote Cell Wall:

Christian Gram 1884 - The Gram stain - Online Experiment Differences between Gram positive and Gram negative cell walls:

The structure of N-acetylmuramic acid:

The structure of murein (peptidoglycan):

Microbial Pathogenesis
Definitions:
Disease is: 'any deviation from or interruption of the the normal structure and/or function of any part of the body'.

A pathogen is: 'a disease causing organism'. Opportunistic pathogens: e.g. Escherichia coli is a normal, 'non-pathogenic' commensal organism in the human gut, but infection of the bladder can result in ascending infection of the ureters & eventually, the kidneys, resulting in renal failure. Most strains of E.coli are inefficient in establishing such infections, but some strains express particular adherence factors which allow them to ascend the ureter & reach the kidney. Obligate pathogens display the most extreme adaptation to their host organisms.
What does a pathogen have to do? Infect (infest) a host Reproduce (replicate) itself Ensure that its progeny are transmitted to another host

Mechanisms of Transmission:
Aerosols - inhalation of droplets, e.g. Rhinoviruses, the 'Common Cold Virus' or Adenoviruses. Faecal-Oral - e.g. Astroviruses, Caliciviruses; these viruses cause acute gastroenteritis. Vector-borne - e.g. in Arthropods such as mosquitos, ticks, fleas: Arboviruses. Close personal contact - especially exchange of bodily fluids: Sex; Blood, e.g. Herpesviruses.

Sites of virus entry:

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Transmission patterns:
Horizontal Transmission: Direct person-to-person spread. Vertical Transmission: Relies on PERSISTENCE of the agent to transfer infection from parents to offspring. Several forms of vertical transmission can be distinguished: Neonatal infection at birth, e.g. gonorrhorea, AIDS. Infection in utero e.g. syphilis, CMV, Rubella (CRS), AIDS. Germ line infection - via ovum or sperm.

The eventual outcome of any virus infection depends on a balance between two processes: PERSISTENCE of the agent vs. CLEARANCE from the host

Host Responses
Mammals have a variety of defences against infection.

Non-specific mechanisms:
Skin Enzymes Fever Mucosal Immunity Humoral Immunity Cell Mediated Immunity

Specific mechanisms - the immune response:

MicrobiologyBytes: Introduction to Microbiology: Microbiology Updated: January 28, Techniques 2007

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Techniques of Microbiology
Microbiology as a discipline is defined by the techniques used to study microorganisms!

Microbiology encompasses the study of:

bacteria smaller fungi smaller algae protozoa viruses

The Techniques:
1. Microscopy and stains: electron microscopy. 2. Sterilization - look at your practical book! 3. Getting a pure culture : aseptic technique + sterile media: a. micromanipulation b. extinction dilution in liquid media c. solid media - streaking out, viable counts d. enrichment culture

4. Composition of culture media, temperature of incubation, osmotic potential of media, pH etc. 5. Anaerobe or aerobe? - microaerophile?

Nutitional Types:
Chemical energy or light energy - chemotrophs, phototrophs, organotrophs, Lithotrophs. Autotrophy or heterotrophy? Sourges of Energy, Hydrogen/Electrons, Carbon: Representative Microorganisms:

Major Nutritional Types:

Photolithotrophic autotrophy:

Light energy Algae; purple & green sulphur Inorganic hydrogen/electron bacteria; blue-green bacteria (H/e-) donor (Cyanobacteria) CO2 carbon source Light energy Organic (H/e-) donor Purple non-sulphur bacteria; Organic carbon source (CO2 green non-sulphur bacteria may also be used) Chemical energy source (inorganic) Inorganic (H/e-) donor CO2 carbon source Sulphur-oxidizing bacteria; hydrogen bacteria; nitrifying bacteria; iron bacteria

Photoorganotrophic heterotrophy:

Chemolithotrophic autotrophy:

Chemical energy source Chemoorganotrophic (organic) autotrophy: Organic (H/e-) donor Organic carbon source

Protozoa; fungi; most nonphotosynthetic bacteria

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Bacterial Growth:
The bacterial growth curve: 1. Lag phase 2. Exponential phase 3. Stationary phase 4. Death phase

During the exponential phase, the number of cells doubles every generation (1, 2, 4, 8, 16...) and the number of cells in the culture (Nf) at any particular time is given by:
Nf = (Ni)2n

where: Ni is the initial size of the population n is the number of generations that have elapsed. This equation is usually written in the form log10 for mathemetical convenience:
log10Nf = log10Ni + 0.301n(log10 2 = 0.301)

From this equation it is possible to ddetermine n (generations elapsed) by knowing the numbers of cells at the beginning and end of a particular incubation period:
n = (log10Nf - log10Ni) / 0.301

This is useful because it enables calculation of the generation time g (the time between the beginning and end of the incubation period:
g + t/n = 0.301 / (log10Nf - log10Ni) Online Experiment: The Bacterial Growth Curve Determine the rules which control bacterial growth.

Microbiology by L.M.Prescott et al. A balanced, comprehensive introduction to all major areas of microbiology. The sixth edition has been updated extensively to reflect the latest discoveries in the field.
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MicrobiologyBytes 2007. MicrobiologyBytes: Microbiology Notes: Bacterial Updated: January 9, Pathogenesis 2007

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Bacterial Pathogenesis
Medical Microbiology: the study of relationships between humans and microbes, including:
Infection Infective disease Colonisation Transmission of infective agents

The central questions:

A. What are infections? B. Which microbes cause them? C. Why do particular individuals get particular infections? D. What influences the outcome of an infection? Pathogenesis (A-D) Epidemiology (C) Diagnosis (A, B) Treatment (D) Control (C, D)

The Germ Theory of Disease

Classification of Bacteria:
Phenotypic:
Morphology Staining properties Growth and biochemical properties. The Gram stain Non-stainable prokaryotes (e.g. Mycobacteria) 16s ribosomal RNA subunit genes

Genotypic:

other genes (e.g. cytochrome c) Pathogens vs Non-pathogens Agents associated with specific diseases

Practical / Medical or Veterinary (this course):

Koch - Isolation of a particular organism associated with particular disease. But the isolation of an organism from an individual does not necessarily imply disease. Many different forms of association between microbes and humans. We are born sterile (microbiologically speaking) After weaning we are hosts to some 1014 bacteria. You are an environment - inhabited by your normal flora comprising several hundred different species of bacteria (+ fungi & protozoa) These organisms are commensals. Pathogens often have some properties in common with commensals but the associations they form with humans progress beyond commensalism to disease.
Stages of infection: 1. Encounter 2. Entry/Establishment 3. Spread 4. Multiplication 5. Damage 6. Outcome

Medical microbiology is primarily concerned with Pathogens and Commensals. Commensals (literally eat at the same table): Some commensals are also pathogens. Specific composition of the commensal /normal flora depends on:
a. Site b. Environmental exposure c. Age d. Short and long term structural abnormalities Exposed/Superficial sites, e.g: Skin Pharynx Gut Internal Sites, e.g: Blood Tissue CSF

Genital tract

Urine

1. We have to know what the normal flora of a particular site is to distinguish between normal and potentially pathological association. 2. Strategy for studying infection at different sites is heavily influenced by whether that site has abonormal flora or not.

Pathogens cause infection and the establishment and outcome of infection is DOSE-RELATED:

ID50 (dose required to produce specified outcome in 50% of target population) is shifted:
by increased virulence - or decreased host-resistance by decreased virulence - or increased host-resistance

An opportunistic pathogen is generally of low virulence (i.e. ID50 is high) and requires decreased host resistance.

SUMMARY:
Suspicion that organisms cause disease (history and context) Recognition of pathogens and non pathogens Stages of infection Normal flora / commensals Infection / infective disease Infection is the multiplication of in a host with associated damage to that host Infective disease results when the damage is sufficient to produce symptoms Capacity of a pathogen to produce disease is referred to as virulence

Opportunistic pathogens

MicrobiologyBytes 2007. MicrobiologyBytes: Microbiology Notes: Koch's Postulates Updated: October 19, 2004 Search

Koch's Postulates
Establishing a specific microbe as the cause of an infective disease:
The Koch - Henle postulates:
1. Isolate the organism from every case 2. Propagate in pure culture in vitro 3. Reproduce disease by injecting the organism into a suitable recipient 4. Re-isolate the organism

OK for major acute diseases like plague, small pox, typhoid. But what about:
chronic or minor conditions multiple causes pathogen can't be grown no suitable model

Pathogens cause infection and the establishment and outcome of infection is DOSE-RELATED:

ID50 (dose required to produce specified outcome in 50% of target population) is shifted :
by increased virulence - or decreased host-resistance by decreased virulence - or increased host-resistance

Any specific attribute of an organism whose loss decreases virulence is called: a virulence factor or virulence determinant.

Formation of microbe human associations:

Encounter may be Exogenous or Endogenous. Factors affecting progress of a pathogen:

Circumstantial Organism -related Host-related

Virulence factors can be related to the stages of infection, e.g:

1. Entry / Establishment:
Motility and chemotaxis Attachment via pili or non-fimbrial adhesins Penetration of epithelia/cells sIgA proteases Capsules Survival inside phagocytes Tissue breakdown by enzymes or toxins Penetration to bloodstream or lymphatics Dissemination in circulating cells

2. Spread:

3. Multiplication:
Iron acquisition (siderophores) Immune evasion Toxins Host "over-reaction" (immun -mediated hypersensitivity)

4. Damage:

It makes sense that these factors could all contribute to virulence. But proof of involvement is important:
Scientifically To target treatment and prevention appropriately

Hence: "Molecular Koch's Postulates":

1. Identify gene (or gene product) responsible for virulence determinant 2. Show gene present in strains of bacteria that cause the disease 3. Not present in avirulent strains 4. Disrupting the gene reduces virulence 5. Introduction of cloned gene into avirulent strain confers virulence. 6. The gene is expressed in vivo 7. Specific immune response to gene protects

The "mug shot " book (3 groups): 1. Distinctive pathogens: Organisms for which the Koch Henle postulates have been fulfilled. 1 microbe ->1 disease, e.g. the causal agents of plague, anthrax, diphtheria, cholera, typhoid etc. 2. Agents of sepsis: Organisms regularly isolated from patients with symptomatic infections. Many microbes ->1 disease, e.g. agents causing pneumonia, wound infections, urinary tract infections, septicaemia 3. Pluripotential pathogens: 1 microbe ->many diseases, e.g. S. aureus, E. coli. Isolation of a pathogen (particularly in groups 2 or 3) does not necessarily mean that a patient has any of the diseases associated with that organism:

Microbes:
Pathogens: Staphylococcus aureus Non-pathogens: Staphylococcus epidermidis Opportunistic pathogens:

Pseudomonas Haemophilus influenzae capsule type b+ E. coli Haemophilus influenzae capsule type bE. coli

Bacillus subtilis

E. coli

List three sources of evidence that could be used to indicate that a particular organism causes a particular disease if there is no suitable laboratory model for the disease.

TRUE OR FALSE?
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1) John Hunter showed that infections could be transmitted by sex: True False

2) Robert Koch was a German GP: True False 3) A pathogen is an organism that is never found on a healthy person: True False 4) The human body is normally host to around: 105 1014 1023 bacterial, fungal and protozoan cells 5) Infection does not always lead to symptoms: True False 6) Some organisms can be pathogens and commensals: True False 7) An opportunistic pathogen has a relatively low infectious dose: True False 8) Damage in infection nearly all results from the production of microbial toxins: True False 9) A particular strain of an organism can be both a pathogen and a non pathogen: True False

10) Endogenous infections are caused by members of the patients normal flora: True False
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MicrobiologyBytes 2007. MicrobiologyBytes: Microbiology Notes: Mycology Updated: April 8, 2009 Search

Introduction to Mycology
All About Fungi

Fungi have the several features that distinguish them from other organisms. They have a filamentous branching system of cells, with apical growth, lateral branching and a heterotrophic nutrition. They are characterized by a life cycle that begins with germination from a spore or resting structure, followed by a period of growth as a substrate is exploited to produce biomass. Finally there is a period of sporulation, where propagules are formed that can be disseminated from the parent mycelium - Fig. 1. Schematic of a fungal life cycle:

MicrobiologyBytes: Fungi

They are vital to the biosphere for many reasons, not least for their decomposing activities on dead substrates that ensure the release of nutrients like carbon, minerals and nitrogen back into the biosphere.

Taxonomy
The origin of the fungi appears to be very ancient. They first appear in the fossil record coincidentally with the appearance of the land plants. This was in the Devonian period around 400 million years ago. The early microscopists found that fungi were simple to study, and they first observed division of cells by watching yeasts under the microscope.

They were one of the earliest groups to be classified, and the earliest scheme was drawn up in 1588. Early biological science was much obsessed with categorizing things and researchers constructed taxonomic systems based solely on structure. However, for the fungi, this created a polyphyletic group that contained microorganisms that had very different ancestors. Taxonomic thinking today now prefers a monophyletic classification, where all groups within a phylum are descendants of one ancestor. Therefore current taxonomy relies on studying characters that are features or attributes of an individual organism that can be used to compare it with another organism. These features can be morphological, anatomical, ultrastructural, biochemical or based on sequences of nucleic acids. Fungi are referred to as the True Fungi, or Eumycota, and are currently divided into four (plus one form class) major phyla on the basis of the morphology and sexual reproduction (see Table 1). New information based on presence and type of mitochondria, and the DNA sequencing of ribosomal RNA, place the members of the fungi into a complex phylogenetic tree. Table 1. Features of the main groups of fungi. Group: Perforate septae Asexual Sexual +/sporulation: sporulation: Lower fungi : Zygomycotina non-motile sporangiospores motile zoospores Higher fungi : Ascomycotina Basidiomycotina Deuteromycotina + + + conidiospores rare conidiospores ascospore basidiospore none zygospore oospore

Chytridiomycotina -

Two of the phyla are sometimes incorrectly described as Lower fungi, the Zygomycotina and the Chytridiomycotina. In the lower fungi vegetative mycelium is non-septate, and complete septa are only found in reproductive structures. Asexual reproduction is by the formation of sporangia, sexual reproduction by the formation of zygospores. We will return to these details later. The other two phyla are sometimes described as the Higher fungi and have a more complex mycelium with elaborate, perforate septa. They are divided into the Ascomycotina and the Basidiomycotina. Members of the Ascomycotina produce asexual conidiospores and sexual ascospores in sacshaped cells called asci. Fungi from the Basidiomycotina rarely produce asexual spores, and produce their sexual spores from club-shaped basidia in complex fruit bodies. A particular problem for some of the fungi is that they do not possess sexual stages, and this lead to the construction of an artificial taxonomic group

based on the asexual, mitosporic stage of the fungus. This group is a form group called the Deuteromycetes, or Imperfect fungi. However, using the current cladistic approaches mycologists are slowly establishing links between these species and other species with sexual stages. It is important to know the taxonomic terms for the kingdom. Fungi Kingdom: Division: (subdivision) Class: Order: Family: Genus:
Species: Eumycota e.g. Basidiomycotina e.g. Basidiomycetes e.g. Agaricales e.g. Agaricaceae e.g. Agaricus e.g. Agaricus bisporus - The mushroom!

Cell biology of the fungi

Their basic cellular unit is described as a hypha. This is usually a tubular cell which is surrounded by a rigid, chitin-containing cell wall. The hypha extends by tip growth, and multiplies by branching, creating a fine network called a mycelium. Hyphae contain nuclei, mitochondria, ribosomes, Golgi and membrane-bound vesicles within a plasma-membrane bound cytoplasm. The sub-cellular structures are supported and organized by micro-tubules and endoplasmic reticulum. If you have forgotten what these structures look like and what they do, please check them in a basic text book. Not all fungi are multicellular, some are unicellular and are termed yeasts. These grow by binary fission or budding, creating new individuals from the parent cell. Simplified diagram of a vegetative yeast cell and a budding cell:

Fungal metabolism
Fungi prefer moist habitats and they are largely mesophyllic, preferring temperatures between 15C and 35C. The carbon needs of fungi for energy metabolism and biosynthesis has to be met heterotrophically by one of three lifestyles:a. parasitism of plants or animals (causing disease) b. saprophytism, growing on dead animal, plant or microbial biomass c. symbiosis, growing together with algae, plants or insects Soluble carbohydrates must enter hyphae by diffusion, and this is followed by active uptake across the fungal membrane. For the saprophytic fungi most carbon in the environment is present as a complex polymer like cellulose, chitin or lignin. These materials must be broken down enzymically before they can be utilized. Therefore fungi release degradative enzymes into their environments including cellulases, chitinases, proteases and multicomponent lignin degrading enzymes, depending on the type of substrate the fungus is growing on. Regulation of these enzymes is by substrateinduction and end-product inhibition. Most fungal carbohydrate metabolism is via glycolysis and the tricarboxylic acid cycle, although both fermentative pathways and anaerobic pathways have been found in specialized fungi. Fungi cannot fix gaseous nitrogen. They can however utilize nitrate, ammonia and some amino acids by direct uptake across the hyphal membrane. More complex nitrogen sources, such as proteins and peptides, can be utilized. once extracellular proteases have degraded them into amino acids. Fungi require both macro and micronutrients, but these are usually available to excess in their environments, and some fungi may have other requirements, for example thiamin and biotin, sterols, riboflavin, nicotinic acid and folic acid. Fungi are capable of the formation and secretion of many types of secondary metabolites. These compounds can be produced by many different metabolic pathways.

Cell wall structure and growth: the structure of the fungal cell wall
The living part of a fungus consists of the apical cell of the mycelium and a few cells that are immediately behind it. Cells that are beyond about 5 cross walls, that is septae, and usually moribund unless they have special survival functions. The cells are very variable in size, but usually fall between 3-10 m wide and 50 m long. However, the apical cell of the mycelium is usually 300 to 400 m long. The septae that separate the cells are usually perforate, and have varying structures to protect the cell from loosing its contents if there was a disruption of a neighbouring cell. In the Ascomycetes the septae are protected by a Woronin body, an osmophilic oily structure which can plug the septum if required, or in the Basidiomycetes there is a complex wall structure to the septum and also a complex of endoplasmic reticulum associated with the septal pore. This is called a dolipore septum. The septae of fungi:

The lower fungi do not have perforate septae, they have complete ones to isolate reproductive or vacuolated regions of mycelium from the rest of their mycelium.

Food material is absorbed from all over this living region, but mostly from around the tip. This creates a concentration gradient around the mycelium, especially from around the tip, and this creates the drive for the mycelium to grow forwards from the tip up the concentration gradient for nutrients and towards fresh substrates. Zones of depletion around a mycelium:

From light and electron microscopy it is apparent that fungal cell walls are made up of a complex of fibrillar materials with an amorphous matrix. Using enzymatic and chemical digestions it has been established that the fibrils in the true fungi are chitin, chains of N-acetyl-glucosamine. This is an acetylated amino sugar derived from glucose. This polymer is also found in insect exoskeletons. The structure of the 1-4 linkages allows for microfibrils to form, with many parallel molecules aligning. These microfibrils can be between 10 and 25 nm in diameter. They are embedded in an amorphous matrix, and the end effect is much like a fibreglass mat. The exact composition of the wall varies between the major taxonomic groups. In the Zygomycetes the outer wall layer contains 1-3 and 1-6 linked amorphous glucans with a chitin microfibrillar inner layer. In the chytrids the outer layer is similar, but the fibrillar component is cellulose. In the higher fungi the outer layer of the Ascomycete cell wall is again 1-3 and 1-6 linked amorphous glucans, but the inner layer contains proteins above the chitin microfibrils. Basidiomycetes differ in that their outer layer is composed of 1-3 linked amorphous glucans above the layer of 1-3 and 1-6 linked amorphous glucans, proteins and cellulose microfibrils. These differences are important, especially in the development of atopy and allergy. Yeast cell walls are rather different. They still contain the amorphous glucans, but there are instead of the linked fibrils of mannans, 1-6 linked polymers which in turn are linked to proteins. This particular combination makes for a much more plastic wall and a spherical rather than tubular shape. It is the ability to move from mycelial to yeast form that allows many of the fungi to become pathogenic. This is called dimorphism, and I return to it later.

Growth

Growth occurs at the hyphal tip by the fusion of characteristic membranebound vesicles derived from the Golgi. These vesicles accumulate in the apical 10 m of the hyphal tip. The organization of these vesicles varies in the different taxonomic groups. Hyphal tips from the three major taxa:

Cell wall synthesis:

As the hypha grows, the tip cell becomes larger and larger as it accumulates biomass. This gives rise to two phenomena. One we have already described, septation, and the other is branching. Branching occurs some way behind the first septum. Branching occurs by the formation of another apical cell growth point within a subapical cell. usually the branches are at a very precise angle to the parent hypha, characteristic of the karyotype of the cell.

Kinetics of growth
When fungi are filamentous their growth rate cannot be established by cell counting using a heamocytometer or by tubidometric measurements which can be used to measure bacterial and yeast growth . However, by measuring mass (M) changes with time (t) under excess nutrient conditions the specific growth rate () for the culture can be calculated using the formula:

dM = M dt

Fungal growth in a given medium follows the growth phases of lag, acceleration, exponential, linear, retardation, stationary and decline phase. Phases of fungal growth:

Exponential growth occurs only for a brief period as a hyphae branches are initiated, and then the new hypha extends at a linear rate into uncolonised regions of substrate. Only hyphal tips contribute to extension growth.

However, older hyphae can grow aerially or differentiate to produce sporing structures. Rates can be very rapid indeed. Some species may extend at 6mm/hour, but more often it is at around 0.1 to 2 mm/hour. It is also possible to observe hyphal growth by microscopy, measuring tip growth and branching rates of mycelium. From this data the hyphal growth unit and the peripheral growth zone can be calculated. The hyphal growth unit (G), which is the average length of hypha which is required to support tip growth, is defined a ratio between the total length of mycelium and the total number of tips. Total number of tips = hyphal growth unit Total length of mycelium In most fungi the hyphal growth unit includes the tip cell plus 2 to 3 subapical compartments. The ratio increases exponentially from germination, but stabilizes to give a constant figure for a particular strain under any given set of environmental conditions. If you destroy the apical compartment of a fungal cell growth of that apex can no longer continue. It can however resume from the sub-apical compartment. The exact length of hyphae needed to maintain that apical growth can be found by making experimental incisions in a colony margin. This so called peripheral growth zone (PGZ)( ) is the region of mycelium behind the tip needed to support maximum growth of the hyphal tip. This zone permits radial extension at a rate equal to that of the specific growth rate of unicells in liquid culture. The mean rate of hyphal extension (E) is a function of the hyphal growth unit and the specific growth rate

E=G

The zone can be as much as 12 cells deep in some fungi, particularly in the lower fungi that have very few septae. Higher fungi with protected perforate septae can have very small PGZs .The radial extension rate (Kr) is a function of the peripheral growth zone and the specific growth rate ( ). Growth is a highly regulated process, and is affected by light, heat, changes in osmolarity, ionic strength of the media and by pressure. Growth is also accompanied by nuclear division and cross wall formation. The apical compartment of the hypha is often mutinucleate, sometimes there are over 50 nuclei. This is because there are repeated mitotic divisions of the nucleus in a parasynchronous wave, beginning at the tip and moving backwards towards the first septum. The mitotic division is very rapid, taking about 4 minutes, and the hypha continues to grow forwards whilst this is happening. Undifferentiated growth does not continue indefinitely. Fungal colonies show a differentiation from margin to the centre of the colony.
1. The extension zone. Hyphae are advancing into fresh media. Branching patterns tend to be at a 90 angle and monopodial. The branches are arranged to give the most effective colonization and utilization of the substrate, and the growth of the hyphae is oriented to avoid the other

Kr =

hyphae. The mechanism by which this occurs is unknown, but probably relates to food gradients and toxin gradients. 2. The productive zone. Here the major increase in biomass occurs. Aerial mycelium is formed, and the hyphae thicken. 3. The fruiting zone. Biomass gain has ceased, and spores are formed. This is equivalent to the stationary phase of batch culture. 4. Ageing zone. Vacuolate or empty hyphae are present, autolysis occurs. This is equivalent to the decline phase of batch culture.

MicrobiologyBytes 2009. MicrobiologyBytes: Infection & Immunity: Protozoa & Helminths Updated: August 23, 2007 Searc h

Protozoa and Helminths

The medically important protozoa and helminths require the invasion of a suitable host to complete all or part of their life cycle. Such organisms are therefore termed parasites and medical parasitology is the study of protozoa and helminth infections of man. The exception are some free-living amoebae that normally live and replicate in the environment but under certain circumstances can infect man. They are not therefore parasites but are included here as examples of protozoa causing human disease. Parasite infections affect millions of people world-wide afflicting considerable human suffering and economic hardship. Far from declining, many parasite infections are increasing throughout the world. The impact of Human Immunodeficiency Virus (HIV) and AIDS has seen the emergence of "new" opportunistic parasites as well as the increased prevalence of other recognised types. Climatic changes induced through global warming has aided the spread of many parasite diseases, whilst starvation and the breakdown in sanitation that accompanies war has seen the re-emergence of others. The appearance of drug resistance has also dramatically influenced the ability to treat and control many parasite diseases. In the United Kingdom parasite infections are relatively uncommon. However, outbreaks of cryptosporidiosis associated with drinking water supplies has been of major concern, and toxoplasmosis remains a serious infection for the foetus when acquired during pregnancy. It is beyond the scope of this brief summary to characterise all protozoal and helminth infections of man. For students wishing to obtain further information on these fascinating and largely unexplored microbes, a few reference books are given at the end of this section. World Wide Web sites also contain useful information as well as diagrams and picture images of the

protozoa and helminths discussed below. Find them through key word searches using one of the several search engines available.

Medically Important Protozoa

Protozoa means "first animal" and refers to simple eukaryotic organisms composed of a single cell (e.g. amoebae). Reproduction may be through simple cell division (e.g. the amoeboflagellates) or sexual involving the fusion of gametes in part of the life cycle (e.g. the apicomplexa) as described below. Some protozoa can form a protective cyst stage capable of withstanding harsh environmental conditions. Amoeboflagellates These use pseudopodia or flagella for locomotion.

Amoebae. These are characterised by a feeding and dividing trophozoite stage that can form a temporary resistant cyst stage. Entamoeba histolytica is the cause of amoebic dysentery producing severe infection of the intestines that can spread to the liver. The organism is characterised by a trophozoite and cyst stage. E. histolytica is an example of a true parasite in that the organism cannot multiply outside of the host. Other amoebae occur naturally in soil and water environments which is their preferred habitat for feeding and replication. These amoebae are termed "free-living" as they have no natural host in which parasitism occurs. They can infect man opportunistically producing severe and often fatal disease. Such free-living amoebae are the Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris, all of which can infect the central nervous system. In addition, Acanthamoeba species can also invade the eye.
Flagellates These organisms have flagella in the trophozoite stage. Trichomonas vaginalis is a common sexually transmitted organism causing trichomoniasis infection of the vagina and urethra. Giardia lamblia causes giardiasis producing symptoms of diarrhoea and other intestinal disturbances. Infection arises from the ingestion of cysts, usually through contaminated water.

Trypanosoma brucei gambiense and T. brucei rhodesiense cause trypanosomiasis, more commonly known as African sleeping sickness. The disease is an arthropod (insect)-borne infections and is spread by the bite of the tsetse fly in which part of the trypanosome life cycle is completed. The eventual invasion of the central nervous system by the trypanosomes gives rise a comatosed state from which the common name for the disease is derived. Trypanosoma cruzi causes Chagas' disease (American trypanosomiasis). The intermediate host in this case are triatomid bugs that feed off the blood of man. Infection results from the inoculation of the bug's faeces that contains the organism into the bite wound. Individuals who survive the acute stage of the disease are frequently left with chronic and progressive

neuronal and smooth muscle lesions in the heart and gastrointestinal tract. T. cruzi has an extensive reservoir in wild and domestic mammals and therefore Chagas' disease is a zoonosis (human infections that can be caught from animals). Leishmania species cause leishmaniasis. The disease is spread by the bite of sandflies in which part of the organism's life cycle is completed. In man, the promastigotes from the bite of the sandfly become ingested by macrophages and multiply within them as amastigotes. Cutaneous leishmaniasis occurs if the region of infection remains localised to the dermis as an open sore. In the Old World (Southern Europe, the Middle East, India, former USSR and parts of Africa) L. major, L. tropica, L. aethiopica and certain subtypes of L. infantum are responsible. In the New World (Mexico southwards and through South America) species responsible include L. braziliensis, L. mexicana and L. amazonensis. If the organism spreads, then mutocutaneous leishmaniasis can occur in which the nose, mouth and palate becomes destroyed. Infection with members of the L. donovani-L.infantum complex produce the systematic disease of visceral leishmaniasis often known as kala-azar that occurs with a global distribution seen in Old and New World leishmaniasis. The parasites multiply within the macrophages of the liver, spleen, bone marrow and other organs. Untreated, the disease is usually fatal. As with trypanosomiasis, leishmaniasis is a zoonosis as many mammals harbour the parasite.
Ciliates These possess rows of cilia around the outside of the body that aid motility. The only member of this group known to infect man is Balantidium coli. This is a cyst forming parasite that is a commensal ("table-sharing" and meaning here a nonpathogenic parasite) of domestic and wild pigs. It does, however, cause severe diarrhoea in humans. Apicomplexa This is a unique group because all members are parasitic. The group includes parasites causing malaria, cryptosporidiosis and toxoplasmosis. They lack any visible means of locomotion (most are intracellular) and have complex life cycles involving sexual and asexual reproduction.. The common feature of all members is the presence of an apical complex in one or more stages of the life cycle. Although the exact components of the apical complex varies among members, it contains enzymes used to penetrate host tissues.

Malaria. Plasmodium species cause malaria. The four principal species are P. falciparum, P. vivax, P. ovale and P. malariae. Malaria means "bad air" and dates from the time when the disease was thought to be spread from stagnant, foul smelling water. The disease is in fact transmitted by the female Anopheles mosquito that inhabits such environments. In the stomach of the female Anopheles male (micro-) and female (macro-) gametocytes

fuse to form a zygote. This in turn forms a motile ookinete that penetrates the midgut wall and develops into an oocyst within which are many thousands of sporozoites. When mature, the sporozoites rupture the oocyst and penetrate the salivary glands. When the mosquito next feeds on man, the sporozoites are passed via the blood stream to infect parenchymal cells of the liver. Here they form pre-erythrocytic schizonts in which several thousand daughter cells, called merozoites. These merozoites enter red blood cells to start the asexual intraerythrocytic cycle and form new gametocytes. The asexual red cell stages are responsible for the pathological changes that occur in malaria (fever, chills, anaemia, liver enlargement, encephalitis renal damage and death). Cryptosporidiosis. Cryptosporidium parvum causes diarrhoeal disease mainly in infants and small children. It is normally self-limiting but in the immunocompromised host the disease can be severe. C. parvum is enzootic in young calves and is usually passed to man in water containing oocysts of the organism. Video Toxoplasmosis. Toxoplasma gondii causes the multi-organ infection of toxoplasmosis. The domestic cat is the definitive host for T. gondii from which man and other mammals can become infected. Infection commonly arises from the consumption of under cooked meat and in the healthy adult is usually asymptomatic. The most devastating form of toxoplasmosis is seen in congenital infection when a pregnant mother passes the organism to the foetus. This can result in severe abnormalities at birth. The life-cycle of T. gondii is complex, involving both sexual and asexual reproduction. Three main life forms of T. gondii occur: (i) the oocyst which is produced from the sexual cycle in the small intestine of the cat and contains sporozoites; (ii) the tachyzoite of the asexual invasive form found in secondary hosts which are derived from pseudocysts; and (iii) the tissue cyst that contains bradyzoites. The life cycle of T. gondii is shown in the following diagram:
Microsporans The medical importance of microsporidial infections has only recently been highlighted by the frequent recognition of these obligate intracellular parasites in material from patients with HIV infection and AIDS. Examples are: Encephalitozoon species, Nosema species and Septata intestinalis. Multi-organ infections occur and S. intestinalis is found in about 2% of all AIDS patients with chronic diarrhoea.
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Medically Important Helminths

In contrast to the protozoa, helminths (worms and flukes) are multicellular with complex reproductive systems and life cycles involving intermediate hosts for the development of larval stages and a definitive host for the adult form. Adults may be dioecious with separate sexes or hermaphroditic. Nematodes -the filariases These are arthropod (insect)-borne infections caused by filarial worms. The classic example is elephantiasis caused by Wuchereia bancrofti. Larvae of W. bancrofti develop in the mosquito and, as in malaria, human infection results from the bite of the insect. When bitten, the larvae (as male and female forms) pass through the lymphatics and mature to thread-like adults, 4-8 cm long, in the lymphatic glands. After mating the females develop eggs and larvae that are released as microfilariae into the peripheral circulation.

The localisation of the adult filariae in the lymph glands causes obstructions in the lymphatic drainage. This then results in the grossly disfiguring condition of elephantiasis that typically involves massive swelling of the legs, scrotum and other extremities. Other arthropod-borne microfilarial infections of man are loaiasis caused by Loa loa. The disease is spread by biting flies and the adult worms migrate along connective tissue, usually reaching the conjunctiva of the eye. Onchocerciasis ("river blindness") is caused by Onchocerca volvulus, a tissue dwelling nematode, the microfilariae of which are predominantly found in the eye and skin.
Soil-Mediated Helminthiases Soil-transmitted helminthic infections are of two types: the hookworms, which undergo a cycle of development in the soil (the larvae being infective), and a group of nematodes that survive in the soil merely as eggs that have to be ingested in order for the cycle to continue.

Hookworms. The most common hookworms are Ancylostoma duodenale and Necator americanus. Adults attach to the walls of the jejunum and females lay large numbers of eggs that are passed out with the faeces. The eggs hatch in the soil and infect man by usually burrowing through the soles of the feet. The larvae then migrate to infect the heart and lungs before passing into the tracheae, pharynx and then the small intestine. Strongyloides stercoralis. Females live in the mucosal glands of the small intestine. Eggs hatch in these glands and the larvae are passed with the faeces into the soil. As with other hookworms, infection results from the larvae burrowing into the skin. The rest of the life cycle is as for A. duodenale and N. americanus. Ascariasis. Adult worms of Ascaris lumbricoides live in the small intestine where they lay large numbers of eggs that are passed out with the faeces. Unlike the hookworms, the eggs are the infectious form in which the larvae develop. When ingested, the eggs hatch in the jejunum, penetrate the mucosa and are carried through the hepatic circulation to the heart and lungs. They again enter the stomach via the tracheae and oesophagus before growing to adulthood in the small intestine. Pneumonitis and intestinal obstruction may accompany heavy infestations. Toxocariasis. The disease results from the accidental infection of man with eggs of the ascarid roundworm of the dog, Toxocara canis, and cat, T. cati. The life cycle is the same as that of Ascaris but the invasive larvae become arrested in various tissues where they are phagocytosed. In the process they induce marked eosinophilia and local tissue reaction commonly involving the liver and eye. Trichurias. Trichuris trichiura ("whipworm") inhabits the caecum where they attach to the mucosa. Eggs from the mature worms are passed with the faeces and develop in the soil. When swallowed, the eggs hatch in the small intestine and the developing larvae pass directly to their attachment sites in the large intestine. Heavy infections can cause abdominal pain and chronic bloody diarrhoea that may result in rectal prolapse.
Snail-Mediated Helminthiases This important group of snail-transmitted helminthiases is all caused by trematodes (flukes) that undergo a complicated cycle involving various species of land or aquatic snails. The most significant of these fluke infections is schistosomiasis and over 200 million people are estimated to be infected world-wide. The three common species infecting man, Schistosoma mansoni, S. japonicum and S. haematobium have similar life cycles. Eggs are passed in the urine (S. haematobium) or faeces (Schistosoma mansoni and S. japonicum) and hatch in natural waters. Miracidia hatch from the eggs, penetrate suitable snails and develop two generations of sporocysts. The last of these then produces fork-tailed cercariae. These cercariae penetrate the skin when a new host comes into contact with the contaminated water. Once through the skin the cercariae shed their tails and become schistosomulae that then migrate through the tissues to the liver. Here male and

female flukes copulate and migrate to either the bladder or rectum where eggs are laid. Schistosomiasis can result in chronic liver, spleen and bladder damage.

Fascioliasis. Fasciola hepatica is found in most herbivores (but primarily sheep) that graze in wet pasturage where the intermediate host, snails of the genus Lymnaea, are found. F. hepatica eggs, shed from the infected primary host, mature into the embryonated form in the environment. These then hatch and release a motile miracidia that seeks out and penetrates the tissue of the intermediate snail host. Cercaria are produced in the snail that when released into the environment can encyst to produce metacercariae. In temperate climates man is often infected by eating wild watercress on which metacercariae have collected. After being ingested, the metacercariae pass through the duodenal wall and penetrate the liver capsule. Following maturation of the young flukes, the adults finally come to lie in the bile ducts or adjacent liver tissue. Here they cause severe damage to the biliary tract and eggs are passed with the bile into the faeces to continue the cycle.
Helminth infections acquired through the gastrointestinal tract Trichinosis. Trichinella spiralis is the cause of trichinosis in man. The nematode circulates between rats and pigs with man becoming infected from eating raw or inadequately cooked pork products. Encysted larvae in the meat excyst (hatch) in the intestine and develop into minute adults in the mucosa. These mature and the females deposit larvae that then migrate through the tissues to reach skeletal muscles in which they encyst. Human infections may be asymptomatic but can include fever, orbital oedema, myalgia and eosinophilia. In the extreme, infection can be fatal through myocarditis or encephalitis.

Enterobiasis. Enterobias vermicularis is a small thread-like "pinworm" mainly infecting young children. The female emerges to the perianal region usually at night and lays some 10,000-15,000 eggs and then dies. In the process they cause severe pruritis (itching). The embryonated eggs are infectious on ingestion and hatch in the duodenum. The larvae pass to the caecum where they mature into adults. Because of the pruritis, children often re-infect themselves from eggs under their fingernails. Bedding is also a source of infection and can be a means of spreading the organism in families and institutions such as orphanages and boarding schools. Taenia solium (pork tapeworm). The adult lives in the small intestine of man that is the definitive host. Segments of the worm pass through the anus and release large numbers of eggs that can survive for long periods outside of the body. When ingested by pigs, the eggs hatch and each releases an onchosphere that migrates through the intestinal wall and blood vessels to reach striated muscle where encystment occurs. When inadequately cooked pig meat is eaten by man, excystment occurs in the small intestine and an adult cestode (worm) develops. If the eggs are released into the upper intestine of man (e.g. through regurgitation) they can invade the host setting

up a potentially dangerous larval infection known as cysticercosis in muscle and other sites. T. saginata (beef tapeworm). This also infects man through cattle. The life cycle is similar to T. solium and in both species the adult tapeworm can grow up to 10 meters in length. Hydatidosis. This is caused by the tapeworm Echinococcus granulosus. The adult worm inhabits the small intestine of dogs from which the eggs of the species are passed. These eggs can be ingested by herbivorous animals and hatch in the duodenum. The embryos enter the circulation where they are carried to various sites to develop into cysts. Dogs become infected when they eat contaminated offal. Humans are infected if they accidentally ingest eggs from infected dogs and the liver is the most common site of infection in which hydatid cysts form.
References Baker, J.R. and Muller, R (Eds). Advances in Parasitology. Academic Press, London. Knight, R. (1982). Parasites Diseases in Man. Churchill Livingstone, London. Peters, W. and Gilles, H.M. (1995) Colour Atlas of Tropical Medicine and Parasitology. Mosby-Wolfe.

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