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10/5/2013 LECTURE PRESENTATIONS For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark Chapter 2 Lectures by John Zamora Middle Tennessee State University A Brief Journey to the Microbial World © 2012 Pearson Education, Inc. I. Seeing the Very Small • • • • 2.1 Some Principles of Light Microscopy 2.2 Improving Contrast in Light Microscopy 2.3 Imaging Cells in Three Dimensions 2.4 Electron Microscopy © 2012 Pearson Education, Inc. 1 10/5/2013 2.1 Some Principles of Light Microscopy • Compound light microscope uses visible light to illuminate cells • Many different types of light microscopy: – – – – Bright-field Phase-contrast Dark-field Fluorescence Animation: Light Microscopy © 2012 Pearson Education, Inc. 2.1 Some Principles of Light Microscopy • Bright-field scope (Figure 2.1a) – Specimens are visualized because of differences in contrast (density) between specimen and surroundings (Figure 2.2) • Two sets of lenses form the image (Figure 2.1b) – Objective lens and ocular lens – Total magnification = objective magnification ocular magnification – Maximum magnification is ~2,000 © 2012 Pearson Education, Inc. 2 10/5/2013 Figure 2.1a A light microscope Ocular lenses Specimen on glass slide Objective lens Stage Condenser Focusing knobs Light source © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI Figure 2.2 Bright-field photomicrographs of pigmented microorganisms © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 3 10/5/2013 Figure 2.1b Path of light through a compound light microscope. Besides 10x, ocular lenses are available in 15–30x magnifications Magnification Light path 100, 400, 1000 10 Visualized image Eye Ocular lens Intermediate image (inverted from that of the specimen) 10, 40, or 100 (oil) Objective lens Specimen None Condenser lens Light source © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.1 Some Principles of Light Microscopy • Resolution: the ability to distinguish two adjacent objects as separate and distinct – Resolution is determined by the wavelength of light used and numerical aperture of lens – Limit of resolution for light microscope is about 0.2 m © 2012 Pearson Education, Inc. 4 10/5/2013 2.2 Improving Contrast in Light Microscopy • Improving contrast results in a better final image • Staining improves contrast – Dyes are organic compounds that bind to specific cellular materials – Examples of common stains are methylene blue, safranin, and crystal violet Animation: Microscopy & Staining Overview Animation: Staining © 2012 Pearson Education, Inc. Figure 2.3 Staining cells for microscopic observation I. Preparing a smear Spread culture in thin film over slide Dry in air II. Heat fixing and staining Pass slide through flame to heat fix Flood slide with stain; rinse and dry III. Microscopy Slide Oil Place drop of oil on slide; examine with 100 objective lens © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 5 10/5/2013 2.2 Improving Contrast in Light Microscopy • Differential stains: the Gram stain • Differential stains separate bacteria into groups • The Gram stain is widely used in microbiology (Figure 2.4a) – Bacteria can be divided into two major groups: gram-positive and gram-negative – Gram-positive bacteria appear purple and gramnegative bacteria appear red after staining (Figure 2.4b) © 2012 Pearson Education, Inc. Figure 2.4a The Gram stain - Steps in the procedure Step 1 Flood the heat-fixed smear with crystal violet for 1 min Result: All cells purple Step 2 Add iodine solution for 1 min Result: All cells remain purple Step 3 Decolorize with alcohol briefly — about 20 sec Result: Gram-positive cells are purple; gram-negative cells are colorless Step 4 G- Result: Gram-positive (G+) cells are purple; gram-negative (G-) cells are pink to red © 2012 Pearson Education, Inc. Counterstain with safranin for 1–2 min G+ Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 6 10/5/2013 Figure 2.4b Microscopic observation of gram-positive (purple) and gram-negative (pink) bacteria © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.2 Improving Contrast in Light Microscopy • Phase-Contrast Microscopy – Invented in 1936 by Frits Zernike – Phase ring amplifies differences in the refractive index of cell and surroundings – Improves the contrast of a sample without the use of a stain – Allows for the visualization of live samples – Resulting image is dark cells on a light background (Figure 2.5) © 2012 Pearson Education, Inc. 7 10/5/2013 2.2 Improving Contrast in Light Microscopy • Dark-Field Microscopy – Light reaches the specimen from the sides – Light reaching the lens has been scattered by specimen – Image appears light on a dark background (Figure 2.5) – Excellent for observing motility © 2012 Pearson Education, Inc. Figure 2.5 Cells visualized by different types of light microscopy. The same field of cells of the baker’s yeast Saccharomyces cerevisiae visualized by (a) bright-field microscopy, (b) phase-contrast microscopy, and (c) dark-field microscopy. Cells average 8–10 µm wide. © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 8 10/5/2013 2.2 Improving Contrast in Light Microscopy • Fluorescence Microscopy – Used to visualize specimens that fluoresce • Emit light of one color when illuminated with another color of light (Figure 2.6) – Cells fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye like DAPI – Widely used in microbial ecology for enumerating bacteria in natural samples © 2012 Pearson Education, Inc. Figure 2.6 Fluorescence microscopy. Same cells are observed by bright-field microscopy in part a and by fluorescence microscopy in part b. Cells fluoresce red because they contain chlorophyll a and other pigments. Fluorescence photomicrograph of cells of Escherichia coli made fluorescent by staining with the fluorescent dye DAPI. © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 9 10/5/2013 2.3 Imaging Cells in Three Dimensions • Confocal Scanning Laser Microscopy (CSLM) – Uses a computerized microscope coupled with a laser source to generate a three-dimensional image (Figure 2.8) – Computer can focus the laser on single layers of the specimen – Different layers can then be compiled for a threedimensional image – Resolution is 0.1 m for CSLM © 2012 Pearson Education, Inc. Figure 2.8 Confocal scanning laser microscopy (a) Confocal image of a microbial biofilm community cultivated in the laboratory. The green, rod-shaped cells are Pseudomonas aeruginosa experimentally introduced into the biofilm. Other cells of different colors are present at different depths in the biofilm. (b) Confocal image of a filamentous cyanobacterium growing in a soda lake. Cells are about 5 µm wide. © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 10 10/5/2013 2.4 Electron Microscopy • Electron microscopes use electrons instead of photons to image cells and structures (Figure 2.9) • Two types of electron microscopes: – Transmission electron microscopes (TEM) – Scanning electron microscopes (SEM) © 2012 Pearson Education, Inc. Figure 2.9 The electron microscope Electron source Evacuated chamber Sample port Viewing screen © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 11 10/5/2013 2.4 Electron Microscopy • Transmission Electron Microscopy (TEM) – – – – Electromagnets function as lenses System operates in a vacuum High magnification and resolution (0.2 nm) Enables visualization of structures at the molecular level (Figure 2.10a and b) – Specimen must be very thin (20–60 nm) and be stained Animation: Electron Microscopy © 2012 Pearson Education, Inc. Figure 2.10a Electron micrographs. (a) Micrograph of a thin section of a dividing bacterial cell, taken by transmission electron microscopy (TEM). Note the DNA forming the nucleoid. The cell is about 0.8 µm wide. Cytoplasmic membrane © 2012 Pearson Education, Inc. Septum Cell wall DNA (nucleoid) Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 12 10/5/2013 2.4 Electron Microscopy • Scanning Electron Microscopy (SEM) – Specimen is coated with a thin film of heavy metal (e.g., gold) – An electron beam scans the object – Scattered electrons are collected by a detector and an image is produced (Figure 2.10c) – Even very large specimens can be observed – Magnification range of 15–100,000 © 2012 Pearson Education, Inc. Figure 2.10c Scanning electron micrograph of bacterial cells. A single cell is about 0.75 µm wide. © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 13 10/5/2013 II. Cell Structure and Evolutionary History • 2.5 Elements of Microbial Structure • 2.6 Arrangement of DNA in Microbial Cells • 2.7 The Evolutionary Tree of Life © 2012 Pearson Education, Inc. 2.5 Elements of Microbial Structure • All cells have the following in common: – Cytoplasmic membrane – Cytoplasm – Ribosomes © 2012 Pearson Education, Inc. 14 10/5/2013 2.5 Elements of Microbial Structure • Eukaryotic vs. Prokaryotic Cells – Eukaryotes (Figures 2.11b and 2.12c) • DNA enclosed in a membrane-bound nucleus • Cells are generally larger and more complex • Contain organelles – Prokaryotes (Figures 2.11a and 2.12a and b) • No membrane-enclosed organelles, no nucleus • Generally smaller than eukaryotic cells © 2012 Pearson Education, Inc. Figure 2.11b Internal structure of eukaryotic cells Cytoplasmic membrane Endoplasmic reticulum Ribosomes Nucleus Nucleolus Nuclear membrane Golgi complex Cytoplasm Mitochondrion Chloroplast Eukaryote © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 15 10/5/2013 Figure 2.12c Electron micrograph of sectioned eukaryotic cells Eukaryote Cytoplasmic membrane Nucleus Cell wall Mitochondrion Eukarya © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI Figure 2.11a Internal structure of prokaryotic cells Cytoplasm Nucleoid Ribosomes Plasmid Cell wall Prokaryote © 2012 Pearson Education, Inc. Cytoplasmic membrane Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 16 10/5/2013 Figure 2.12a Electron micrograph of sectioned prokaryotic cells Prokaryotes (a) Bacteria © 2012 Pearson Education, Inc. Prokaryotes Archaea Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.5 Elements of Microbial Structure • Viruses – Not considered cells – No metabolic abilities of their own – Rely completely on biosynthetic machinery of infected cell – Infect all types of cells – Smallest virus is 10 nm in diameter © 2012 Pearson Education, Inc. 17 10/5/2013 2.6 Arrangement of DNA in Microbial Cells • Genome – A cell’s full complement of genes • Prokaryotic cells generally have a single, circular DNA molecule called a chromosome – DNA aggregates to form the nucleoid region (Figure 2.14) – Prokaryotes also may have small amounts of extra-chromosomal DNA called plasmids that confer special properties (e.g., antibiotic resistance) © 2012 Pearson Education, Inc. Figure 2.14 The nucleoid © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 18 10/5/2013 2.6 Arrangement of DNA in Microbial Cells • Eukaryotic DNA is linear and found within the nucleus – Associated with proteins that help in folding of the DNA – Usually more than one chromosome – Typically two copies of each chromosome – During cell division, nucleus divides by mitosis – During sexual reproduction, the genome is halved by meiosis © 2012 Pearson Education, Inc. 2.6 Arrangement of DNA in Microbial Cells • Escherichia coli Genome – – – – 4.64 million base pairs 4,300 genes 1,900 different kinds of protein 2.4 million protein molecules • Human Cell – 1,000 more DNA per cell than E. coli – 7 more genes than E. coli © 2012 Pearson Education, Inc. 19 10/5/2013 2.7 The Evolutionary Tree of Life • Evolution – The process of change over time that results in new varieties and species of organisms • Phylogeny – Evolutionary relationships between organisms – Relationships can be deduced by comparing genetic information in the different specimens – Ribosomal RNA (rRNA) is excellent for determining phylogeny – Relationships visualized on a phylogenetic tree (Figure 2.16) © 2012 Pearson Education, Inc. Figure 2.16 Ribosomal RNA (rRNA) gene sequencing and phylogeny. (a) DNA is extracted from cells. (b) Many identical copies of a gene encoding rRNA are made by the polymerase chain reaction DNA DNA sequencing Aligned rRNA gene sequences Gene encoding ribosomal RNA Cells Isolate DNA © 2012 Pearson Education, Inc. PCR Sequence analysis Generate phylogenetic tree Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 20 10/5/2013 2.7 The Evolutionary Tree of Life • Comparative rRNA sequencing has defined three distinct lineages of cells called domains: – Bacteria (prokaryotic) – Archaea (prokaryotic) – Eukarya (eukaryotic) • Archaea and Bacteria are NOT closely related (Figure 2.17) • Archaea are more closely related to Eukarya than Bacteria • Eukaryotic microorganisms were the ancestors of multicellular organisms (Figure 2.17) © 2012 Pearson Education, Inc. Figure 2.17 The phylogenetic tree of life as defined by comparative rRNA gene sequencing BACTERIA ARCHAEA EUKARYA Animals Entamoebae Green nonsulfur bacteria Mitochondrion GramProteobacteria positive bacteria Chloroplast Euryarchaeota Methanosarcina MethanoExtreme Crenarchaetoa bacterium halophiles Thermoproteus Methanococcus Thermoplasma Pyrodictium Fungi Plants Ciliates Thermococcus Cyanobacteria Flavobacteria Slime molds Marine Crenarchaeota Pyrolobus Flagellates Methanopyrus Trichomonads Thermotoga Microsporidia Thermodesulfobacterium Aquifex © 2012 Pearson Education, Inc. LUCA Diplomonads (Giardia) Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 21 10/5/2013 III. Microbial Diversity • • • • 2.8 Metabolic Diversity 2.9 Bacteria 2.10 Archaea 2.11 Phylogenetic Analyses of Natural Microbial Communities • 2.12 Microbial Eukarya © 2012 Pearson Education, Inc. 2.8 Metabolic Diversity • The diversity in microbial cells is the product of almost 4 billion years of evolution • Microorganisms differ in size, shape, motility, physiology, pathogenicity, etc. • Microorganisms have exploited every conceivable means of obtaining energy from the environment © 2012 Pearson Education, Inc. 22 10/5/2013 2.8 Metabolic Diversity • Chemoorganotrophs – Obtain their energy from the oxidation of organic molecules (Figure 2.18) – Aerobes use oxygen to obtain energy – Anaerobes obtain energy in the absence of oxygen • Chemolithotrophs – Obtain their energy from the oxidation of inorganic molecules (Figure 2.18) – Process found only in prokaryotes © 2012 Pearson Education, Inc. 2.8 Metabolic Diversity • Phototrophs – Contain pigments that allow them to use light as an energy source (Figure 2.18) – Oxygenic photosynthesis produces oxygen – Anoxygenic photosynthesis does not produce oxygen © 2012 Pearson Education, Inc. 23 10/5/2013 Figure 2.18 Metabolic options for conserving energy Energy Sources Chemicals Light Chemotrophy Phototrophy Organic chemicals Inorganic chemicals (glucose, acetate, etc.) (H2, H2S, Fe2+, NH4+, etc.) Chemoorganotrophs Chemolithotrophs Phototrophs (glucose O2 CO2 H2O) © 2012 Pearson Education, Inc. (H2 O2 H2O) (light) Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.8 Metabolic Diversity • All cells require carbon as a major nutrient – Autotrophs • Use carbon dioxide as their carbon source • Sometimes referred to as primary producers – Heterotrophs • Require one or more organic molecules for their carbon source • Feed directly on autotrophs or live off products produced by autotrophs © 2012 Pearson Education, Inc. 24 10/5/2013 2.8 Metabolic Diversity • Organisms that inhabit extreme environments are called extremophiles • Habitats include boiling hot springs, glaciers, extremely salty bodies of water, and high-pH environments © 2012 Pearson Education, Inc. 2.9 Bacteria • The domain Bacteria contains an enormous variety of prokaryotes (Figure 2.19) • All known pathogenic prokaryotes are Bacteria • The Proteobacteria make up the largest phylum of Bacteria – Gram-negative • Examples: E. coli, Pseudomonas, and Salmonella • Gram-positive phylum united by phylogeny and cell wall structure – Cyanobacteria are relatives of gram-positive bacteria © 2012 Pearson Education, Inc. 25 10/5/2013 Figure 2.19 Phylogenetic tree of some representative Bacteria Spirochetes Deinococcus Green sulfur Planctomyces bacteria Green nonsulfur bacteria Chlamydia Cyanobacteria Thermotoga Gram-positive bacteria OP2 Aquifex Proteobacteria © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.9 Bacteria • Many Other Phyla of Bacteria – Green sulfur bacteria and green nonsulfur bacteria are photosynthetic – Deinococcus is extremely resistant to radioactivity – Chlamydia are obligate intracelluar parasites © 2012 Pearson Education, Inc. 26 10/5/2013 2.10 Archaea • Two Phyla of the Domain Archaea – Euryarchaeota (Figure 2.28) • Methanogens: degrade organic matter anaerobically, produce methane (natural gas) • Extreme halophiles: require high salt concentrations for metabolism and reproduction • Thermoacidophiles: grow in moderately high temperatures and low-pH environments © 2012 Pearson Education, Inc. 2.10 Archaea – Crenarchaeota (Figure 2.28) • Vast majority of cultured Crenarchaeota are hyperthermophiles • Some live in marine, freshwater, and soil systems © 2012 Pearson Education, Inc. 27 10/5/2013 Figure 2.28 Phylogenetic tree of some representative Bacteria Marine group Crenarchaeota Halobacterium Natronobacterium Marine group Euryarchaeota Sulfolobus Methanobacterium Halophilic methanogens Methanocaldococcus Thermoproteus Pyrococcus Methanosarcina Thermoplasma Pyrolobus Methanopyrus Desulfurococcus Hyperthermophiles © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.11 Phylogenetic Analyses of Natural Microbial Communities • Microbiologists believe that we have cultured only a small fraction of the Archaea and Bacteria • Studies done using methods of molecular microbial ecology, devised by Norman Pace – Microbial diversity is much greater than laboratory culturing can reveal © 2012 Pearson Education, Inc. 28 10/5/2013 2.12 Microbial Eukarya • Eukaryotic microorganisms include algae, fungi, protozoa, and slime molds (Figure 2.32) – Protists include algae and protozoa • The algae are phototrophic (Figure 2.33a) • Protozoa NOT phototrophic (Figure 2.33c) – Fungi are decomposers (Figure 2.33b) – Algae and fungi have cell walls, whereas protozoa and slime molds do not © 2012 Pearson Education, Inc. Figure 2.32 Phylogenetic tree of some representative Eukarya Flagellates Diplomonads Trichomonads Slime molds Ciliates Animals Green algae Plants Red algae Fungi Diatoms Brown algae Early-branching, lack mitochondria © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 29 10/5/2013 Figure 2.33a Microbial Eukarya - Algae © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI Figure 2.33b Microbial Eukarya - Fungi © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 30 10/5/2013 Figure 2.33c Microbial Eukarya - Protozoa © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 2.12 Microbial Eukarya • Lichens are a mutualistic relationship between two groups of protists (Figure 2.34) – Fungi and cyanobacteria – Fungi and algae • Microbial eukaryotes are comprehensively discussed in Chapter 20 © 2012 Pearson Education, Inc. 31 10/5/2013 Figure 2.34 Lichens. (a) An orange-pigmented lichen growing on a rock, and (b) a yellow-pigmented lichen growing on a dead tree stump, © 2012 Pearson Education, Inc. Marmara University - Enve 303 Env. Eng. Microbiology - Prof. BARIŞ ÇALLI 32