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Professor Michael Kotiw...

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BIO1104 - Medical Microbiology/Immunology Tuesday, 8 March 2011 3:09 PM

Lecture 11 - Microbial Genetics 1: Objectives To have a brief overview of how prokaryotic and eukaryotic DNA

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differs  

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To become aware of how bacteria grow To understand how bacteria can exchange genetic material Lecture 11 Summary Introduced genetic differences between prokaryotes and eukaryotes Discussed bacterial replication by binary fission Discussed how bacteria can acquire antibiotic resistance (R factors) by  Mutation  Transformation  Conjugation  Transduction Practice Questions Q1. Bacteria divide by a process called: A. Meiosis B. Mitosis C. Binary fission D. Sexual budding E. All the above Q2. Lambda is a bacteriophage. This means it is: A. A bacteria with a virus B. A bacterial enzyme C. A protist with a virus D. A fungal virus E. None of the above Q3. Plasmids are best described as: A. Bacterial viruses B. Extrachromosomal circular DNA C. Bacteriophages D. Cephalosporin enzymes

E. Betalactamase enzymes Q4. Describe the transduction, conjugation and transformation methods of DNA transfer in bacteria.

Lecture 12 - Microbial Genetics 2 objectives  

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To overview bacterial genomic replication To examine in detail the mechanisms by which bacteria can exchange genetic material by: 1.Transformation 2.Transduction 3.Conjugation To provide a brief overview of the nature of mobile elements Practice Questions Q1. The origin of replication in bacteria: (a) Occurs at multiple sites in the bacterial chromosome. (b) Does not involve semi-conservative replication. (c) Involves rolling circle replication. (d) Is not found in plasmid DNA. (e) Involves unidirectional DNA replication. Q2: Describe the nature of genetic transfer by conjugation. Your answer should differentiate between F+, Hfr and F’ conjugation processes Q3. Describe the nature of transduction. Your answer should include discussion of generalized and specialized transduction.

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Lecture 13 - Microbial Pathogenesis To define medical microbiology To overview some infectious diseases caused by  Bacteria  Fungi  Viruses  Parasites To examine the nature of infectious disease To examine microbial factors that lead to disease Lecture 13 - Summary

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We have overviewed aspects of infectious diseases

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We have defined the term medical microbiology We have defined different levels of pathogens We have looked at some representative bacterial, fungal, viral and parasitic infections

Practice Questions Discuss the differences between exotoxins and endotoxins Define and discuss with examples the term ‘microbial virulence factors’  Define Koch’s postulates and their purpose.  Discuss, with examples what is meant by the following terms when referring to disease –causing microbes  Pathogen  Opportunistic Pathogen  Commensal microbe  

Lecture 14 - Microbial Pathogenesis 2 Examine case studies in bacterial infections:  Bacterial pneumonia  Bacterial epiglottitis  Bacterial meningitis  Sepsis Practice Questions Write notes on bacterial pneumonia

Lecture 1: Microbiology and Immunology 1: Introduction Monday, 7 March 2011 4:02 PM

Today's Topics:  History and Diversity of Microorganisms Announcements:  Homework:

Lecture Topic: History and Diversity of Microorganisms During the lecture, take notes here.

OVERVIEW 

What is Microbiology?

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- Study of organisms that are too small to be seen by the unaided eye Main Organisms Studied: - Bacteria, fungi, viruses, micro-algae, protozoa and helminths (worms) - Size range from 3mm (protozoa) to 30nm (viruses)

THE SCOPE OF MICROBIOLOGY 

Microbiology is a very diverse and complex discipline, because of the wide range of microorganisms studied and their many characteristics...

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Includes different disciplines such as agriculture, immunology, genetics, ecology, industry and genetic engineering.

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Microbiology has numerous practical uses particularly in medicine and industry

(Taxonomy - Place within the naming system)

THE IMPACTS OF MICROORGANISMS ON EARTH  

Life on earth cannot exist without microorganisms They live in diverse and extreme habitats Eg. Oceans where micro algae and cyana-bacteria contribute to more than 50 % of the photosynthesis on earth...

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They also have a massive impact on energy and nutrients flow - What they lack inside, they make up for it in huge numbers and effects...

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Refer to examples of microbes in different environments - What others can you think of? * Algae in a pond, fungi in a decomposing fruit and etc….

APPLICATIONS USING MICROORGANISMS 

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There is evidence of microorganisms at work in everyday life - How many can you identify? * Composting, flavouring in foods, beer, wine, cheese and yoghurt, Sauerkraut, Vegemite Genetic Engineering (would not be possible without bacteria) Bioremediation Metal exclusion Industrial Fermentors - Antibodies, food additives, washing powder, enzymes….

INFECTIOUS DISEASES OF HUMANS AND PLANTS 

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Although microbes are useful there are also over 2000 pathogenic types that causes disease on humans and others which attack plants and animals Pathogenic: Disease causing organism Microorganisms cause food spoilage and food poisoning (Salmonella) The malnourished and unvaccinated are the most vulnerable - one-third of the world population

HISTORICAL FOUNDATIONS OF MICROBIOLOGY 

Hippocrates (Greek physician, 400 B.C) prepared ethical standards for the practice of medicine that are still in use today.

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He was a keen observer and realised that: - Diseases could be transmitted by objects such as clothing - People recovered from the plague could take care of plague victims without being infected again

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Microbiology is a rather "newer" discipline when we compare the other disciplines such as physics for example.

Invention of the Microscope 

What was the most significant invention for the development of microbiology? - YES! The Microscope….

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Because organisms in microbiology were not visible to the unaided eye, people could not prove or connect the fact that microorganisms were causing diseases. It wasn't until there was some sort of advances in microscopy that they could actually start linking the two together.

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Linkage occurred in Mid 1600s

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Robert Hooke (English Physicist) - Built one of the first compound microscopes and observed cork material - Coined the word "Cells" because it reminded him of cells. - Observed 'cells' - the dead material with the walls left behind - Considered to be one of the first applied microbiologist

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Antonie Van Leeuwenhoek (Dutch Linen Merchant) - He was the first person to make specific lenses at a power which was able to see things with a good resolution. - Made microscopes and lenses to look at flaws in fabric but became fascinated by the microscopic 'animalcules' he saw - First to describe bacteria and protozoa * Considered to be the 'Father' of bacteriology and protozoology

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Louise Pasteur disproved the belief of Spontaneous Generation using scientific method - Unequival support to what is known as the Germ Theory of Disease (eg. microorganisms [germs] can invade other organisms and cause disease)

How he disproved Spontaneous Generation: 

Using a round bottom flask which had a swan neck in it, the liquid in it was heated by fire in a Bunsen flame and heated it to the point where it was sterile.

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What he thought was that if he did not have any organisms coming down from the atmosphere, they would settle in the area in the swan neck and therefore unable to travel up to the sterile liquid.

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once that was broken it would then allow the organisms down into the round bottom flask thus making the flask unsterile.

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Robert Koch devised a series of important postulates that provided proof of the germ theory in disease.

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It helped prove etiology (the casual agent) of many diseases. These postulates are still in use today.

Koch's postulates are: 

The Microorganisms must be regularly isolated from the diseased host (constant associations between microorganisms and the diseased host).

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It must be isolated and grown in pure culture in vitro (Done outside the host whereas in vivo means inside the host). Its cultural characteristics must be described (what is looks like such as: Size, colour etc...). (Macroscopic: Able to be viewed with the naked eye)

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The pure culture of the organism must be inoculated into healthy tissue of the same host species and produce the same disease.

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From the experimental host, the same organism must be reisolated and again grown in pure culture, with characteristics matching those of the first isolation.

* There are a few instances where Koch's Postulates could not be proven. Eg. Can the Microorganism grow in pure culture? Some cannot be isolated. Are there any ethical considerations? For example you cannot inoculate someone for step 3.

TAXONOMY 

Carl Von Linne (Limmaeus) laid down basic rules of taxonomy - That is the formal system of organising, classifying and naming living things

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Bionomial System: Latin names with two parts Eg. * Vitis vinifera (Grape vine) * Equus caballus (domestic horse) * Homo sapiens (us) *Escherichia coli (intestinal bacteria) - Always Italics however underlined when handwritten - MUST be the correct spelling

LEVELS OF CLASSIFICATION 

Prokaryotes divided into two major groups - Bacteria - Achaea (or Archaebacteria, which are organisms which are not as evolved as the bacteria) = MONERA

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How do they fit into the five kingdom system? - Plants - Animals - Fungi - Protists - *Monera

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Characteristics used in bacterial classification: 1. Microscopic morphology 2. Macroscopic morphology 3. Physiological/Biochemical characteristics 4. Chemical composition of cells 5. Serological analysis 6. Genetic and molecular analysis

Summary After class, use this space to summarize the main points of this Lecture Topic.  Overview of Microbiology  Impacts of Microorganisms on earth  Infectious Diseases  Historical foundations on Microbiology  "Postulates" by Koch  Taxonomy Study Questions After the lecture, create questions based on the notes. Koch's postulates: Can the organism be grown in pure culture?

At the end of this topic you should be able to: Appreciate the importance of microorganisms in our everyday

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lives  

Know the significance of microbes to the ecology of life on Earth Know the different types of microorganisms studied (i.e. bacteria, fungi, viruses….)

Know some important uses and activities of microorganisms

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Be able to describe some of the important stages in the history of microbiology and the contribution of some key players (van Leeuwenhoek, Pasteur & Koch)

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Know the basis of the scientific method and the difference between inductive and deductive reasoning

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Know the key terms such as fermentation, spontaneous generation, germ theory of disease, Koch’s postulates

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Understand why it is necessary to classify organisms and the major ways this is done for prokaryotes

Lecture 2: Methods for Visualizing Microorganisms Monday, 7 March 2011 4:02 PM

Today's Topics:  Units of Size  Properties of Light  Compound Microscopes Announcements:  Homework:

Lecture Topic: Methods for Visualizing Microorganisms During the lecture, take notes here.

UNITS OF SIZE Review of exponential notation

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Fig 3.2 is an indication of the relative sizes of both macroscopic and microscopic organisms. Microbiologists are mainly concerned with: 

Micrometre (mm) is a thousandth of a millimetre (mm) or a millionth of a meter (m). Exponentially this is 1 x 10-6m

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Nanometre (nm) is ten-thousandths of a millimetre (mm) or exponentially 1 x 10-9m

PROPERTIES OF LIGHT Light behaves both as particles called photons and waves

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Wavelength: the distance between two adjacent crests or two adjacent troughs = lambda λ The colour of visible light is determined by its wavelength

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Electro-magnetic radiation EMR Ranges from short to long wavelengths (Gamma rays to radio waves)

Light microscopy only makes use of visible part of EMR

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Black 7th Ed Fig 3.4 p 55 

Light Microscopy uses many different properties of light:

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Reflection: Light hitting a specimen can be reflected and we can observe the reflected light

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Transmission: Light entering a specimen and which comes out the other side transmitted Absorption: Light entering a specimen is absorbed and does not

A. leave.

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Refraction: Light is bent at an angle so it is transmitted at a different angle relative to it entering the specimen.

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Fluorescent: Light is absorbed by a specimen which molecules within it (usually of shorter λ). Filters in the pathway of the light coming out of the specimen, can block the exciting light so that we can only see the fluorescent light

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Phosphorescence: Similar to except light emission by the specimen continues after illumination (excitation) has stopped.

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Luminescence: Is the emission of light by a specimen.

THE COMPOUND LIGHT MICROSCOPE 

This is the one we use in the lab - Why is it called a 'compound light' microscope?

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It has a compound set of lenses It uses light as a its illumination source

MAGNIFICATION 

Total magnification is calculated by the magnification of the objective by the magnification of the eyepiece - scanning (4x) x (10x) = 40x magnification - low power (10x) x (10x) = 100x magnification - high ‘dry’ (40X) x (10x) = 400x magnification - oil immersion (100x) x (10x) = 1000x magnification

RESOLUTION AND RESOLVING POWER  

Resolution is the ability to see two separate objects The unaided eye cannot separate two points less than 0.2mm apart - i.e The human eye is inadequate for most microorganisms

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Resolving power or resolution (R.P) = Wavelength of Light (λ) / 2 X Numerical Aperture (NA) of Lens

EFFECT OF λ ON RESOLUTION 

Resolving power/resolution (R.P) is limited by the wavelength of light used by the instrument THEREFORE: The smaller the wavelength = smaller the resolution or resolving power = smaller the objects we see.

WHAT IS NUMERICAL APERTURE? 

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NA is the widest cone of light that can enter the lens - Greater the NA of objective lens, the better and smaller the RP (Resolving Power/Resolution) (i.e. the > NA the more light gets in the lens) Each objective has a defined NA (Written on the side)

NA AND OIL IMMERSION 

x100 objective is used with immersion oil placed between specimens and lens - WHY??? * Because…. Cut down Light Loss as reflected or refracted light - HOW???

* Immersion Oil has the same refractive index as glass i.e. it improves the NA of the lens

NOTE: Even the best light microscope have RP of no smaller than 200nm (0.2µm) and this is limited by the wavelength of light

VARIATIONS IN MICROSCOPY TECHNIQUE     

Bright field (most common type): transmitted light is observed directly through the specimen Fluorescence Dark Field Phase Contrast Differential interference contrast (Nomarski) Latter 3 used on living cells (observe sub cellular structures)

THE ELECTRON MICROSCOPE (EM) 

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Electron Microscope uses a beam of electrons instead of light - WHY??? *Shorter λ = better resolution of smaller objects (Re : eqn for RP) (λ e @ 0.005nm c/f light @ 500nm) EM can resolve objects 0.2nm apart

PREPARING SPECIMENS FOR LIGHT MICROSCOPY 

Wet mounts - Simplest method - Drop of culture on a slide overlaid with a cover slip

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Hanging drop - Drop of culture 'hangs' from the cover slip - Good for viewing microbial motility Hanging Drop Technique (Below)

PRINCIPALS OF STAINING 

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Use stains to colour cells artificially How do stains work? - Stains are salts, composed of +ve (basic) or -ve ions (acidic) Bacteria are slightly -ve (acidic), therefore stain with basic dyes +ve (basic)

SIMPLE STAINS 

Solution of a single (basic) stain usually alcohol in water Eg. Crystal violet, methylene blue and safranin

DIFFERENTIAL STAINS  

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Distinguish different kinds of bacterial cells or 'differently' stain parts of cells Three main stains - Gram stain - Ziehl-Neelsen acid-fast stain - Schaeffer-Fulton endospore stain Gram Stain - Developed in 1884 by Hans Christian Gram, important because it divides bacteria into 2 groups - Gram Positive: stain with crystal violet - Gram Negative: don't stain with crystal violet but take up counter stain safranin Based on cell wall composition o Gram positive : thick peptidoglycan (a thick cell wall) o Gram negative : thin peptidoglycan (a thin cell wall)

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Acid Fast Stain - Identifies bacteria within the genus Mycobacterium Stain is carbolfuchsin which colours cells red, and is not easily removed Counter stain is Methylene blue

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Schaeffer-Fulton endospore stain - Endospores are internal survival structures (Bacillus genera) Highly resistant to usual staining techniques

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Black 7th Ed Fig 3.2 p 54

Summary After class, use this space to summarize the main points of this Lecture Topic.  Units of size  Light: Wavelength, Crest, Trough,  Properties of Light  Properties of a Compound Microscope  Magnification  Resolution  Stains Study Questions After the lecture, create questions based on the notes.

At the end of topic 2 you should be able to:  Explain the necessity for the use of microscopy in this discipline 

List the units of measurement used for microorganisms

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Understand the properties of light and be able to define: reflection, transmission, absorption, luminescence, fluorescence, phosphorescence, refraction, wavelength

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Define resolution, numerical aperture; understand the reason for the use of oil with the x100 objective

Lecture 3: Cell Structure and Function Wednesday, 16 March 2011 3:01 PM

Today's Topics:  Announcements:  Homework:

Lecture Topic: During the lecture, take notes here.

THE CELL CONCEPT  

Individual microorganisms (except viruses) is an independent unit that is able to sustain itself and IS alive, IS a CELL All living cells are similar - Nucleus: genetic information - Cytoplasm - Cell membrane * Semi-permeable - Cell Walls

BASIC CELL TYPE 

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Two distinct types: - ...