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Unit II - Microbiology Sources: • Pelczar M.J. and Chan E.C.S., Elements of Microbiology, McGraw-Hill, Inc. 1981 • Paustian, T. , http://www.bact.wisc.edu/MicrotextBook/ University of Wisconsin-Madison 2002 (Textbook on the web) Student Objectives: • 1. To become familiar with the ubiquitous nature of microorganisms. • 2. To learn some basic bacteriological lab techniques. • 3. To understand how bacteria grows. • 4. To become familiar with major groups of microorganisms. . • 5. To know how microorganisms can be controlled. • 6. To be familiar with diseases caused by microorganisms. Section One •History of Microbiology The First Microscope. • Invented in late 1500’s • A simple microscope = like a magnifying lens The discovery of microorganisms. • Leeuwenhoek = Dutch naturalist in late 1600’s – Ground lenses and made microscopes. – First to record observations of microscopic organisms seen in rainwater. – Called the little organisms “animalcules”. • Basic kinds of bacteria as observed by Leeuwenhoek –spirilla – spiral-shape • cocci – spherical • bacilli – rod-shaped Spontaneous Generation • Belief that organisms arose from non life • Disproved by use of beef broth experiments • Louis Pasteur – the gooseneck flask Pasteur Schulze acid base Schwann’s flame Schroeder’s cotton plug Pasteur’s Fermentation • Used for many years. • Pasteur discovered microbes were responsible Disease • 1540’s – first theories that small organisms caused disease. Key Discoveries • Pasteur isolated anthrax – causing microbes • Koch discovered anthrax was caused by bacilli bacteria • Koch developed culture media Koch’s postulates • A specific microbe is associated with a given disease. • That microbe can be isolated and grown. • The pure culture can be used to infect a host organism. • Microbes can be “recovered” from infected organism Koch Immunization • Any process that develops resistance in a host to a specific disease. Pasteur discovered immunization accidentally. • By using old cultures, injected chickens remained healthy. • He injected a new culture in those chickens and again they remained healthy. Source: http://www.tutorvista.com/content/biology/biology-iv/immunesystem/vaccination-and-immunisation.php • Source: http://www.bbc.co.uk/schools/gcsebitesize/history/shp/modern/indrevmethodsrev2.shtml This source says Charles Chamberland (France: 1880) discovered by chance (when he left bacteria exposed to air) that injecting chickens with an attenuated (weakened) form of chicken cholera gave them immunity to the disease (ie he discovered the principle of inoculation) . Pelczar and Chan credit this discovery to Pasteur. Other breakthroughs • Jenner and smallpox immunization • Vaccinations (from vacca = cow) • Rabies vaccines. • Antisepsis • Many more !! Today’s Microscope. • Bright field – dark objects in bright area • Compound – multiple lenses. Up to 2000 x • Electron – maximum magnification because of short wavelengths. Up to 400,000 x The oil immersion lens. • is 100 X • Requires oil to prevent light refraction • Gives maximum magnification for compound light microscope. Quiz – History of Microbiology • COMING SOON • GET READY !!!! Section 2 • Laboratory Techniques Microscopes • Light microscopes – max 2000 X • Electron microscopes – max 400,000 X Bright-field Microscopy • Light background and dark objects. The Compound Light Microscope • Main components for directing light. –Eyepiece, objectives, stage, condenser, iris diaphragm, fine and course adjustment knobs, base, mirror, etc. Resolving Power • Resolving Power = wavelength NA objective + NA condenser • NA = numerical aperature • Wavelength rane is 400 to 700 nm • NA for high dry is about 0.85 • NA for oil-immersion objective: 1.2 to 1.4 • Whatever the resolving power, if two objects are closer together than the numerical resolving power, they appear as one. • “The resolving power of a microscope is the ability of the devise to distinguish between two objects. A light microscope can only resolve two objects that are bigger than 250 nanometers because objects smaller than this fall well below the size of a wavelength of visible light (500 – 650 nanometers). Non lens system can ever resolve two dots that are closer together than half the wavelength of the light that is used to view them.” Source: http://www.blurtit.com/q571512.html • • • • • • • • No doubt you have seen a sunrise and a sunset. The sky turns red, orange, yellow and even purple because the colored wavelengths of light travel farther through the air (atmosphere). When the sun is low in the sky, this long journey through the atmosphere means the colors with shorter wavelengths, like blue, have already scattered or bounced off in numerous directions. Orange sunsets (yellow and red light waves) appear when the air is clean. Sunsets that are the most spectacular occur when red wavelengths reflect off of overhead clouds. Spectators continue to see light in the sky long after it has turned dark on the ground. Why is this? Because night doesn't "fall". It actually rises from the ground as the sun goes farther below the horizon. Civil twilight occurs when the sun is 6° below the horizon. This is from the time that the sun drops below the horizon until artificial lights (street or home) are needed. Astronomical twilight occurs when the sun is 18° below the horizon. This is when there is no sunlight on the western horizon and stars can be seen. Twilight is shorter in the tropics because the sun's path is more perpendicular to the Earth's plane and it takes less time to go from 6° to 18° below the horizon at this angle. White nights occur in extreme northern latitudes where the evening twilight merges with the morning twilight. http://sci.odu.edu/sci/Scire/05Edition/sunset.html Preparation of Specimens • Wet mount - short term mount – high dry • Hanging Drop – high dry • Stained smears – best for bacterial form - oil immersion Wet mount source: http://www.greatscopes.com/act005.htm Hanging Drop Slide Source: http://homepage.smc.edu/wissmann_paul/microbiology/motility.html Negative Staining • Stains background and makes bacteria stand out more clearly. –Nigrosine stain Staining the Bacteria (positive) • Stains bacteria and not the background. – Main stains are methylene blue, gentian violet (crystal violet), and carbol fuchsin. The Staining Process Three basic steps. • Smearing • Fixing • Staining Step One – the smear • A very clean slide • Draw circle on back of slide and label back of slide. • Sterilize your loop. • Place very small drop of sterile water on slide • Sterilize loop. • Obtain sample on loop – Remember Bacteria are very small !! • Smear bacteria in water Step two – Fixing the smear • Air dry the smear • Pass slide through burner flame up to 3 times • Do not flame the slide until all water has disappeared !!! Step three - staining • Cover the smear with stain for a few seconds. • Wash all the stain off the slide. • Dry using filter paper Special Staining • Gram Stain procedure– divides bacteria into two groups. • Gram positive – organisms that are stained purple. • Gram negative – organisms that retain counterstain color. • Acid-fast staining – to classify those bacteria which retain stain when washed with acids or alcohol. Growing Bacteria in the Lab • Culture and Media Growth Requirements: • energy source • a source of carbon • required nutrients, • proper O2, pH, temp, etc. Culture: • A population of microorganisms cultivated in a medium Culture media: • are designed to provide all the essential nutrients in solution for bacterial growth . Pure Culture • Culture containing only one species of organism. Types of Media • Liquid – for pure batch cultures • Solid – isolation of pure cultures. Agar. Assignment: • Read online textbook section: “Culture Media for the Growth of Bacteria” Page 16 of Word document on CD Section 3 • Survey of the Microbial World Terminology: • Microbiology – study of small living organisms of microscopic size. • Protist – a microorganism in the Kingdom Protista. (includes most microorganisms) • Prokaryote - a cell in which the nuclear substance is not enclosed in a membrane. (includes the bacteria) • Eukaryote – a cell that has a definitive or true nucleus (these cells make up the bodies of all non protist organisms) • Virus – obligate intracellular parasitic organism that is smaller than bacteria. They can only reproduce in cells of host organisms. The Cell – A review • General Structure: –Membrane –Nucleus – contains nucleoproteins –Cytoplasm (cyto= cell, plasm = formed substance) • Characteristics of all living things: – Reproduce – Energy needs and growth – Elimination of waste – Response to stimuli – Susceptible to mutations • Look for the 3 general structures in each of the following three organisms. Paramecium – a representative cell A Typical Bacterial Cell Euglena – another typical cellular organism Prokaryotic cells • No internal membranes • Division by fission not by mitosis • Cell wall with mucopeptide material. Some Prokaryotic cells • have capsule or slime layer covering cell wall • have flagella (“whip”) for motility Prokaryotic cytoplasm May contain: • Ribosomes – proteins and RNA • Granules - chemicals • Nuclear material - DNA • Mesosomes - folds of Cytoplasmic membrane Eukaryotic cells • Contain internal membranes - called Endoplasmic Reticulum (ER) – distinguishing it from the prokaryotic cells. Eucaryotic cell structures • ER • • • • • Nucleus Golgi apparatus Mitochondria Chloroplasts Vacuole • Microtubules • Microfilaments • Flagella • Cilia • Cell walls Groups of Microorganisms • Prokaryotic protists • Eucaryotic protists • Viruses Please note: • Classification schemes often change. • Some literature lists Bacteria as a member of Kingdom Monera and not Protista. • Others split bacteria into two kingdoms called Archaebacteria and Eubacteria. Prokaryotic Protists • Bacteria – • 3 basic shapes • Size range from 0.5 mm to 2.5 mm • Cyanobacteria – • Contain chlorophyll and photosynthesize Eucaryotic Protists • Fungi • Rigid cell walls, no chlorophyll • Protozoa • Include flagellates, amebas, ciliates, and sporozoa – more on that later ! • Algae • Contain chlorophyll Paramecium – a protozoan Viruses • Not cellular • Obligate parasites • 20 nm to 300 nm in size • Host specific Section 4 • - Protozoans Protozoa introduced • Classified by locomotion –Flagellates –Amebas –Ciliates –Sporozoa Flagellates • Cell is surrounded by pellicle – gives the organism shape. Disease-causing Flagellates • Giardia lablia that nasty intestinal parasite • Trypanosoma (African sleeping sickness – spread by tsetse flies) • Trichomonas - vaginitis, spread as venereal disease Amebas • Name comes from Greek word meaning “change” • Pseudopodia – “false feet” are protoplasmic extensions Disease-causing Amebas • Entamoeba histolytica – cause of amebic dysentery. • (yes there are non disease causing Amebas like Ent. Gingivalis and Ent. Coli) Ciliates • Contain at least one macronucleus and one micronuclei per cell. • May have partial covering of cilia or complete covering like Paramecium Disease-causing Ciliates • Only one !! • Balantidium coli – causes “bloody diarrhea” which is transmitted by infected food or water from swine feces. Sporozoans • All are parasitic. • Feed on host’s cells or body fluids. • All produce spores – a resistant or dormant cell. Disease-causing Sporozoans • Plasmodium - Malaria-causing sporozoans • Toxoplasma gondii - mild infection usually requiring no treatment but can cause birth defects if mother is infected. The changing face of Protozoans • Cyst formation • Trophozoites - organism in stage of active growth, also called vegetative stage. Section 5 •Algae Algae Shapes • Single cells may be: –Spherical –Rod-shaped –Club-shaped –Spindle-shaped Algae Growth • May grow as single cells. • May grow in multicellular colonies. • Colonies may be made of identical cells. • Colonies may be made of different cells that specialize. Algae Cellular Structure • Algae is Eukaryotic • Contain chloroplasts. • If motile, they have flagella. • May have stalks or spines for anchoring. Classification of Algae • Based on: – Pigment – Chemistry – Flagella – Cell wall features – Cell organization (single or colony) – Life cycles and reproduction. Some major Divisions • Chlorophycophyta – green algae • Rhodophycophyta – red algae • Chrysophycophyta – golden algae • Bacillariophycophyta - diatoms Diatoms Blue-green Bacteria/algae Reproduction • Assexual – By binary fission (cell division producing two daughter cells) – Spore formation. • Sexual –Gametes –Zygote Growth Requirements • Algae are aerobic – need oxygen • Algae are photosynthetic – need light • Algae require moisture – need water • Algae require nutrients