Microbiology Chapter Notes
Microbiology Chapter Notes Bio 4401
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Lecture 4: Classification of Bacteria 1. Classification (taxonomy): a. Taxonomy is to name, describe, then classify organisms Put in groups based on similarities b. Used to: Identify See relationships to increase understanding to solve problems Control 2. Artificial: based on a few easily recognizable characteristic a. Problem: may not show common ancestry b. Example: Linnaeus classification 2 kingdoms Plants and animals Everything fit into 2 kingdoms Fungi and bacteria put into Plants Protozoa put into animals c. Whittaker – separate prokaryotes and eukaryotes All prokaryotic cells are bacteria One kingdom – monera/prokaryotae 3. Natural: shows relationships with sexual reproduction, common ancestry a. Want to show relationships from common ancestry b. Carl Woese – use DNA/RNA technology to determine relationships, realized that not all prokaryotes are closely related, so developed 3 domains 4. 3 Domains a. Woese show relationships b. Shows that Archaea more closely related to Eukarya than to Bacteria 5. Bergey’s Manual of Systematic Bacteriology a. 1 edition i. Still used in health field ii. Separate bacteria based on cell wall by gram staining 1. Prokaryotes divided into G+ or G iii. 4 different Phyla (divisions) for bacteria based on cell wall chemistry 1. Gracilicutes: thin skin a. Cell wall consistent with being gram negative. All don’t stain gram neg, but all consistent Ex: Ecoli (diarrhea), Klebsiella pneumonia (nosocomial pneumonia), Neisseria (gonorrhea, meningitis), Rickettsiae (rocky mountain spotted fever), Chlamydia (STD), Borrelia (lyme disease), Treponema (syphilis), 2. Firmicutes: thick skin a. Gram positive consistency Ex: Bacillus (anthrax), Staphylococcus (boils, carbuncles), Streptococcus (pneumonia, sore throat), Clostridium (tetanus), Mycobacterium (tuberculosis), Corynebacterium (diphtheria) Mycobacterium doesn’t stain well 3. Tenericutes: soft skin No cell wall, stains pink (gram neg) Ex: Mycoplasma (pneumonia) 4. Mendosicutes: faulty skin No murein (peptidoglycan) in cell walls, gram positive or gram negative Ex: archaebacteria * Artificial classification doesn’t always show common ancestry nd b. 2 edition of Bergey’s Manual i. new relationships based on DNA and RNA (NATURAL) ii. ~40 different phyla – keep domains, not kingdoms 1. Cyanobacteria: bluegreen a. Photosynthetic b. Stains gram c. Converted world from anaerobic to aerobic 2. Actinobacteria a. Cell wall consistent with g+ b. High G+C (guanine – cytosine) ratio i. Problem: different ratio shows different groups; same ratio doesn’t mean same group ii. Not that good for differentiating iii. Ex: Mycobacterium (TB, leprosy) and corynebacterium (diphtheria) 3. Firmicutes a. Gram +, but low G+C ratio b. Staphylococcus (boils), Streptococci (sore throat, pneumonia), Bacillus (anthrax), Clostridium (tetanus), mycoplasma (pneumonia) i. Surprised to see mycoplasma here because it has no cell wall 4. Proteobacteria a. Gram – b. Escherichia coli (diarrhea), Klebsiella (cause type of pneumonia, resistant to antibiotics) Neisseria (meningitis and gonorrhea), rickettsiae (rocky mountain spotted fever) 5. Chlamydiae a. Chlamydia trachomatis, Gram b. STD 6. Spirochetes a. Gram – b. Ex: Treponema (syphilis) and Borellia (lyme disease) Questions 1. Bacteria with faulty skins (i.e. without murein) according to Bergey’s first edition, are classified into which Division? Mendosicutes 2. True or False? An example of an artificial classification would be Whittaker's five kingdom system. True 3. According to the Woese 3 domain system and Bergey’s 2 edition, which of the following are more closely related? a. E. coli and methanogens b. Staphylococci and mycoplasma c. Staphylococci and Treponema d. Streptococci and Corynebacterium e. Chlamydia and Rickettsiae Lecture 1. The Scope of Microbiology 1. Definitions Microbiology: the science or study of microscopic life Science: the study of, or a body of knowledge of Taxonomy: name, describe, classify Morphology: shape, other characteristics such as spores, flagella, capsules Ex: cocci is a shape Physiology: enzymes Genetics: (molecular biology) DNA Ecology: influence of microorganisms on environment Microscopic Life *study units for exams and quizzes* 1 mm= 1000 micrometer 1 micrometer= 1000 nm 1nm= 1000 picometer or 10 Angstroms Macroscopic: limit of resolution, can be seen with the naked eye; 0.2 mm= 200 micrometer Anything above .2mm, you can see Resolution = resolving power: what is the smallest thing you can see? The smaller the object, the higher the resolution Rhizopus Stolomifer = bread mold Sporangium: 250350 micrometers This is macroscopic because its above 200 micrometers Q: what’s the limit of resolution of a light microscope? Light Microscope: 0.2 micrometer = 200 nm(under best conditions), but usually only 0.5 micrometers; fungi some algae, unicellular (primitive) protozoa Rickettsia: very tiny bacteria could still see Mycoplasma: even tinier bacteria, usually not visible Electron microscope: 0.5 nm Q: can you see e.coli using a microscope? 2. Microscopic life a. Linnaeus Carolus Linneus / Carl Von Linne: Categorized all living things between 2 kingdoms Plants: green (photosynthesis), cell wall, spores, no independent movement. Later came: Ex. Bacteria (cell wall), Fungi (cell wall, spores) Animals: no photosynthesis, no cell wall, no spores, independent movement Later came: Ex. Protozoa b. Whittaker (1969): Categorized cells into 2 groups (prokaryotic/eukaryotic) KNOW THE DIFFERENCE Prokaryotic: have ribosomes, no membranous organelles, no nucleus, mitochondria, chloroplasts( have a double membrane so are considered organelles, Endoplasmic Reticulum, or Golgi bodies(not organelles bc single membrane). Ex. Monera (Prokaryotae), all bacteria Bacteria: most are absorptive heterotrophs Eukaryotic: have membranous organelles and ribosomes Ex. Protista, Fungi, Plants, Animals Know which groups have what Plants Fungi Animals Protista Monera Whittaker organized this order of evolution Humans are actually more related to fungi than plants Q: How would Linnaeus do it? How would Whittaker do it? How would Woese? Q: All of the following are prokaryotes except… review readings c. Carl Woese (1978): Categorized all living things into 3 domains, used DNA and RNA to differentiate 1) Bacteria: prokaryotes, contain Murein in cell wall, cell membranes bonded by ester linkages. Ex. Vibrio cholerae cholera Mycobacterium tuberculosisTB Shigella Bacillary dysentery diarrhea with blood tinged Is vibrio prokaryotic? Does it have ribosomes? Mycoplasma and mycobacterium are different species How would Linnaeus classify Shigella? It’s a plant, because the bacteria fit in with the plants 2) Archaea: prokaryotes, no Murein in cell wall, ether linkages. Ex. Hyperthermophils (80120˚C), methanogens(produce gas), halophils (salt loversup to 36% salt) 3) Eukarya: cells with membranous organelles a: Protista: eukaryotes, simple plants (Ex. Algae such as Kelps), simple animals (Protozoa such as Malaria, Giardia, Amoebic dysentery), and simple fungi (such as water moldsSaprolegnia) Q: Where would malaria be according to Whittaker? It wouldn’t be in a domain because Whittaker didn’t do domains b: Fungi: absorptive heterotrophs, mostly multicellular (have hyphae) Mushrooms Molds: athletes foot, ringworm Yeast: Candida albicans (can be from taking antibiotics), thrush c: Plants: photosynthetic autotrophs, uses inorgan2 CO to make food, ALL multicellular, examples: bryophytes (moss), gymnosperms (pines), angiosperms (flower) Have cellulose Q: Does a palm tree have ribosomes? What kingdom? d: Animals: multicellular, ingestive heterotrophs, use organic molecules for food. Ex. Sponges, worms, up to mammals Helminths: worms that cause infection (2 categories) Round worms: pinworms, ascaris Flat worms: tape worms, blood flukes (Schistosoma, over 100 million cases each year worldwide) Have chitin Viruses: acellular, not alive, no cell membranes, no ribosomes, contain RNA or DNA Ex. Flu, common cold, measles, herpes, ebola, AIDS Measles: virus, not prokaryote or eukaryote 3. Subdiciplines (study of…) • phycology algae • mycology fungi (mushrooms, molds, yeast) • protozoology protozoa • virology viruses • bacteriology bacteria • medical microbiology relationship between disease and microorganisms • parasitology protozoa, helminths, some insects (ones that transfer disease—mosquito) • immunology defense mechanism, antibodies, antigens, allergy/hypersensitivities • molecular biology DNA/RNA sequencing. Identification, relationships • microbial ecologist how organisms act in the soil, water, or other habitats, carbon cycle, nitrogen cycle • microbial physiologist metabolic pathways, fermentations 4. Beneficial Aspects of Microorganisms • Decomposers*—absorptive heterotrophs (saprophytes live off dead material or saprobes) Number one beneficial aspect!!! Most important • Antibiotics chemical from microorganisms that will kill or stop the growth of another micro organism in small concentrations i.e. Penicillium (a mold found on bread and other things) gives us the antibiotic penicillin—used to treat gram + infections (streptococci bacteria) “chemotherapeutic agents”/magic bullets it affects the pathogen but not the host (penicillin affects the murein in the cell wall, we don’t have cell walls). Ciprofloxacin (not a magic bullet but it is a chemo agent) works on DNA synthesis. Penicillin is a chemotherapeutic agent and a magic bullet because it treats strep • Other Chemicals and drugs Acetic acid, alcohol, insulin (from E.coli) • Foods—cheese, bread, beer, wine • Research tools—bacteria have 1 chromosome so easy to work with 5. Detrimental Aspects • Spoilage—bacteria/fungi don’t know the difference between good and bad. They spoil food, shower curtains, walls • Cause plant and animal disease Questions 1. In this field of microbiology the aim is to monitor and control the spread of disease in communities. a. immunology b. epidemiology c. dairy microbiology d. biotechnology e. industrial microbiology 2. Since most microorganisms encountered in microbiology will fall between 100 µm and 10 µm in overall dimension, observation would require use of which of the following techniques? a. human eye only b. light microscope only c. light microscope and electron microscope d. electron microscope only e. human eye and light microscope 3. The scientific names of bacteria are always a combination of a. the kingdom name followed by the division name b. the class name followed by the order name c. the family name followed by the genus name d. the generic name followed by the common name e. the generic name followed by the species name 4. The system for organizing, classifying, and naming living things is a. nomenclature b. taxonomy c. taxa d. identification e. classification 5. The traditional mainstay of bacterial identification has been: a. biochemical characteristics b. serological analysis c. G + C base composition d. DNA analysis using genetic probes e. Nucleic Acid Sequencing and rRNA analysis. 6. Yeast have which of the following characteristics? a. prokaryotic, unicellular b. prokaryotic, multicellular c. eukaryotic, unicellular, absorptive heterotroph d. eukaryotic, multicellular, absorptive heterotroph e. eukaryotic, multicellular, photosynthetic autotroph 7. This discipline is the study of protozoal and helminth diseases. a. phycology b. mycology c. protozoology d. parasitology e. immunology 8. The most important role of microorganisms is a. they cause 5 million deaths annually from diarrheal disease b. they are used as research tools for production of hormones such as insulin c. they are saprobes and break down organic matter into inorganic chemicals d. production of foods such as beer and yogurt e. destruction of crops 9. Which of the following is NOT a distinguishing characteristic of Bacteria? a. Their DNA is not enclosed within a membrane. b. They lack membranebounded organelles. c. They have cell walls containing peptidoglycan. d. They are unicellular. e. None of these. Lecture 3. Techniques for Observing Microorganisms II 4. Fluorescence Microscopy a. Basic Setup Intense Light Source Mercury Vapor Lamp Gives off a lot of heat and can dry the specimen Exciter Filter Filters out anything that is greater than 400nm [>400nm]. So it lets through anything less than 400nm (Ultraviolet light). Barrier Filter Filters out anything less than 500nm [<500nm]. Why? UV light is damaging to the eye. If there’s no specimen on the slide, you see black because you’re seeing light less than 500 and greater than 400 nm. Fluorescent Dye Fluorochrome is a fluorescent dye that will be hit by UV light on the dyed specimen, and will thus will be reemitted (give off) as visible light. The fluoresced energy made when the light hits the dye, and gets converted to visible light which is what you see b. Appearance of Field and specimen The field will be dark and black, the dyed specimen will shine. If the fluorescent dye is absorbing energy, and it then releases it (fluoresces), are the wavelengths longer or shorter than the original? Longer, because longer wavelengths take less energy; anything longer than 500nm (different colors based on dye used) will show. Lets UV light through c. Use Mycobacterium Microorganisms have a very limited use because with fluorescent microscopy, you must stain organisms with high lipid (wax) contents, which is mostly just mycobacterium. Mycobacterium tuberculosis (rod shaped) causes Tuberculosis When grown on an agar plate, it looks like dried up wax and easily absorbs the dye. In this case, we use Auramine O Wax. o The Auramine O dye will stick to the mycobacterium and fluoresce under the microscope. o You can tell you have TB when your saliva comes out very thick and waxy. o Fluoresces yellow. Is yellow light UV light? No its visible light. o Auramine O sticks to waxy materials o Only need to use high power whereas acid fast needs oil immersion Acid Fast Technique You take a sample of your organism, stain it with Acid Fast, and then use oil immersion to observe the organism. Harder to focus in oil immersion (so it’ll take a longer time to complete) smaller field of view. Not a fluorescent dye: a dye dissolving in very waxy saliva Developed specifically for TB and not used elsewhere TB will come out pink(mycobacterium) Anything else in saliva comes out a different color (ex staphylococci, streptococci) d. Immunofluorescence AKA Fluorescent Antibody Technique Allows you to see organisms that do NOT have a waxy content Used for Steptococcus pyogenes Strep Throat Used for Treponema pallidum Syphilis One method for this is a direct method. A syphilis organism can be grown to have its antibodies extracted. This will produce “anti syphilis antibodies” (Y shaped), and a dye will be attached to the antibody. This forms a complex and allows the anti syphilis antibodies to stick to the syphilis organisms, and if the bright lights flash under the microscope, then that means the patient has syphilis. Dye sticks to antibody antibody sticks to organism lights Would you use this for TB? No Would you use a fluorescent microscope for mycoplasma? No If its not syphilis, the dye can’t stick Antibody v. Antigen: o Antigen= anything foreign to your body o Antibody= fights against antigens 5. Differential Interference Contrast Microscope (DIC) a. Basic Setup Uses prism to break up the light, and another to bring light back together. b. Appearance of Field and Object Very similar to phase contrast microscope (provides high contrast), also 3Dimensional view, Creates false colored specimen because of the prisms c. Use Used on live specimen (to see inclusions) Earthworms are adapted to both dry earth and wet conditions, so white blood cells are affected by cytoskeleton when it changes and adapts B. Electron Microscopes 1. Transmission Electron Microscopy (TEM) a. Basic Setup Uses electrons, which have 0.1nm 0.2nm wavelengths = higher resolutions 1000x better than our lab microscopes Can’t use glass lens because glass is an insulator for electrons, so they use a condenser magnet and objective magnet instead. Can’t use ocular because electrons will shoot into your eyeball, so they use a projector magnet to project it onto a fluorescent screen. This happens in a vacuum so that electrons can flow through. Resolution = 0.5nm = 500 pm = 5 angstroms (5 Å) [very high resolution] b. Appearance of Field and Object With nothing in the field of view, it will be very bright and white The less dense the material, the less you see; the more dense the material, the more you see because there will be more electrons that will scatter, and so the material will appear darker c. Use Viruses Inclusions of bacteria DNA and large proteins 2. Scanning Electron Microscopy (SEM) a. Appearance of Field and Object Uses 0.01µm = 10nm resolution 10x better than our lab microscopes b. Use Surface detail of bacteria 3D view Questions 1. In this technique ultraviolet light absorbed by the specimen. a. Compound light microscope b. Phase contrast microscope c. Darkfield microscope d. Fluorescent microscope e. Electron microscope 2. Normal bacteria (those not possessing large amounts of wax in their cell walls) may be seen with a Fluorescent microscope using what technique? immunofluorescence 3. True or False? A Differential Interference Contrast microscope provides a high degree of contrast between the object and the field of view due to the presence of an exciter filter which focuses the incident rays of light. False LECTURE 5 – ANATOMY AND CHEMICAL COMPOSITION I 1. BASIC FORM (MORPHOLOGY = SHAPE) A. COCCUS (plural/ pl: COCCI) o Spherical o Many names of bacteria are based on the basic form of the bacteria o Streptococcus pneumoniae chain of cocci – look like a string of pearls (typical pneumonia, meningitis) o Staphylococcus aureus – clusters of cocci look like a cluster of grapes (boils, carbuncles) o Neisseria – 2 cocci together (diplococci) (meningitis, gonorrhea) o Smith will test on the diseases that don’t have the shape in the name B. BACILLUS (pl. BACILLI) o Rod shaped, elongated o Bacill s anthracis (anthrax) Streptobacilli – joined in chains o Clostridium tetani (tetanus) o Escherichia coli (diarrhea) o Klebsiella pneumoniae (nosocomial pneumonia) o Corynebacterium diphtheriae (diphtheria) – have a palisade arrangement C. SPIRAL SHAPED o Spiral shaped have at least 1 bend/twisted in them, curved o Vibrio – a genus name but also a shape A rod shape but with a bent in it, but sometimes look rod shape under microscope Vibrio cholerae: Cholera (diarrhea) ; one of the worst ones. You can die if untreated because you lose so much water o Spirochetes – corkscrew shape Treponoma pallidum: Syphilis Borrelia: Lyme disease Both of these diseases slowly destroy your body 2. SIZE Cocci: diameter~ 1 micrometer (twice the size of a microscope – ours sees 0.5 micrometers) Bacilli: 0.5 micrometer wide x 2 micrometer long (best) Older cultures will be short and look like cocci Long bacilli: broth Short bacilli: agar Spirals: 0.5 micrometer wide x 1 to 14 micrometer long (spirochetes = longer ones) 3. GLYCOCALYX A. DEFINITION: outermost layer of bacteria (glycocalyx > cell wall > cell membrane) o Extracellular: not an integral part of the cell, like your hair (if you shave it, you won’t die) o Nonliving o 2 basic types (both are gooey) Slime layer is loosethin layer of slime. It is easily washed away. Staphylococcus aureus: Boils, carbuncles Ex if you smeared Vaseline all over your body Capsule is much thicker and larger. It is not readily removed. Klebsiella pnemoniae: Nosocomial pneumonia Streptococcus pneumoniae: typical pneumonia, meningitis (bacterial) Streptococcus pyogenes: Strep sore throat Bacillus anthracis: Anthrax Haemophilis influenza***: Bacteria that cause meningitis, frequently isolated after someone got the flu but doesn’t cause the flu (a flu is caused by a virus). This is a secondary infection of the flu. o Strep neumo and niseria also cause influenza (check) Looks like mucus: gooey and sticky B. CHEMISTRY: made of proteins or polysaccharides (starch) or a combination of the two o Typically a capsule looks like mucous because that is the consistency of polysaccharides and proteins. Used to think that it was waste product because of its mucousy look. C. REGULATION: o Genetic: some bacteria have the genes to code for a slime layer, while others have the genes to code for a capsule. Some even lack the genes to code for either of them Need the gene to produce glycocalyx o Nutrition: if a bacterium is growing on a correct, nutrient rich medium, it can develop the capsule more effectively. ENVIRONMENT influenced. You need the proper env’t and nutrition to show it. In lab, you are given klebsiella pneumoniae (has a tiny capsule bc less nutrition). However, if you took directly from a host (human), then you see mucous and larger capsules. D. COLONIAL APPEARANCE: (something you see with your eyes on the plate/ macroscopic) – F. 4.10a o Capsules: mucoid (mucus) on plate; looks like someone sneezed on plate – sticky, stringy (Ex: Klebsiella) o Slime layer: wet and shiny, looks like shiny leather pants E. MICROSCOPIC APPEARANCE: (better) o Because proteins/polysaccharides repel dye, capsules don’t stain well o Negative stain: Similar to blood smear. You start off with India ink on it, take a sample from the plate and mix it with the ink. Take a 2 slide and back it up into there – spreading/ coating the slide with India ink. Once it dries, under the microscope it will be solid black. Next, organisms are spread out on the slide and take up space on the India ink. How do we see it? Take a dye (crystal violet) to stain the organism. Now the slide will be colored black except where the capsule is. You’re painting the slide and staining the organism (inside of the bacterium will be violet, outside the cell will be black), but the capsule is not stained. Capsule will be clear/shine like a halo. Slime layer have a smaller halo than a capsule. o Quick review of negative stain: Capsule doesn’t stain (clear), due to the proteins/polysaccharides which repel dye Example: Klebsiella (rod shaped) Capsule = clear area Bacteria = purple Only through the gram stain can you tell gram positive and gram negative F. IMPORTANCE IN IDENTIFICATION: o Go through 3 levels to identify genus and species Gram staining: Gram + or Gram –? Shape/morphology: cocci or rod/ bacilli? Biochemical tests: looks for enzymes (sugar – ex: lactose); can organism break down X? o We have lots of varieties so do Antigen Antibody Reaction and so we come up with SEROVARS (serological varieties) o SEROVARS (serology) – serological varieties= races= strains (classification by serotypes based on glycocalyx) All the organisms have different proteins/ polysaccharides (called antigens). We will produce different antibodies against it. Ex: More than 90 different serovars for Streptococcus pnemoniae that can affect humans. Anthrax has over 2000 serovars Antigens are very expensive Q: Why do we do this? Does everyone die from steptococus pnemoniae? No, but some do bc some serovars are resistant to antibiotics. You have to identify what kind of antibiotic sensitivity it may have. But some do. Important in development in vaccines. Best kind of vaccine will be to include everything. o Vaccines are not 100% affective bc it doesn’t include all the strains. o May affect a few certain serovars. Ex: two common vaccines: PPV23 (Pneumococcal polysaccharide vaccine) adult form that treats 23 of the most common serovars for strep. pnemoniae and PCV13 (Pneumococcal conjugate vaccine) originally for children but now also approved for adults over 50 years old G. FUNCTIONS: o Capsule helps form microcolonies bc capsules help bacteria stick together in groups. Groups provides protection, those inside the center are protected by the those outside from: Drying (organisms can stay up to 2 months on a dry bench) and chemicals (Ex: alcohol) Phagocytosis: white blood cells have a difficult time engulfing the larger microcolonies o Hyaluronic acid (chemical within the capsule) We have it, like a cement between our cells Disguises itself from the white blood cells, so we don’t recognize it as foreign (wolf in sheep clothing) Ex: Streptococcus pyogenes (strep sore throat) The hyaluronic acid produces a chemical that will protect it from phagocytosis and help the bacteria stick together. The capsule and slime layers allow bacteria to adhere to inanimate objects (like anything inserted into the body,e.g. catheter – urinary catheters/ IV needles) and adhere to the hosts (bad practice of aseptic technique) o Over 100k Americans die/ year from infections o Virulent (pathogenic) = Has a capsule, able to adhere and cause diseases o Avirulent (nonpathogenic) = lost it’s capsule bc when you grow it in a lab it’s not in the best conditions. Less chance of disease because not sticking. Questions 1. The cells of bacteria can be categorized according to their shape. Clostridium has what shape? a. cocci b. bacilli c. spirochete d. vibrio e. spirilla 2. Which of the following is NOT true about the glycocalyx? a. It may be composed of polysaccharide. b. It may be composed of polypeptide. c. It may be responsible for virulence. d. It is used to adhere to surfaces. e. None of these. Lecture 5. Anatomy and Chemical Composition-I 1. Basic Form 2. Size 3. Glycocalyx a. Definition b. Chemistry c. Regulation d. Colonial appearance e. Microscopic Appearance f. Importance in Identification g. Functions LECTURE 2 – TECHNIQUES FOR OBSERVING MICROORGANISMS A. LIGHT MICROSCOPES resolution (resolving power) of Human Eye: 0.2mm (200m) “What is the smallest thing you can see?” He will ask for numbers. Keep in mind units use VISIBLE LIGHT. A type of electromagnetic radiation. Travels in waves measured by wavelength wavelength is the distance from one peak to another peak Visible light is 400800 nm (not exact) Visible light includes the colors of the rainbow: red, orange, yellow, green, blue, indigo, violet Thus each color is classified by wavelengths and its energy is inversely proportional to wavelengths. 5 types: brightfield, darkfield, phase contrast, fluorescence, differential interference contrast Electromagnetic radiation Wavelength (nm) V I B G Y O R | | | | Xrays UV Visible light infrared microwaves Gamma, electrons 100400nm 400 800 nm heat rays radio waves < 1 nm Shorter wavelengths longer wavelengths More energy less energy Resolving Power is the same for every single light microscope 1) BRIGHTFIELD: a. Basic setup Glass lenses used/help to focus & magnify: Ocular = 5,10,15,20x Most common ocular (used in lab) 10x Objective(s) = 4x (scanning, top of viewing range like MOLD), 10x (low power), 40x (high power), 100x (oil immersion, decreases resolution) Specimen Condenser (glass lens) uses VISIBLE light used in lab b. Appearance of field and object Circle of white light = seen without specimen, usually colorless Specimen = needs to be darker for contrast (provided through staining techniques) (ex. Gram staining: more purple is better) Need contrast; best way to see bacteria in a Bright Field Microscope is to stain them Iodine used Purple makes it easier to see c. Magnification = ocular magnification x objective magnification Highest possible magnification = 20 x 100 = 2000x Our microscopes (10x) so max is 1000x in lab because of resolving power The smaller the magnification the higher the resolution RP wavelength() 2NA d. Resolving power (aka resolution) = NA = numerical aperture (how much the light bends, considered a constant, except with oil immersion) Ex Visible light (800400 nm) If I put 400 in numerator, it’ll be a smaller number and larger resolving power. If I put 800 in the numerator, it’ll be a larger number and smaller resolving power oil immersion lens requires use of oil for maximum effect oil reduces light refraction and improves resolution If you leave the oil off, the RP number will get larger (and thus resolution is less) If its not sitting in the oil you’re decreasing the resolution The numerical value of RP is not relative to how much better an object can be seen. Higher resolution = smaller number value The higher the NA or the smaller the wavelength you get better resolution (smaller RP number) = clearer image The shorter the wavelength the smaller the resolving power The smallest object we can see (0.2 mm or 200µm) 1. For microscopes: 0.2 µm* or 200nm (ours is .5 µm) (*the best resolving power with LM) 2. Difference is 1000 between our eyes and the microscope Violet = 400 nm for smallest object, the lower the resolution Smaller wavelength= Higher resolution which means violet light is better than red light in terms of resolution Smaller the wavelength, the higher the resolution e. Uses Large (eukaryotic) microscopic objects Can be used for anything large (yeasts, molds, fungi, algae, protozoa, helminths) Prokaryotes/most bacteria (except for mycoplasmas, the smallest bacteria) Viruses cannot be seen This is the microscope we use in lab 2) DARK FIELD a. Basic setup Similar to a bright field but it is modified by a dark field stop just below the condenser, just above the light source (not an example of a light field microscope). Middle blocks the light, the only light that goes through is around the edges and bends. (allows light to go in at an angle and then be reflected off the top) Prevents light from going up the center Light comes in at an angle Pitch black without specimen total darkness Object is illuminated against a black background, SHINES (like stars in the night) Did you increase resolution? No, we increased contrast b. Uses Increased contrast not increased resolution (which is the same for all microscopes you cannot increase the resolution, only the contrast) No need for stains – don’t use stains Can be used for alive specimens able to examine motility, morphology (shape), Increased contrast, no artifacts (no differing of shape) But can’t see inclusions (internal details) No artifacts. In lab, when you add dyes and enzymes it can distort the shape of an organism Examples Treponema pallidum (Syphilis: an STD) a. Spirochete: like a corkscrew, spiralshaped, lesions/rash is symptom b. hard to stain because of its layer of lipids c. Usually alive and motile d. Cant see on the inside You can now see mycoplasma due to increased contrast, they glide (like amoeba) because no cell wall 3) PHASE CONTRAST a. Basic Setup The setup is similar to a bright field except for 2 modifications: Annular Diaphragm – dark circle surrounded by another dark circle. A ring of light is generated which initiates the effect of phase contrast. Located below condenser (lets through a ring of light that helps the light to bend) Phase shifting element – this takes the light and focuses it by either advancing or retarding it, different refractive index Located in objective Increased contrast Focuses the direct ray (direct ray straight up, incident rays are diffracted) Controls speed/brightness depending on index of refraction Advance – to make brighter by speeding up the light Retard – to make darker by slowing down the light b. Appearance of Field and Object Looks similar to bright field but objects are more sharply defined because it focuses the direct rays of light off the specimen and helps to eliminate the scattering of the light. c. Uses Hospitals, cancer research to see details of cells Cells have to be alive Look at unstained, living specimens (morphology and motility) Can clearly see inclusions (endospores in bacteria) Can be used even with mycoplasma Why can we see the mycoplasma? Did we increase the resolution? No, contrast Dean Murphy Questions 1. Resolving power is: a. ability to enlarge objects b. ability to show detail c. the change in angle of the light ray as it passes into the lens d. use of a single magnifying lens. e. use of a second magnifying lens. 2. In bright field microscopy, all of the following statements are true EXCEPT a. objects may be magnified 1,000 to 2,000 times their diameter b. resolution is better using red light than blue light c. objects appear darker than the field of view d. the limit of resolution is 200 nanometers e. this technique is not useful for the study of most viruses 3. This technique is useful in diagnosis of syphilis. a. Bright Field light microscope b. Phase contrast microscope c. Darkfield microscope d. Differential Interference Contrast microscope e. Transmissin Electron Microscope Lecture 2. Techniques for Observing MicroorganismsI Microbes are found in all natural habitats LECTURE 6: ANATOMY AND CHEMICAL COMPOSITIONII 4. Cell Wall glycocalyxcell wallcell membrane (outside to inside of cell) a. General Properties The cell wall is not considered alive, nonliving The glycocalyx was considered extracellular while the cell wall is not considered extracellular this means that it is necessary for the survival of the organism; essential Without it, the organism would die unless you would let it grow under special conditions Cell wall makes 2040% of the dry weight of the organism Memorize properties b. Functions 1) Cell wall prevents plasmoptysis (bursting/lysis) Plasmoptysis water flows in and the cell becomes more turgid/firm Bacteria live in a low osmotic environment (hyposmotic/low pressure) Inside the bacteria, bacteria are hypertonic = have higher solute concentration inside o Normally, any inside of the plant is hypertonic o Solute is anything that influences the osmotic pressurecould be salt, sugar, proteins o If it’s hypertonic then its hyperosmotic means high osmotic pressure Environment around the bacteria is a hypotonic one = has lower solute concentration, hyposmotic, low osmotic pressure Question: Which way will water normally flow? Into the cell, flowing from a hypotonic to hypertonic o Normally water flows into the cell if there a lot of salt in the bacteria, this means that there is lower water in there water is greater outside the cell thus, the water will flow from low solute to high solute concentration o This is so because the concentration of solutes inside the cell is high and a low concentration of water. The environment/outside of the bacteria is hypotonic and has a high concentration of water and low solute concentration o Water will flow from hypotonic into hypertonic, from low to high solute concentration Water will flow into the cell and push against the inside of the cell the cell membrane regulates the water flowing in/out Cell wall is the rigid structure, what holds everything in place Turgor (turgid) pressure water is flowing into the cell water pushes against the cell wall it is the cell wall that prevents bacteria from plasmoptysis/burst open one specific chemical of the cell wall = murein that provides the support against plasmoptysis Main function for cell wall in bacteria is to prevent plasmoptysis, but it cant prevent plasmolysis penicillin will destroy the cell wall o Some antibiotics work interfere with the murein and with no cell wall With no murein to support the cell wall against plasmoptysis, the cell wall isn’t rigid, water fills up the cell and they burst/die o If the cell were isotonic = less water flows in = not as firm/limp = flaccid o Cell wall does not shrink with plasmolysis (only cell membrane shrinks) ie: if you make the outside high in sugarwater will flow from inside to outside of cell and the cell shrinks Bacteria = hypotonic/has lower solute concentration Environment = hypertonic/has higher solute concentration High osmotic pressure 2) The cell wall is responsible for the shape of the bacteria If you remove the cell wall, the l organisms cells would be spherical because the spherical shape requires the least amount of energy Even the plants would be spherical (take cellulase to break down cellulose, the cells look spherical) 3) Cell wall provides an anchor/attachement for flagella The flagella rotates in a wheel 4) Acts as a sieve/filter Sieve is like a mosquito screen, has holes that keep the big things out c. Chemistry 1) Backbone Consists of murein sugars and proteins/amino acids Murein is also known as peptidoglycan (the rigid part that prevents plasmolysis) 2 types of amino sugars (glucose modified with NH2 group) NAM = Nacetylmuramic acid NAG = Nacetyalglucosamine o Bacteria have both NAM and NAG but NAM is unique only to bacteria, meaning that NAM is only found in bacteria and no other microorganisms o NAG is found in chitin (fungi, crustaceans, shrimp) Chitin is a polysaccharide in fungi and anthropods Alternating rows of NAM and NAG are held together by covalent bonds; peptide interbridges connect NAM’s together What interferes with peptidoglycan? Penicillin Penicillin targets the murein in the cell walls of the bacteria o Specifically It prevents the building of the peptide interbridges between the NAM causes the murein to be weakened (weaker cell wall) bacteria undergo plasmoptisis/burst o This works best when bacteria are growing but also works during cell wall maintainance (remodeling their cell walls) When the bacteria are making the cell wall (especially when actively growing), you have enzymes in there called Penicillin Binding Proteins (transpeptidases) o How is it that penicillin works is that the penicillin will sit in the active site of the transpeptidase and prevent buildup of the peptide interbridges of murein (the penicillin binds to the active site of the transpeptidase) o Competitive inhibition Cephalosporin Do the same thing as penicillin Prevent the formation of peptide interbridges Are chemotherapeutic agents (and in this case also magic bullets) o Chemicals that work against the cell wall formationgoal of chemotherapeutic agents is to break down the cell wall of the bacteria because if they don’t have a cell wall, then the bacteria will burst and die Lysozyme Lysozyme is an enzyme Can also destroy murein but in a different way: o In order for penicllin and cephalosoporin to work, you need a bacteria where the cell wall is forming. They prevent the formation of the cell wall. The lysozyme, however, will work on an already mature cell o Lysozyme works by breaking down the link between the NAM and NAG Lysozyme is found in our tears and saliva Alcohols, detergents Both affect the cell wall Don’t work on the murein in the bacteria cell wall or the peptidoglycan attach the outer/second membrane but not the peptide interbridges Cell walls found in all bacteria except for mycoplasma 2) Gram reaction Hans Christian Gram stained cadaverswashed skin with purple dye, alcohol to remove the dye, pink dye Pink = gram negative (retains safranin – pink) Purple = gram positive (retains crystal violet) Question: Why do different organisms stain differently if they all have murein in their cell wall? 1. Is there a specific chemical that stains gram positive/purple the first theory has never been proven Gram Gram Type of… positive negative Teichoic acid + Carbohydrate/polysaccharide Opolysaccharide + carbohydrate Lysine + Amino acid + Amino acid Diaminopimelic acid Murein (% of cell 5090% 20% The structure the prevents wall) plasmoptysis Thickness of 2080 nm 10 nm murein layer Outer (2 ) + membrane Porins + proteins Periplasmic space Thin Thick 2. Chemical that allows different affinities thickness of murein layer could possibly be true because the gram + have more murein but yeast stain gram positive o problem: yeast are fungi = have chitin but not murein o yeast are eukaryotic. Also Archaea do not have murein but still some stain G+ and some G. 3. Permeability hypothesis Why is the purple dye retained in the gram positive? o Due to thickness of cell wall. Yeast have very thick cell wall. further proven by the fact that mycoplasma (lack a cell wall) stain gram negative permeability phenomenabased on the thickness of the cell wallsyeast would have very thick walls compared to bacteria Does the cell membrane include peptidoglycan? Yes Does it include the cell wall? Yes Can the cell membrane prevent plasmoptysis? o Outer Membrane Only found in gram negative bacteriaacts as a barrier while murein is more structure Represents 80% of a Gram negative cell wall Cell membrane is made up of phospholipoprotein Outer membrane made up of LPS = lipopolysaccharides Similar to phospholiprotein in that it regulates materials going in and out Not stiff
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