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Microbiology

MODULE TITLE: INTRODUCTION TO MEDICNE

MODULE CODE:BM-IM401/1 Course title: Microbiology

Course Instructor: Meka A. MSc.

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Course Objectives

At the end of the course the students should be able to:

Describe the historical development of microbiology and the natural history of microbial diseases

Explain the rationale for classifying microbes into bacteria, fungi viruses, parasites

Classify microorganisms and illustrate their cellular/anatomic characteristics in general

Discuss the Unique differentiating features of eukaryotes and prokaryotes

Identify the source and spread of microbes

Describe The nature of bacteria Discuss the morphological differences and Growth requirement of

bacteria, nomenclature and classification of bacteria Explain biology of protozoa Describe medically important helminths, ectoparasites Discuss the nature and properties of viruses Explain brief appraisal of pathogenicity of viruses Describe nature of fungi : basic structures and classification Identify sterilization and disinfection methods List the different mechanisms of disinfection and sterilization

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Introduction

Microbiology Is the study of microorganisms Microorganisms are all single-celled microscopic organisms and include

the viruses, which are microscopic but not cellular Microbial cells differ in a fundamental way from the cells of plants and

animals microorganisms are independent entities that carry out their life

processes independently of other cells

The science of microbiology revolves around two interconnected themes:

(1) understanding the living world of microscopic organisms,

(2) applying our understanding of microbial life processes for the benefit of humankind and planet Earth

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Microorganisms

The main types of medically important microorganisms:

1. Bacterium,

2. Fungus,

3. Virus,

4. Protozoan,

5.Helminth.

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Historical development of microbiology and the natural history of microbial diseases

Microorganisms are visible with the help of magnifying

lenses (ex. Microscope)→ Microscope is the tool of microbiologist. Microbiology started after the discovery of magnifying

lenses. Microscope has two major roles:

i. Magnification- Enlarging the sizeii. Resolution – showing the fine details

(scattering)

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Progress In Microscopy 1590 Hans & Zacharius Janssen: Lense makers 1665 Robert Hooke- views and describes fungi 1676 Anthony van Leeuwenhoek observed

first microscopic organism, blood cells and protists

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Robert Hooke and early microscopy

The first descriptions ofMicroorganisms by Robert Hooke in Micrographia in 1665

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Microorganisms were first observed by Antonie van Leeuwenhoek, using a primitive microscope

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(a) A replica of Antoni van Leeuwenhoek’s microscope. (b) Van Leeuwenhoek’s drawings of bacteria, published in 1684.Even from these simple drawings we can recognize severalshapes of common bacteria: A, C, F, and G, rods; E, cocci;H, packets of cocci

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Photomicrograph of a human blood smear taken through a van Leeuwenhoek microscope. Red blood cells are clearly apparent

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Progress in Tools

• 1883 Carl Zeiss and Ernst Abbe make

advancements in microscopy lenses and techniques.

• 1931 Ernst Ruska- first electronic microscope

Christian Grham – the use of stains

Summary of types of Microscope

Microscopy Bright Field Light Microscopy Dark Field Microscopy Phase Contrast Microscopy Fluorescent Microscopy Electron Microscopy Transmission Electron Microscopy - TEM Scanning Electron Microscopy - SEM

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Investigator Nationality Date Contributions

Robert Hooke English 1664 Discovery of microorganisms (fungi)

Antoni van Leeuwenhoek

Dutch 1684 Discovery of bacteria

Edward Jenner English 1798 Vaccination (smallpox)

Louis Pasteur French Mid- to late 1800s

Mechanism of fermentation, defeat of spontaneous generation, rabies and othervaccines, principles of immunization

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Investigator Nationality Date Contributions

Joseph Lister English 1867 Methods for preventing infections during surgeries

Ferdinand Cohn

German 1876 Discovery of endospores, preventing culture media from contamination

Robert Koch German Late 1800s Koch’s postulates, pure culture microbiology, discovery of agents oftuberculosis and cholera

Martinus Beijerinck Late

Dutch 1800s to 1920 Enrichment culture technique, discovery of many metabolic groups of bacteria,concept of a virus

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Factors that contributed to the dev’t of Microbiology

1. Controversy between the supporters ofAbiogenesis (Theory of spontaneous generation) and

Biogenesis

2. The Germ Theory of Diseases

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The controversy

Spontaneous GenerationLiving things arise from non-living matter

spontaneously

BiogenesisLiving things arise from pre-existing life form

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Theory of Spontaneous generation

Early belief that some forms of life could

arise from vital forces present in nonliving or decomposing matter.

ex. flies from manure, etc

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Theory of Biogenesis

Some of the Supporters of Biogenesis

Francesco Redi

Lazaro Spallanzani

Louis Paster

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Challenges to spontaneous generation

Does Spontaneous Generation work? Francesco Redi’s Experiment (1600’s) Problem: Did rotting meat produces

maggots spontanously? Experiment: Used 3 jars:

1.Covered2.Uncovered3.Meshed & closed

Result: No growth in boiled

and closed jar.

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Lazaro Spallanzani’s Experiment

Spallanzani's Problem :What causes microbes to form in decaying broth?

Hypothesis: Microbes come from the air. Boiling will kill microorganisms.

Experiment: Spallanzani put broth into four flasks Flask 1 was left open Flask 2 was sealed Flask 3 was boiled and then left open Flask 4 was boiled and then sealed

Result: No growth only in Flask 4. Conclusion: Life arise from pre-existing form.

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Spallanzani is best known for his experiments to disprove abiogenesis. He showed having boiled a broth and then sealed the container, no microorganisms would grow

That is, broth did not spontaneously produce microorganisms

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Louis Pasteur’s Experiment

Louis Pasteur did an experiment to show bacteria do not arise spontaneously.

He showed microbes caused

fermentation and spoilage,

and disproved spontaneous

Louis Pasteur’s Experiment

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Louis Pasteur’s Exp.

Pasteur’s work also led to the development of effective sterilization

Food science also owes a debt to Pasteur, as his principles are applied today in the preservation of milk and many other foods by heat treatment (pasteurization).

Pasteur’s fame from his rabies research was legendary and led the French government to establish the Pasteur Institute in Paris in 1888

Originally established as a clinical center for the treatment of rabies and other contagious diseases,

the Pasteur Institute today is a major biomedical research center focused on antiserum and vaccine research and production

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Significances of the controversy

Existence of microorganism in two forms: Vegetative - heat labile Spore form – resistance to heat

Spores and sterilizationAseptic techniqueSterilization techniques

“Pasteurization” Heat Chemical

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History of Microbiology cont…

Spores and sterilization

• Some microbes in dust and air were resistant to high heat.

• Spores were later identified.

• The term “sterile” was introduced which meant completely eliminating all life forms from objects or materials.

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Microbiology as a science

Developed through accumulation of knowledge following scientific methods.

Scientific Method involve: Identification of problem/gap of knowledge

• Hypothesis

• Experimentation

• Results

• Conclusion or theory

Natural History of Microbial Diseases

Even as early as the sixteenth century it was thought that something that induced disease could be transmitted from a diseased person to a healthy person.

After the discovery of microorganisms, it was widely believed that they were responsible, but definitive proof was lacking Improvements

In sanitation by Ignaz Semmelweis and Joseph Lister provided indirect evidence for the importance of microorganisms in causing human diseases

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Do Microbes Cause Disease?

• 1546 Girolamo Fracastoro wrote about“contagion” – communicable disease

Mainly philosophical as the existence of MOs were not known Proposed that disease are transmitted by:

1. direct contact2. through air 3. through inanimate objects such as clothes

1835 Agostino Bassi de Lodi linked a fungi with a silkworm disease– the first recognized contageous agent of animal disease!

but it was the work of a German physician, Robert Koch (1843–1910) that give experimental support to the concept of infectious disease

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Germ theory of disease

Germ Theory is the concept that micro-organisms can cause disease, and this theory is the foundation of modern medicine.

Many diseases are caused by the growth of microbes in the body and not by sins, bad character, or poverty, etc.

Robert Koch was a man who dedicated his life to finding the causes of infectious diseases

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Koch was convinced that microbes caused some diseases However, to test this idea, he needed to isolate the causative agent. Almost all samples from diseased animals or any natural surface

contained many different microbes and it was impossible to tell which one was the problem.

A method was needed to separate these different bacteria. The most common method of isolation was to continually dilute a sample in liquid broth in hopes only one type of microbe would be found

A major contribution to bacterial techniques was the development of methods using solid medium for the cultivation of bacteria.

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Robert Koch’s Experiment

Robert Koch (1843-1910) verified (realized)the Germ theory (and formulated Koch’s postulates).

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Experiment

Figure

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Koch’s Postulates

In 1876 Robert Koch – cultivates Anthrax usingblood serum and published postulates:

1. The disease agent must be present in every case, and absent in healthy individuals.

2. The agent must be isolated and cultured in vitro (i.e. cultivated in a laboratory environment).

3. Disease must be produced when a pureculture is inoculated into susceptible host

4. The agent must be recoverable from infected host

Exceptions to Koch’s postulates

1. Many healthy people carry pathogens but do not exhibit symptoms of the disease.

2. Some microbes are very difficult or impossible to grow in vitro(in the laboratory) in artificial media. Eg. Treponema pallidum

3. Many species are species specific. Eg. Brucella abortus cause abortion in animals but no report in humans.

4. Certain diseases develop only when an opportunistic pathogen invades immunocompromised host

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Contribution of Koch discoveries

The establishment of the fact that disease are caused by microorganisms,

Koch's laboratory also developed methods of pure culture maintenance and aseptic technique.

Aseptic technique involves: the manipulation of pure cultures in a manner that prevents

their contamination by outside microorganisms. Equally important, aseptic technique prevents their spread

into the environment

Contribution of Koch disc…

The postulate not only offered a means for linking the cause and effect of an infectious disease,

but also stressed the importance of laboratory culture of the putative infectious agent

These discoveries led to the development of successful treatments for the prevention and cure of many diseases,

thereby greatly improving the scientific basis of clinical medicine and human health and welfare

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Contribution of Koch disc…..

Koch announced his discovery of the cause of tuberculosis M. tuberculosis in 1882

and published a paper on the subject in 1884 in which his postulates are most clearly stated.

For his contributions on tuberculosis, Robert Koch was awarded the 1905 Nobel Prize for Physiology or Medicine.

Koch had many other triumphs in medicine, including discovering the organism responsible for the disease cholera

and developing methods to diagnose exposure to M. tuberculosis (the tuberculin test)

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History con…..

Martinus Beijerinck (1851–1931)&Sergei Winogradsky (1856–1953) greatest contribution to the field of microbiology was their clear formulation of the enrichment culture technique

In 1929 Alexander Fleming observed that molds can produce a substance that prevents the growth of bacteria.

His discovery, an antibiotic called penicillin, was later isolated and produced commercially to protect people against the harmful effects of certain microorganisms.

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The Modern Era of Microbiology

In the 1940s microbiology expanded into the fields of molecular biology and genetics.

Viruses were found to be simple microbes that could be studied quantitatively, and they were used to study the nature of DNA

In the early 1970s, genetic researchers discovered recombinant DNA.

Scientists found that DNA could be removed from living cells and spliced together in any combination.

They were able to alter the genetic code dictating the entire structure and function of cells, tissues, and organs.

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The development of early techniques in microbiology

Year Event

1664 Robert Hooke is the first to use a microscope to describe the fruiting structures of molds. He

also coined the term cell when using a microscope to look at cork, as the dead plant material in cork reminded him of a jail cell.

1673 Anton van Leeuwenhoek, a Dutch tradesman and skilled lens maker, is the first to

describe microbes in detail.

1872 Ferdinand Julius Cohn publishes landmark paper on bacteria and the cycling of elements.

In it is an early classification scheme that uses the name Bacillus.

1872 Oscar Brefeld reports the growth of fungal colonies from single spores on gelatin and

the German botanist Joseph Schroeter grows pigmented bacterial colonies on slices of potato.

1877 Robert Koch develops methods for staining bacteria, photographing, and preparing

permanent visual records on slides.

1881 Koch develops solid culture media and the methods for obtaining pure cultures of bacteria.

1882 Angelina Fannie and Walther Hesse in Koch's laboratory develop the use of agar as a

support medium for solid culture.

1884 Hans Christian Gram develops a dye system for identifying bacteria [the Gram stain].

1887 First report of the petri plate by Julius R. Petri.

1915 M. H. McCrady establishes a quantitative approach for analyzing water samples using the

most probable number, multiple-tube fermentation test.

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Microbial Taxonomy

Previously living organisms grouped into five kingdoms: plants, animals, fungi, protists, and bacteria.

DNA sequence-based phylogenetic analysis, on the other hand, has revealed that the five kingdoms do not represent five primary evolutionary lines

Instead, cellular life on Earth has evolved along three primary lineages, called domains.

Two of these domains, the Bacteria and the Archaea, are exclusively composed of prokaryotic cells.

The Eukarya contains the eukaryotes, including the plants, animals, fungi, and protists.

Microbial Taxonomy con…

Definition Taxonomy : is a system for organizing, classifying & naming of living things.

• Primary concerns of taxonomy are:classification, nomenclature, and identification

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Microbial Taxonomy con…

Classification is the organization of organisms into groups on the basis of either phenotypic similarity or evolutionary relationships

The hierarchical nature of classification is that species is made up of one to several strains, and similar species are grouped into genera (singular, genus). Similar genera are grouped into families, familiesm into orders, orders into classes, up to the domain, the highestlevel taxon.

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Microbial Taxonomy cont….

Nomenclature – giving a two word name (Binomial) (Genus and species name)

Identification – assigning to the corresponding taxa or group using the existing system of

classification set

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Microbial Taxonomy cont….

Nomenclature: • Binomial nomenclature: naming organisms using

the genus and species name together It is (scientific)

• Genus – always capitalized(Ex. Bacillus, )

• species – use lowercase (ex. subtilis)

• Both italicized or underlined

-Bacillus subtilis, or

- Bacillus subtilis, or

- B. subtilis

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Microbial Taxonomy cont…

Levels of Classification:• Kingdom/Domain

• Phylum or Division

• Class

• Order

• Family

• Genus

• species

The polyphasic approach to taxonomy uses three kinds of methods—phenotypic, genotypic, and phylogenetic—for the identification and description of bacteria

Phenotypic analysis examines the morphological, metabolic,

physiological, and chemical characteristics of the cell Genotypic analysis considers characteristics of the genome These two kinds of analysis group organisms based on

similarities They are complemented by phylogenetic analysis, which seeks

to place organisms within an evolutionary framework

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Microbial Taxonomy cont…

Domains ►Developed after the five-kingdom system

1. Eubacteria -true bacteria, with true peptidoglycan

2. Archaea –odd bacteria that live in extreme environments, high salt, heat,etc

3. Eukarya- have a nucleus, & organelles

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Microbial Taxonomy cont….

Approaches in Microbial Taxonomy

Classical (Phenotypic)

Molecular (Genotypic)

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Classical Approach to microbial taxonomy

Cell morphologyCell shapes, cell grouping

Physiological characteristicsTolerance to salt, pH, temperature

Biochemical characteristicsCarbohydrate fermentation, utilization of nitrogen

source, etc

SerologyAntigen type

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Molecular (Genotypic) Approaches

Plasmid profiling

DNA-DNA hybridization

DNA sequencing

►All involve Molecular techniques

PCR (Polymerase Chain Reaction)

DNA/Plasmid Extraction, purification, etc

Bacteria:

Bacteria: This domain includes the kingdom of the heterotrophic

eubacteria and includes all human pathogen bacteria. The other kingdoms, for instance that of the

photosynthetic cyanobacteria, are not pathogenic. It is estimated that bacterial species on Earth number

in the hundreds of thousands, of which only about 5500 have been discovered and described in detail.

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Eucarya:

Eucarya: This domain includes all life forms with cell possessing

a genuine nucleus. The plant and animal kingdoms (animales and

plantales) are all eukaryotic life forms Pathogenic eukaryotic microorganisms include fungal

and protozoan species

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Bacteria

• No membrane bounded organelles

• Cell wall made of Peptidoglycans

• Antibiotic sensitivity

• Reproduction by Binary fission

• DNA with no Histones

• 70S ribosome Some are parasitc, others are beneficials (food,

antibiotic, industry, agriculture, environment, medical), some are pathogenic (Salmonella, Shigela), still some others are toxin producing(Clostridium, S.aurus),

Fungal general characteristics

They are achloropyllus Without true root, stem, and leaf, no vascular system Heterotrophic mode of nutrition(they digest and ingest) They follow both sexual and asexual mode of reproduction Some are parasitic;(smuts, rust, athletes foot, ringworm); others are beneficial(antibiotic, food, industry), some form associations(lichen, Mycorrhiza, termites), some produce toxins which is potent chemical to human, animal and

other vertebrates, Some are still poisonous(Amanita spp.) Some are still disease causing Mycosis (athletes foot, ring worm,)

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Major groups of fungi

Fungi• Classification based on:

– Morphology

– Reproduction

_Nutrition, Habitat, life cycle, growth

Molecular

The major Fungal divisions are:

Zygomycetes

Ascomycetes

Basidiomycetes

Deutromycetes /fungi imperfecta/ mitosporic

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Medically important protozoa

• Amoeboid protozoa

– Causes brain infections

• Flagellated protozoa

– Example: Giardiasis

• Apicomplexan protozoa

– Example: Malaria