Michigan State University Main Themes in Microbiology Questions

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Homework 2 Please use this document to take notes/draft your answers. Submit your final answers for grading to the “Homework 2 Final Answers” file to be graded. All answers must be in your own words. Answers copied directly from the textbook, PowerPoint, or an online source will not be given credit. Each question is worth 2 points. 1. What characteristics do all living organisms have? Do you agree that viruses are not living? Why or why not? 2. How can fimbriae make a bacterial cell more virulent? How can a capsule make a bacterial cell more virulent? 3. What is a biofilm and where might you find one? 4. How does a Gram Stain work? Describe each of the 4 reagents used and how they act on a Gram-positive cell vs. a Gram-negative cell. 5. How does Penicillin work? What type of bacteria would it be most effective against? 6. Compare and contrast prokaryotic and eukaryotic cells. Describe at least two ways they are similar and two ways they differ. Topic 3 PPT Cells – Prokaryotes in Particular Outline • Characteristics of life • Two types of cells • Size, Shape, and Arrangements of Prokaryotic Cells • Parts of a Prokaryotic Cell – External – Cell Membrane – Internal Characteristics of life? • Made of cells – Has DNA for storage and transmission of genetic information • Metabolism – Takes in nutrients, uses enzymes, creates energy (ATP), and excretes waste • Reproduction – Asexually or sexually • Growth and development • Movement and/or irritability – respond to internal/external stimuli; self-propulsion of many organisms • Cell support, protection, and storage mechanisms – cell walls, vacuoles, granules and inclusions Characteristics of Life 4 Cells • Cell Theory of Life – “All life is composed of cells and all cells can carry out the basic life processes (metabolism and reproduction).” • Two types of Cells: • Prokaryotic Cells – lack nucleus and other membrane-bound structures (organelles) – Domains of Archaea and Bacteria are both prokarytotes • Eukaryotic Cells – have a nucleus and organelles – Domain of Eukarya or eukaryotes – Includes protozoa/protists, fungi, helminthes, and algae Before moving on… • Brainstorm what you know about prokaryotes and eukaryotes. – What is the same? – What is different? • Can you diagram it? See the next slide for an example. Anything that is unique to eukaryotes would go in the left circle. Anything unique to prokaryotes in the right circle. Any structures, processes, or traits they share would go in the portion that intersects in the middle. Similarities between Prokaryotic and Eukaryotic Cells (these would go in the middle portion of your circles) • DNA • the genetic material, made up of A, T, C, and G, and arranged in a double helix • Plasma membrane • a phospholipid bilayer with proteins that separates the cell from the surrounding environment and functions as a selective barrier for the import and export of materials • Cytoplasm • the rest of the material of the cell within the plasma membrane, excluding the nucleoid region or nucleus, that consists of a fluid portion called the cytosol and the organelles and other particulates suspended in it • Ribosomes • the organelles on which protein synthesis takes place • Reproduction • Both make exact copies of themselves • Metabolism • Both obtain nutrients, make energy, and expel waste • Ability to maintain internal homeostasis Differences Between Prokaryotic & Eukaryotic Cells (these would go in one circle or the other) • DNA location – Prokaryotes have a central region called the nuclear region where genetic material is concentrated, but not bound by a membrane – Eukaryotes have a membrane bound organelle called the nucleus that contains the genetic material • DNA structure – Prokaryotes have a singular, circular chromosome – Eukaryotes have multiple, linear, paired chromosomes • DNA storage (histone proteins) – Present in Eukaryotic cells due to the massive amount of genetic material present. Histones help condense it so it fits in the nucleus. – Not present in Prokaryotes • Extrachromosomal DNA – Present in the mitochondria and chloroplasts of Eukaryotic cells. – Present in plasmids in prokaryotic cells. More Differences Between Prokaryotic & Eukaryotic Cells • Cell Walls • Found in Prokaryotic cells and made of peptidoglycan. Helps the cell maintain shape and avoid lysis via osmosis. • Found in some Eukaryotic cells like plants (cellulose) and fungi (chitin). Absent in animal cells. • Locomotion • Some Prokaryotes are mobile via one or more flagella. The flagella are thin and rotate like a propeller. • Some Eukaryotes are mobile via flagella. The flagella are thick and move side to side. Some eukaryotes have cilia. • Reproduction • Prokaryotes use binary fission. • Eukaryotes use mitosis and meiosis. How many similarities and differences did you come up with? • Did you miss some that are on the list? – Add them to your diagram. • Did you come up with some that are not on the list? – Nice job! • For many students, reading the information alone (in the form of a textbook or PPT) is not enough for them to learn the information. Try to organize the information in a way that lets you see the connections (like the activity you just completed) to help you better understand the material. Size of Prokaryotes • prokaryotes are the smallest of all organisms • they typically range 0.5 – 2.0 µm in diameter and 1.0 – 60 µm in length (Note: 1 µm = 1 millionth of a meter) • • • In Microbiology there is often an exception to the general rule! Most prokaryotes are very small, but Epulopiscium fishelsoni is huge in comparison. (Compare its size to the E. coli cell pictured.) It is a bacterial symbiont of sturgeon fish (80 µm in diameter and 600 µm in length. Relative Size of Living Things Magnification Why are prokaryotes so small? SA/V= 6 SA/V=3 SA/V = 1.5 Small size is an advantage! A larger Surface Area (SA) to Volume (V) ratio = easier diffusion of nutrients in to any part of the cell and quick diffusion of waste products out of the cell. Bacterial Shapes – Coccus – spherical – Bacillus – rod – Coccobacillus – very short and plump (too long to be a cocci, too short to be a bacilli) – Vibrio – gently curved, comma shaped – Spirillum – helical, twisted rod, rigid – Spirochete – spring-like, flexible 16 Common bacterial shapes 17 Pleomorphism • Variation in cell shape and size within a single species • Bacteria of the genus Mycoplasma are noted for their pleomorphism – they lack a cell wall to hold a single shape 18 Bacterial Arrangements • • Arrangements happen when the cell walls of bacteria remain attached following binary fission (reproduction). There is no exchange of cytoplasm or other materials between the connected cells. • Cocci: • • • • • • Singles – no arrangement Pairs - diplococci Groups of four - tetrads Irregular cluster - staphylococci Chains - streptococci Cubical packets - sarcina – Bacilli: • Singles – no arrangement • Pairs - diplobacilli • Chains - streptobacilli 19 What arrangement is this? (1) • Neisseria (22,578X) • Notice how the cells are still attached via the cell wall (yellow) and how there is no sharing of cytoplasm (blue.) Streptococcus (9605X) What arrangement is this? (2) What arrangement is this? (3) What arrangement is this? (4) • Bacilli divide only on the axis of the narrow ends • Sometimes they separate and you have random arrangement of single bacilli. • Sometimes they remain attached in chains. Bacillus megaterum (10,000X) • They never divide along *Rods in chains the wide sides – or Streptobacilli therefore you will never see a cluster of rods. Medically Important Bacteria Structure of a bacterial cell An Overview of Structure of bacterial cells 1. External Structures: Capsules, slime layers, flagella, fimbriae, and pili 2. Cell envelope: cell wall and cell membrane 3. Internal structures: Cytoplasm with ribosomes, nuclear region, and in some cases granules and/or vesicles External Structures • Appendages • Those used for motility – flagella – axial filaments (periplasmic flagella) • Those used for attachment or as channels – fimbriae – pili • Glycocalyx – surface coating • Capsules • Slime layers 27 Flagella • How to classify them based on number and location? • What are they made of? • How do they make a bacterial cell move? • Can a bacterial cell actually “steer” itself towards something it wants? Naming Flagellar Arrangements Monotrichous – single flagellum at one end Lophotrichous – small bunches emerging from the same site Amphitrichous – flagella at both ends of cell Peritrichous – flagella dispersed over surface of cell 29 1. One polar flagellum located at one end or pole Monotrichous flagella = monotrichous flagellum Pseudomonas (3,300X) Amphitrichous flagella Spirillum (694X) 3. Two or more flaggela at one or both ends = Lophotrichous flagella Lophotrichous flagella 4. Flagella all over the surface = Peritrichous flagella (1) Peritrichous flagella Salmonella (1200X) Peritrichous flagella (2) Atrichous Parts of the Flagella • 3 parts: – Filament – long, thin, helical structure composed of protein flagellin – Hook – curved sheath – Basal body – stack of rings firmly anchored in cell wall 36 Flagella movement (1) • Bacterial flagella do not move side to side like eukaryotic flagella do instead they rotate like a spinning propeller • When it rotates counter clockwise the bacterium moves in a straight line referred to as a “run” • When it rotates clockwise it causes the bacteria to “tumble” and change diretionrandomly • Usually bacteria move in a random run and tumble pattern • Very fast, about 10 body lengths/sec! Flagella movement (2) Flagella movement (3) • Sometimes flagellar movement is nonrandom movement towards a chemical (Chemotaxis) or towards light (Phototaxis) • Substances often exist in gradients: areas of higher to lower concentration • Bacteria can sense these gradients by “counting” the number of a particular substance that hit receptors on the cell surface Flagella movement (4) • If the substance is desirable such as nutrient it is called an attractant and the bacterium will move towards it. • If the substance is harmful, it is considered a repellent and the bacterium will move away from it. • The bacterium cannot steer itself in one direction or another. Rather it continues to “run” if it is going in a favorable direction or initiates a “tumble” if it is going in an unfavorable direction. Flagella movement (5) • What do you notice about the bacterium in each of the three conditions? – Is there net movement to one side or the other? – When does the bacteria “run” for a longer period of time? – What makes it “tumble?” Axial Filaments or Periplasmic Flagella • Found in spirochetes • Attached to cell membrane at each end of the cytoplasmic cylinder that forms the body between the cell wall and an outer sheath • Twisting action causes the rigid body to rotate like a corkscrew Axial filaments or Endoflagella Leptospira interrogans (50,000X) Cross section of Axial Filaments • Fine, proteinaceous, hair-like bristles emerging from the cell surface • Function in adhesion to other cells and surfaces • Makes bacteria more dangerous as they are able to adhere and colonize a surface instead of being washed away by mucous, urine, etc. Fimbriae 45 E. coli (14,300X) Conjugation Pili • Attach two cells and form a pathway for the transfer of genetic material (conjugation) • Found only in gramnegative cells • Leads to genetic variety • Problematic for us because this is one of the ways antibiotic resistance genes are spread among bacterial populations Glycocalyx • • Coating of molecules external to the cell wall, made of sugars and/or proteins Two types: 1. Slime layer - loosely organized and attached 2. Capsule - highly organized, tightly attached 47 Capsule • Complex polysaccharide molecules arranged in a loose gel • Secreted by cell wall • Only certain bacteria form capsules and may not do so under all conditions – I.e. outside a host Bacillus anthracis does not form a capsule and inside a host it will – Why??? – Because encapsulated bacteria are able to evade host defense mechanisms (phagocytosis) because the capsule covers up all the bacterial structures the immune system would recognize as foreign Slime Layer • Less tightly bound to the cell wall and is usually thinner than a capsule • Protects the cell against drying, traps nutrients and binds cells together (biofilm) • Allows for adherence to rock surfaces or root hairs of plants to keep them near nutrients or oxygen • Even allows for adherence to tooth surfaces to cause plaque Biofilms • The slime layer helps Biofilms form. This picture is of a biofilm of bacteria growing on tooth enamel. • What is a biofilm? How does the ability to form a biofilm makes a bacterial species more virulent (able to cause disease.) The Cell Envelope • Under the capsule and the slime layer, external covering outside the cytoplasm • Composed of two basic layers: – Cell wall and cell membrane • Maintains cell integrity • Two different groups of bacteria demonstrated by Gram stain: – Gram-positive bacteria: thick cell wall composed primarily of peptidoglycan and cell membrane – Gram-negative bacteria: outer cell membrane, thin peptidoglycan layer, and cell membrane 51 Prokaryotic Cell Wall • Main functions – Maintains characteristic shape of the cell • I.e. if the cell wall is removed the cell will take on a spherical shape – Prevents the cell from bursting from osmotic shock • Osmotic shock is a sudden change in the solute concentration around a cell, causing a rapid change in the movement of water across its cell membrane. – Under conditions of high concentrations of either salts, substrates ,or any solute in the environment surrounding the cell, water is drawn out of the cells through osmosis. This also inhibits the transport of substrates and cofactors into the cell thus “shocking” the cell. Alternatively, at low concentrations of solutes, water enters the cell in large amounts, causing it to swell and either burst or undergo apoptosis. – Does not regulate entry of materials into and out of the cell (job of cell membrane) • is rigid yet extremely porous – Differences in the properties of cell walls result in different appearances when stained and the ability to distinguish between G-, G+, and acid-fast bacteria. Structure of Cell Walls • Peptidoglycan is the primary component: – Unique macromolecule composed of a repeating framework of long glycan chains cross-linked by short peptide fragments – The Antibiotic penicillin is effective because it interferes with synthesis of peptidoglycan components 53 Gram-Positive Cell Wall – thick peptidoglycan layer – Includes teichoic acid and lipoteichoic acid: function in cell wall maintenance and enlargement during cell division; move cations across the cell envelope; stimulate a specific immune response – Some cells have a periplasmic space, between the cell membrane and cell wall 54 Gram-Negative Cell Wall • Inner and outer membranes and periplasmic space between them contains a thin peptidoglycan layer • Outer membrane contains lipopolysaccharides (LPS) • Lipid portion (endotoxin) may become toxic when released during infections • G- are less susceptible to penicillin because this outer membrane prevents against its entry into the cell • Contain porin proteins in upper layer – regulate molecules entering and leaving cell 55 Structures of Gram-Positive and Gram-Negative Bacterial Cell Walls 56 Another view of Gram-Positive and Gram-Negative Bacterial Cell Walls Comparison of Gram-Positive and Gram-Negative Cell Walls 58 What does the cell wall tell us about bacteria? • Differential stains (I.e. the Gram Stain or the acid fast stain) allow us to differentiate between types of bacteria based on differences in their structures The Gram Stain (1) The Gram Stain (2) How does the Gram Stain work? • G+ bacteria have very thick peptidoglycan and they retain the crystal violet = purple • G- bacteria have very thin walls of peptidoglycan lose the crystal violet easily during the decolorization stage and are counter-stained with safranin = red • Very good stain…been around for 150 years! • But some bacteria, gram-variable and acid-fast must be stained using other methods Acid-fast bacteria • Mycobacteria – Have a different cell wall composition • Thick like G+ bacteria • 60% lipid (mycolic acid) and contains much less peptidoglycan • These lipids make the bacteria highly impermeable to most stains as well as protect them from acidic and alkali solutions – Slow growers because this relatively impermiable membrane also impedes entry of nutrients into the cell as well – Acid fast staining uses carbolfuchsin which binds to the cytoplasm then resists removal upon addition of an acidic solution – Why is it so important to have a difinitive way to identify these organisms? • Mycobacterium tuberculosis and Mycobacterium leprae are both acid-fast bacteria that cause serious disease in humans. Acid Fast Cell Wall Wall-Deficient Organisms • Bacteria that belong to the genus Mycoplasma have no cell walls • Protected from osmotic swelling and bursting by a strengthened cell membrane that contains sterols • One example of a sterol is cholesterol. Animal cells also use this molecule to strengthen their cell membranes. Practice • Draw the cell membrane and all parts of the cell wall of the following bacterial types: – Gram-positive – Gram-negative – Acid-fast Selective Toxicity • Antibiotics can have selective toxicity – meaning they are harmful to micorbes without causing damage to the host. Typically this is due to them affecting a structure that is present in microbes and absent in the host. – Penicillin inhibits peptidoglycan synthesis and therefore prevents dividing bacteria from making a functional cell wall and they die from osmotic shock. – Can penicillin be harmful to humans? Do you consider this drug to have good selective toxicity? Prokaryotic Cell Membrane • Phospholipid bilayer with embedded proteins • Represented by the fluid mosaic model • Functions in: – Providing site for energy reactions, nutrient processing, and synthesis – Passage of nutrients into the cell and discharge of wastes – Cell membrane is selectively permeable 68 • Phospholipids (keeps anything besides gasses and water from getting into or out of the cell) • Hydrophilic (waterloving), polar, phosphate head group • Hydrophobic (waterfearing), non-polar fatty acid chains as tails • Proteins serve as gates to allow nutrients in and wastes out Selective Permeability Inside a Prokaryotic Cell • Cytoplasm • Ribosomes • Nucleoid – Chromosome – Plasmids • Storage Structures • Cytoskeleton • Endospores Cytoplasm • Semi-fluid substance inside the plasma membrane • 4/5 water • 1/5 materials dissolved or suspended • • • • Enzymes and other proteins Carbohydrates Lipids Inorganic ions • Site of many chemical reactions Bacterial Ribosome – Site of protein synthesis – Found in all cells – Made of 60% ribosomal RNA and 40% protein – Consist of two subunits: large and small – Prokaryotic ribosomes differ from eukaryotic ribosomes in size and number of proteins • Some antibiotics, streptomycin and erythromycin, disrupt bacterial protein synthesis by binding to the 70S prokaryotic ribosomes but leaving the 80S eukaryotic ribosomes unaffected. Nucleoid • Chromosome – Single, circular, doublestranded DNA molecule that contains all the genetic information required by a cell • Plasmids – Free small circular, doublestranded DNA – Not essential to bacterial growth and metabolism – Used in genetic engineering readily manipulated and transferred from cell to cell Bacterial Internal Storage (1) • Inclusions and granules – Intracellular storage bodies – Vary in size, number, and content – Bacterial cell can use them when environmental sources are depleted 74 Bacterial Internal Storage (2) – 1. Granules • Not membrane-bound • Materials so densely compacted that they do not easily dissolve in cytoplasm • I.e. glycogen, phosphate – 2. Inclusions • Specialized membrane enclosed structures • I.e. gas vacuole used by aquatic organisms to regulate depth at which they float and therefore intensity of light that reaches them • I.e. containing the lipid p-B-hydroxygutyrate which is used as an energy store • I.e. containing iron (magnetosomes) enable the bacteria to respond to the earth’s magnetic fields Spores -Sporulation cycle 76 Endospores (1) • Dehydrated, metabolically inactive • Spore-formation is induced following nutrient depletion – Can also be found to occur in normal conditions and may be a way that the bacteria hold troops in reserve so as not to be wiped out by sudden onset of poor conditions • Resistant to ordinary cleaning methods and boiling – Pressurized steam at 120oC for 20-30 minutes will destroy • Some common spore forming bacteria are Bacillus species and Clostridium species • Are you familiar with Bacillus anthracis, Clostridium tetani, Clostridium botulinum, and Clostridium difficile? 77 • How does the ability to form spores make these organisms more dangerous to humans? Endospores (2) • NOTE: bacterial endospores are not the same as fungal spores which are means of reproduction, endospores are a means of survival Activity: Can you name each structure? Can you briefly describe its function? You may want to organize the information in a table for study purposes. How are prokaryotes and eukaryotes related? • • The first prokaryote appeared approximately 3.5 billion years ago The first eukaryote appeared approximately 1.7-2.2 billion years ago • What led to this increase in cellular complexity??? – Evidence suggests that large prokaryotes engulfed smaller prokaryotes, lived together with them in symbiosis creating a larger, more complex eukaryotic cell. – This is called the Endosymbiotic Theory and was proposed by Lynn Margulis in the 1960s. (Read 5.1 Making 80 Connections in your textbook on page 124) The Endosymbiotic Theory • The term "endosymbiosis" means "to cooperate inside". – – • The smaller cells were likely what we know of as mitochondria and chloroplasts today. – – – • • Advantage to the smaller cell is…protection. No other cells will engulf (and digest!) them. Advantage to the larger cells is…the smaller cells produce energy which can be used by the larger cells. Both of these organelles have their own DNA and their own ribosomes. The DNA is in a single circular chromosome and the ribosomes are 70S. (Remind you of prokaryotic DNA and ribosomes?) In fact, the DNA in the chloroplast is very similar to photosynthetic bacteria called cyanobacteria. The DNA in the mitochondria is most like that of the bacteria that causes typhus. Both of these organelles divide inside the eukaryotic cell independently of the cell cycle in a process that looks very similar to binary fission. (Remind you of prokaryotic cell division?) When the larger cell that had engulfed the smaller cells went to divide, copies of the smaller prokaryotes inside were made and passed down to the daughter cells. Eventually, the smaller prokaryotes that had been engulfed adapted and evolved into some of the organelles we know of today in eukaryotic cells like the mitochondria and chloroplasts. Other organelles eventually arose from these first organelles, including the nucleus where the DNA in a eukaryote is housed, the endoplasmic reticulum and the Golgi Apparatus. In the modern eukaryotic cell, these parts are known as membrane bound organelles. They still do not appear in prokaryotic cells like bacteria and archaea, but are present in all organisms classified under the Eukarya domain. What do you think? • Is the endosymbiotic theory a plausible explanation for the appearance of eukaryotic cells on earth? Topic1 – Main Themes in Microbiology Overall Course Goals ▪ To share knowledge from different sources to give you a healthy appreciation of the microscopic world around you. ▪ To introduce or reintroduce you to scientific concepts that you will encounter in future science courses and/or as a healthcare professional. ▪ To help you distinguish between a good source and a poor source of scientific information. Help you become a good “consumer” of science. Outline ▪ What is microbiology? ▪ Why study microbiology? ▪ Nomenclature (scientific names) of bacteria as well as other living organisms ▪ Classification of living organisms ▪ Historical contributions to the development of microbiology ▪ Fields in microbiology What is Microbiology? ▪ Microbiology = study of microorganisms or microbes ▪ Microorganisms = organisms too small to be seen with the naked eye ▪ Can you give an example of a(n)… ▪ ▪ ▪ ▪ ▪ ▪ Bacteria Algae Fungi Protozoa Helminthe Virus Can you give an example of… ▪ Microbes that are harmful to humans? – Disease causing microbes – Microbes that cause food to spoil ▪ Microbes that are helpful to humans? – Normal microbiota – Microbes used to produce food/beverages As you go through the following slides, you can use this table to help you condense what you know about the 7 classes of microorganisms. Class of Microorganism Prokaryotic Single or /Eukaryotic Multicellular? or Neither? Nutrition type? Examples? Other info? Bacteria ▪ Very small, relatively simple, single-celled ▪ Prokaryotic cells – Lacks a nucleus and other membrane bound organelles ▪ Ubiquitous in Nature ▪ Absorb nutrients from their environment ▪ Diverse metabolic capabilities ▪ Reproduce by Binary Fission ▪ Many are pathogens, but despite the large amount of attention devoted to diseasecausing bacteria less than 1% of all known bacteria are harmful! Archaea ▪ ▪ ▪ Very small, simple, exist as single cells Prokaryotic When first discovered, they got a reputation as extremophiles because they live in what we would consider unlivable conditions – ▪ ▪ Even more metabolically diverse than bacteria Not known to be pathogenic, but many are part of our normal flora – ▪ Bottom of the ocean, in lakes full of sulfuric acid deep in caves, hot springs created by volcanoes, etc. Normal flora are all of the microbes that live in an on our bodies More closely related to eukaryotic cells like us than other prokaryotic cells like bacteria! Where do Archaea fit in? ▪ Eukaryotic cells Algae – Unlike prokaryotic bacteria eukaryotic cells have a nucleus and membrane bound organelles ▪ Most are microscopic, singlecelled organisms ▪ Some are large, multicellular organisms ▪ Autotrophs (self feeders) vs. all the other classes of microbes that are Heterotrophs (other feeders) – Photosynthetic: important oxygen producers in the ecosystem ▪ Found in fresh and salt water ▪ Few are harmful – Red Tide What is Red Tide? Red tide is a result of population explosion or algal bloom ▪ Organisms implicated include (protists) dinoflagellate species Gymnodnium and Gonyaulax ▪ The dinoflagellates produce a powerful neurotoxin that affects vertebrates ▪ The shellfish that eat them are not affected but the fish, seals, or even humans that eat the shellfish get sick Red Tide Continued ▪ Does sufficient cooking of shellfish which have fed on Gonyaulax (the protist dinoflagellate that produces red tide) protect us from getting sick? ▪ Unfortunately, no. ▪ There is no way to visually tell if your shellfish is affected. It doesn’t look or taste differently. We are protected because the shellfish we buy at the store or eat in a restaurant is regularly tested for toxin levels. ▪ You would have to be more vigilant if you were to go out and harvest your own wild shellfish. Fungi ▪ Eukaryotic organisms (nucleus) ▪ Can be single-cellular like yeast or multicellular like mushrooms ▪ Cell walls – most have chitin ▪ Saprophytic (feeds off dead or decaying organic matter) ▪ Found everywhere – especially soil & water ▪ A few species are harmful to humans – i.e. yeast, ringworm, maduromycosis. Fruiting bodies, budding yeast, candiasis of oral cavity, & mandura foot Protozoa ▪ Eukaryotic cells – unicellular ▪ Ingest or engulf food from surroundings ▪ Many can resist drying out/death by going into a dormant state called a cyst ▪ Many known as pathogens: cause malaria, amoebic dysentery, African sleeping sickness, toxoplasmosis Helminthes (Worms) ▪ Eukaryotic cells ▪ Multicellular organisms ▪ But many have microscopic lifestyles, such as dog heartworm, swimmer’s itch, tapeworms Viruses ▪ ▪ ▪ ▪ Very small Noncellular (acellular) No metabolism The ultimate parasite must use a host to reproduce ▪ All groups of organisms are infected by viruses ▪ Many are harmful – HIV, herpes, polio, common cold, measles, rabies, hepatitis, Ebola, etc., etc., etc. Microbial Diversity: 6 of the 7 Types of Microbes Why aren’t archaea always included in the classes of microbes? ▪ Depending on your source you may or may not see archaea included in the types or classes of microbes. ▪ Archaea were only discovered as being distinct from bacteria in the late 70s. ▪ There are no known archaea that are human pathogens. ▪ There are recently discovered archaea found to live in the human gut! ▪ For the purposes of this course, we will consider archaea the 7th class of microbes. Microbial Structure • Two cell lines – Prokaryote – microscopic, unicellular organisms, lack nuclei and membrane-bound organelles – Eukaryote – unicellular (microscopic) and multicellular, nucleus and membrane-bound organelles • Viruses - Acellular, parasitic particles composed of a nucleic acid and protein Top Microbial Causes of Death Why Study Microbiology(1)? Helpful…. – Microbes cover the human body inside and out and are an important part of human health – They outnumber us 10 bacterial cells to 1 human cell! – Important in protecting us from pathogen colonization ▪ Take up space in the body ▪ Compete for nutrients ▪ Even produce antimicrobial compounds Harmful…. – On the other hand, pathogens are microbes too – And even the “helpful” microbes can become harmful if they find a way into an otherwise sterile part of the body (opportunistic pathogens) Why study Microbiology (2)? ▪ Understanding gained from studying complex life processes like metabolism in simple single-celled organisms can be applied to higher organisms ▪ Microbes play an important role in the ecosystem both as a part of many cycles as well as in recycling dead material. N Cycle Why study Microbiology(3)? ▪ Microbes are used in research and medicine – Much simpler than maintaining mice, rats, etc. – Great numbers in little time (E. coli can double in 20 minutes) – Microbes are used to produce large amounts of antibiotics, insulin, etc. at low cost What is life? All life is… ▪ Composed of cells ▪ Carries out similar functions like metabolism – Obtaining nutrients and eliminating wastes ▪ Able to grow and reproduce How do we name living organisms (Taxonomy?) ▪ Carolus Linnaeus (1707-1778) devised the Binomial Nomenclature System – Each distinct species is given a scientific name by scientists that is standard around the world in place of common names that may differ and cause confusion. – Scientific name consists of Genus & species ▪ Escherichia coli, Escherichia is the genus and coli is the species. ▪ The Genus is always capitalized and the species is always lower case. ▪ It is set apart from the rest of the text by putting it in italics if typed or underlined if handwritten. ▪ It must be spelled out in its entirety the first time used. Subsequent uses can be abbreviated with just the first letter of the Genus and the entire species. (E. coli) Sample Taxonomy How do we classify living organisms? We can use the Five-kingdom classification system or the Three-domain classification system. Imagine that you are a scientist living in the 1700s… ▪ You identify a new organism. ▪ How would you classify it? ▪ What tools would you have available to you? Five-Kingdoms ▪ The answer…not many! You would make most of your observations with your eyes alone. You’d note similarities and differences that you could see – otherwise known as morphology. ▪ You would probably group anything green that thrives in sunlight in the “plants” branch. Anything that moved and ate other things in the “animals” branch. Five-Kingdoms Continued ▪ For the small, single celled organisms, anything with a nucleus was placed in “Protists.” While anything without a nucleus was placd in “Monera.” ▪ Monera includes eubacteria (most of the bacteria relevant to human health), cyanobacteria which used to be called blue-green algae because of their photosynthetic nature, as well as archaeobacteria (the extremists) which live in seemingly uninhabitable environments (salty, hot, acidic, cold, under great pressure) New inventions ▪ In 1977, Carl Woese looked at the genetic makeup of living things and found that plants, fungi, animals, and protists were remarkably similar . ▪ On the other hand, there were two very distinct groups of prokaryotes lumped into Monera. ▪ He devised the Three Domain system of classification to better represent how closely related one organism is to another genetically. Three Domains Bacteria - true bacteria Archaea - odd bacteria that live in extreme environments, high salt, heat, etc. Eukarya - have a nucleus and organelles Three Domains Continued ▪ The closer two organism are to one another = the greater their genetic similarity. ▪ So if you look at the end of the Eukarya branch (shown in blue) you can see how closely humans(animals) are related to fungi! ▪ On the other hand, if you look at E. coli (found on the green, Bacteria, branch), you can see how distantly related it is to Methanogens (found on the orange, Archaea, branch.) Antonie van Leeuwenhoek (1632-1723) ▪ Dutch linen merchant ▪ First to observe living microbes ▪ Single-lens magnified up to 300X ▪ Made detailed drawings of microbes he observed in drops of water and most famously from scrapings taken from teeth (see his quote on the next slide) Anton van Leeuwenhoek Quote “For my part I judge, from myself (howbeit I clean my mouth like I’ve already said), that all the people living in our United Netherlands are not as many as the animals that I carry in my own mouth this very day.” Leewenhoek’s “little animals” History of Microbiological Thought ▪ Two opposing schools of thought: – Spontaneous Generation is an early belief that some forms of life could arise from vital forces present in nonliving or decomposing matter ▪ The theory of Spontaneous generation would argue that microbes arise in the body as a RESULT of disease state instead of as the CAUSE of the disease state – Theory of Biogenesis - the idea that living things can only arise from other living things ▪ This theory states that the microbe invades which CAUSES the disease state. Francesco Redi ▪ Experimental Design – Three jars with a piece of meat in the bottom – One left uncovered, one corked, and the last covered with gauze – Where are maggots present? – Did this experiment confirm or refute spontaneous generation? Redi’s Experiment Continued ▪ Redi’s experiment refuted spontaneous generation. – There were maggots present on the meat in the container left open to the air (and the flies that lay egg which develop into maggots.) – There were no maggots found on the meat in the sealed jar. – There were maggots on top of the gauze in the third jar (flies were fooled by being able to sense the meat.) – If spontaneous generation were true there would be maggots on all samples of meat. Louis Pasteur ▪ Louis Pasteur – boiled infusions in flasks and then drew glass tops into swan-necks ▪ No microbes grow in broth ▪ Why did this work? ▪ Microbes can fall but not fly…therefore they get trapped in the dip of the swan neck and never reach the broth to grow ▪ If the swan neck is broken off Microbes will grow Others tried to repeat Pasteur’s work – but failed. Were they wrong? Lack of skill? Or something else? Spore forming microbes contaminated many of these flasks. Pasteur did not have these in his laboratory. Spore forming bacteria concentrate their genetic material into a hard calcium shell that can resist desiccation (drying out), nutrient deprivation, and heat treatments such as boiling or even some autoclaves. (Do not confuse bacterial spores with fungal spores which are used for reproduction. Bacterial spores are merely used for survival in an unfavorable environment. So does that mean that bacterial spores are similar to protozoa cysts?) Once put into a favorable environment bacterial spores can regain their vegetative state and reproduce Pasteur Continued ▪ ▪ ▪ ▪ Portrait painter Teacher Professor of Chemistry Wine and silkworm industries ▪ Found yeast produced good wine and bacteria-contaminate wine was sour ▪ Identified three different pathogens of silkworms and associated each with a specific disease ▪ Pasteurization of wine ▪ Heating in the absence of oxygen ▪ Any other food products pasteurized? ▪ Development of vaccines – rabies in particular ▪ Used the dried spinal cord from rabbits infected with rabies Robert Koch ▪ Identified bacterium causing anthrax: Bacillus anthracis; tuberculosis: Mycobacterium tubuculosis; cholera: Vibrio cholerae. ▪ Recognized active versus dormant cells (spores) ▪ Developed techniques for studying cells in vitro ▪ Pure cultures – found a way to grow specific bacteria separated from other bacteria by streaking (Help from Angelina and Walther Hesse) ▪ Koch’s Postulates – most outstanding achievement ▪ Research on malaria, typhoid fever, sleeping sickness, and more. Koch’s Postulates: One organism-one Disease ▪ 1. The specific causative agent must be found in every case of the disease. ▪ 2. The disease organism must be isolated in pure culture. ▪ 3. Inoculation of a sample of the culture into a healthy, susceptible animal must produce the same disease. ▪ 4. The disease organism must be recovered from the inoculated animal. Koch’s Postulates Continued Angelina & Walther Hesse ▪ Walther was a colleague of Koch. ▪ Angelina suggested that Koch add agar, a cooking thickener, to broth. ▪ Use of agar was a turning point in studies of bacteria because it allowed for isolation of pure culture necessary to identify one organism to one disease in Koch’s Postulates. Hospital Sanitation ▪ Ignaz Philipp Semmelweis – encouraged sanitary practices in hospital ▪ Joseph Lister – Father of antiseptic surgery which reduced the incidence of infections Early Immunology ▪ Immunology – The science of immunology deals with the host’s response to microbial invasion. This science led to the development of vaccines ▪ Jenner – discovered that cowpox protects against smallpox. The word vaccine comes from the Latin vacca for cow. ▪ Pasteur – developed vaccines for rabies and cholera. Using the Scientific Method to Eradicate Small Pox (1) Using the Scientific Method to Eradicate Small Pox (2) Early Virology Virology – special techniques were necessary for isolating, propagating, and analyzing ▪ Porcelain filters originally developed to remove bacteria from water. Used to isolate viruses! ▪ Beijerinck determined that filtrate could be infectious and used the term virus (from the Latin word for poison). ▪ Electron microscopes were developed in the 1930s and first virus seen in 1939. ▪ Hershey and Chase showed that viruses contained DNA and that only the DNA had to go into a host cell (bacteria) to cause more viruses to be formed. ▪ DNA is the genetic information Early Chemotherapy ▪ Chemotherapy - development of drugs to treat disease began at least by the first century AD with herbal medicines ▪ Discovery: quinine from tree bark could treat malarial infections. ▪ Paul Ehrlich – first serious researcher in this area who discovered the first successful drug treatment for syphilis. Coined term “chemotherapy” ▪ Fleming discovered penicillin in 1928. “Others” purified and mass produced during WW II. Antibiotics-the wonder drugs ▪ Along with improved working conditions, better sanitation, better hygiene, immunizations, and less crowding, antibiotics can take credit for increasing life expectancy from 47 in the 1900s to 68 in the 1950s ▪ Many thought we had conquered disease and that further research was not imperative ▪ World focus switches to Cancer in the 1970s ▪ In 1981 the emergence of AIDS was the wake up call ▪ More recently we are dealing with the increase in antibiotic resistant strains of bacteria (another wake up call) Early Molecular Biology ▪ Molecular Biology – youngest branch of microbiology. ▪ Watson and Crick discovered the double helix structure of DNA in 1953. ▪ Microbial genetics led to many important genetic discoveries including the three domain classification system Areas of Study: Fields of Microbiology ▪ Organismal groups: bacteriology, phycology (studies algae), mycology (studies fungi), protozoology, parsitology, virology. ▪ Processes or functions: microbial metabolism, microbial genetics, microbial ecology. ▪ Health Related Fields: immunology, epidemiolgy, etiology, infection control (nosocomial infections), chemotherapy. ▪ Applications: disease related; food & beverage technology, environmental; industrial, pharmaceutical, genetic engineering. Tools of the Microbiology Lab Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. The Microscope Key characteristics of a reliable microscope are: • Magnification – ability to enlarge objects • Resolving power or resolution– ability to show detail 2 Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Magnification Magnification is the enlargement of an image due to an interaction between visible light waves and the curvature of a lens. 3 Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Magnification in Two Phases – The objective lens is found on the “nose” of the microscope. Our lab has microscopes with 4X, 10X, 40X, and 100X objectives. – The ocular lens is found in the eyepiece of the microscope. Our oculars have a magnification of 10X. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Total Magnification • Total magnification of the final image is a product of the separate magnifying powers of the two lenses objective ocular total x = power power magnification Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Resolution • Resolution is the capacity to distinguish or separate two adjacent objects • The level of detail we can see • depends on the wavelength of light that forms the image along with characteristics of the objectives • In the picture on the right, you can see image B is better resolved because the separate fingers are visible Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. The Purpose of Oil is to increase the resolving power of a microscope by. In our lab it is used with the 100X objective. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Bright-field Microscopy • most widely used • specimen is darker than surrounding field • used for live and preserved stained specimens Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Electron Microscopy • Forms an image with a beam of electrons (instead of light) that can be made to travel in wavelike patterns when accelerated to high speeds • Electron waves are 100,000 times shorter than the waves of visible light • Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength • Magnification between 5,000X and 1,000,000X Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Thermal worm – found in deep sea vents…smaller than a bacterial cell and only visible with an electron microscope. What else is out there that we haven’t seen yet?!?! Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Specimen Preparation for Optical Microscopes • Wet mounts and hanging drop mounts – allow examination of characteristics of live cells: size, motility, shape, and arrangement • Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Staining • Dyes are used to create contrast by imparting color to either the microbe (positive stain) or to the background (negative stain) – Simple stains – one dye is used; reveals shape, size, and arrangement – Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain) – Structural stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Staining Examples Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. The 6 I’s of Culturing Microbes (1) Inoculation – introduction of a sample into a container of media to produce a culture of observable growth Isolation – separating one species from another Incubation – under conditions that allow growth Inspection Information gathering Identification Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. The 6 I’s of Culturing Microbes (2) Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Isolation • If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cells— a colony. A colony consists of one species. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Isolation Techniques – Streak plate technique – Pour plate technique – Spread plate technique Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Inspection • If a single species is growing in the container, you have a pure culture but if there are multiple species than you have a mixed culture. • Check for contaminants (unknown or unwanted microbes) in the culture. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Ways to Identify a Microbe: • Cell and colony morphology or staining characteristics • DNA sequence • Biochemical tests to determine an organism’s chemical and metabolic characteristics • Immunological tests Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Media: Providing Nutrients in the Laboratory Media can be classified according to three properties: 1. Physical state – liquid, semisolid, and solid 2. Chemical composition – synthetic (chemically defined) and complex 3. Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Physical States of Media Liquid – broth; does not solidify Semisolid – contains solidifying agent Solid – firm surface for colony formation Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Agar – The most commonly used solidifying agent – Solid at room temperature, liquefies at boiling (100oC), does not re-solidify until it cools to 42oC – Provides framework to hold moisture and nutrients – Not digestible for most microbes Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Chemical Content of Media • Synthetic – contains pure organic and inorganic compounds in an exact chemical formula • Complex or nonsynthetic – contains at least one ingredient that is not chemically definable • General purpose media – grows a broad range of microbes, usually nonsynthetic • Enriched media – contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Examples of Enriched Media Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Selective & Differential Media Differential Selective media: contains one or more agents media:that allows inhibit growth of some microbes and encourage growth of several growth of the desired microbes types of microbes and displays visible differences among those microbes Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Some media can be both Selective & Differential Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education.
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1. What characteristics do all living organisms have? Do you agree that viruses...


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