8/23/2016 Microbes Units of Measurement Learning Objectives Invisible Invaders List the metric units of measurement that are Amazing Allies used for microorganisms Chapter 3 Observing Microorganisms through a Microscope Metric system-standard unit of length is the meter Microorganisms are too small to be seen with the naked eye. Units are related by factors of 10 Microbes and their components are measured in Various types of microscopes micrometer and nanometer are used to visualize microorganisms Table 4.1 Metric Units of Length Today's microscopes use ac和 magnifications ten to thousand 39nabout a yard times greater clarity to that of a van Leeuwenhoek's 1n 11m.101m 3其 1tm。0m 039it1in-254 om Duneer ol amuhooncoe 12 cn Staining procedures of 000m.10"n microorganisms are used to observe features of the microorganisms as well to aid 9010w5e4,Z5 visualizing the microbe
8/23/2016 1 Invisible Invaders Amazing Allies Chapter 3 Observing Microorganisms through a Microscope 1 Microorganisms are too small to be seen with the naked eye. Various types of microscopes are used to visualize microorganisms Today’s microscopes use magnifications ten to thousand times greater clarity to that of van Leeuwenhoek’s Staining procedures of microorganisms are used to observe features of the microorganisms as well to aid visualizing the microbe 2 Units of Measurement Learning Objectives • List the metric units of measurement that are used for microorganisms 3 • Metric system – standard unit of length is the meter • Units are related by factors of 10 • Microbes and their components are measured in micrometer and nanometer 4
8/23/2016 Units of Measurement Key Characteristics of a Microscope 1m=106m= Magnification-ability to enlarge objects 103mm 1nm=109m= Resolving power (resolution) 106mm ability of the lenses to show 1000nm-1m fine detail and structure 10 nm t m -ability of the lenses to 0.001m:1nm distinguish two points a specified distance apart -shorter the wavelength of light-the greater the resolution NA double helix -example:if a microscope has a resolving power of 0.4 um,it can distinguish two points if they are 0.4 um apart Microscopy:the Instrument Light Microscopy Learning Objectives Light microscopy use of any kind of microscope that uses visible light to observe Diagram the path of light through a compound specimens microscope Define total magnification and resolution Light microscope use long wavelengths and has Identify a use for dark-field,fluorescence,and a resolving power of 0.2 um confocal microscopy,and compare each with Simple microscope uses one lens bright-field microscopy Compound light microscope uses a series of Explain how electron microscopy differs from lenses to magnify the image light microscopy Identify a use for TEM and SEM microscopes Series of lenses forms a clearly focused image that is larger than the specimen itself
8/23/2016 2 Tick Actual siz Red blood cel E. coli bacteria T-even bacteriophag (viruses) DNA double helix 1 µm = 10–6 m = 10–3 mm 1 nm = 10–9 m = 10–6 mm 1000 nm = 1 µm 0.001 µm = 1 nm Units of Measurement Unaided eye ≥ 200 m Light microscope 200 nm – 10 mm Scanning electron microscope 10 nm – 1 mm Transmission electron microscope 10 pm – 100 m Atomic force microscope 0.1 nm – 10nm 5 Learning Objectives • Diagram the path of light through a compound microscope • Define total magnification and resolution • Identify a use for dark-field, fluorescence, and confocal microscopy, and compare each with bright-field microscopy • Explain how electron microscopy differs from light microscopy • Identify a use for TEM and SEM microscopes Microscopy: the Instrument 6 Magnification – ability to enlarge objects Resolving power (resolution) - ability of the lenses to show fine detail and structure - ability of the lenses to distinguish two points a specified distance apart - shorter the wavelength of light – the greater the resolution - example: if a microscope has a resolving power of 0.4 m, it can distinguish two points if they are 0.4 m apart Key Characteristics of a Microscope 7 Light Microscopy • Light microscopy – use of any kind of microscope that uses visible light to observe specimens • Light microscope use long wavelengths and has a resolving power of 0.2 m • Simple microscope uses one lens • Compound light microscope uses a series of lenses to magnify the image • Series of lenses forms a clearly focused image that is larger than the specimen itself 8
8/23/2016 Refractive index is the measure of the light-bending ability of a medium. ·mo finely detailed specimens must be made to contrast sharply with the substance in which they are suspended. The refractive index of specimens are changed by staining After staining,the light rays pass through the two materials with different refractive indexes Oil Immersion Lens To obtain achieve hioh Light Path in a Microscope A microscope must accomplish three things: The light may bend in air so much that it misses the small high- magnify the object you magnification lens after are trying to view passing through the specimen. resolve the details of the To preserve the direction of light object rays at the highest sion oil make these details visible b)The path of light mersion oil has the (Bottom to top) 3
8/23/2016 3 Total magnification = power of objective lens X power of ocular lens 9 A microscope must accomplish three things: - magnify the object you are trying to view - resolve the details of the object - make these details visible Light Path in a Microscope 10 Refractive index is the measure of the light-bending ability of a medium. • To obtain clear, finely detailed image under a light microscope, specimens must be made to contrast sharply with the substance in which they are suspended. • The refractive index of specimens are changed by staining • After staining, the light rays pass through the two materials with different refractive indexes 11 To obtain achieve high magnification (1000x) with good resolution, the objective lens must be small The light may bend in air so much that it misses the small highmagnification lens after passing through the specimen. To preserve the direction of light rays at the highest magnification, immersion oil is placed between the glass slide and the oil immersion objective The immersion oil has the same refractive index as the glass Oil Immersion Lens 12
8/23/2016 Condenser contains an opaque Types of Light Types of Light Microscopy disk that eliminates all light in the center of the beam(light Microscopy that enters the lens directly) Dark-Field Many different types of light microscopy Only light reflected by the Bright-field specimen enters the objective lens Dark-field Light objects are visible against Fluorescence a dark backoround. Used to exomine: live microbes that have little contrast in bright-field microscopy that be microbes that are distorted by staining Types of Light Microscopy Types of Light Microscopy:Fluorescence Bright-Field Uses UV(ultra violet)light ·Usual operating Fluorescent substances absorb UV conditions of a light and emit visible light. compound light Cells may be stained with microscope fluorescent dyes:fluorochromes. ·Dark objects are A principle use in microbiology is a visible against a bright diagnostic technique called background. immunofluorescence A fluorescent antibody binds a ·Light reflected off specific microbe-labeling the the specimen does not enter the objective microbe with fluorescence Pathogenic microbes are detected orange lens. within clinical specimens
8/23/2016 4 Types of Light Microscopy Many different types of light microscopy Bright-field Dark-field Fluorescence 13 • Usual operating conditions of a compound light microscope • Dark objects are visible against a bright background. • Light reflected off the specimen does not enter the objective lens. Types of Light Microscopy Bright-Field 14 Types of Light Microscopy Dark-Field • Condenser contains an opaque disk that eliminates all light in the center of the beam (light that enters the lens directly). • Only light reflected by the specimen enters the objective lens. • Light objects are visible against a dark background. • Used to examine: - live microbes that have little contrast in bright-field microscopy - microbes that cannot be stained - microbes that are distorted by staining 15 • Uses UV (ultra violet) light • Fluorescent substances absorb UV light and emit visible light. • Cells may be stained with fluorescent dyes: fluorochromes. • A principle use in microbiology is a diagnostic technique called immunofluorescence • A fluorescent antibody binds a specific microbe - labeling the microbe with fluorescence • Pathogenic microbes are detected within clinical specimens Types of Light Microscopy: Fluorescence A thin blood smear showing trypanosomes stained with acridine orange 16
8/23/2016 Types of Light Microscopy:Confocal Transmission Electron Microscopy (TEM) Used to create 3D microscopic Ultrathin sections of specimens. images of a specimen Electrons pass through specimen and are scattered Uses fluorochromes to stain the specimen Electromagnetic lenses focus the image onto a Uses laser light to illuminate one fluorescent screen or film. thin slice of the specimen at a time Specimens may be stained with heavy metal salts Successive slicing of the whole to improve contrast. specimen reveals a 2D image when Electron beam compiled Computers construct 3D images from scanned planes of the specimen Tetrahymena thermnoohila rotated to view with the cilia shown in green F8for8na折 substances .sho and one o f the throughout a cell. sop Electron Microscopy Transmission Electron Microscopy (TEM) hr地g92es 5.000-100.000￥ resolution 2.5 nm patterns when accelerated to high speeds ro power is a function of wavelength. 638般oabetnmen5i0oxand rochetes cover ing the T4 bacteriophage binding Tw types of el fron microscopes osal epithelic of the 5
8/23/2016 5 • Used to create 3D microscopic images of a specimen • Uses fluorochromes to stain the specimen • Uses laser light to illuminate one thin slice of the specimen at a time. • Successive slicing of the whole specimen reveals a 2D image when compiled • Computers construct 3D images from scanned planes of the specimen • 3D images are rotated to view distribution of substances throughout a cell. Types of Light Microscopy: Confocal Tetrahymena thermophila with the cilia shown in green and one of the organism's two nuclei, shown in blue. 17 Electron Microscopy • Visualizes objects smaller than 0.2 m, such as viruses or internal cell structures • Forms an image with a beam of electrons 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. • Uses electromagnetic lenses to focus a beam of electrons instead of glass lenses • Magnification between 5,000X and 100,000X • Two types of electron microscopes: transmission and scanning Transmission Electron Microscope Scanning Electron Microscope 18 • Ultrathin sections of specimens. • Electrons pass through specimen and are scattered • Electromagnetic lenses focus the image onto a fluorescent screen or film. • Specimens may be stained with heavy metal salts to improve contrast. Transmission Electron Microscopy (TEM) 19 5,000-100,000 resolution 2.5 nm Transmission Electron Microscopy (TEM) T4 bacteriophage binding to the surface of an Escherichia coli bacterium spirochetes covering the mucosal epithelial surface of the biopsied colon 20
8/23/2016 Scanning Electron Microscopy (SEM) Preparation of Specimens for Light An electron gun produces a beam of primary Microscopy electrons that scan the surface of a whole specimen and knock electrons from its surface Learning Objectives Secondary electrons emitted from the specimen Differentiate an acidic dye from an basic dye produce a 3D image. Explain the purpose of simple staining tron gu List the steps in preparing a Gram stain and describe the appearance of gram-negative and gram-positive cells after each step Compare and contrast Gram stain and the acid fast stain Explain why each of the following is used: capsule stain,endospore stain,flagella stain a Scanning Electron Microscopy (SEM) Preparing Smears for Staining 1000-10.000× resolution 10 nm Staining means coloring the microbe with a dye that emphasizes certain structures A thin film of a solution of microbes on a slide is called a smear. Before staining,cells must be fixed to a microscope slide. This kills the microbe and attaches them to the slide while preserving the microbe with minimal distortion ulfed by a oral apparatus 2离h d macrophage cilia L.Preparing a smear 6
8/23/2016 6 • An electron gun produces a beam of primary electrons that scan the surface of a whole specimen and knock electrons from its surface. • Secondary electrons emitted from the specimen produce a 3D image. Scanning Electron Microscopy (SEM) 21 1000-10,000 resolution 10 nm Scanning Electron Microscopy (SEM) Paramecium showing the oral apparatus and the cilia A SEM image of E. coli (green) being engulfed by a macrophage 22 Learning Objectives • Differentiate an acidic dye from an basic dye • Explain the purpose of simple staining • List the steps in preparing a Gram stain and describe the appearance of gram-negative and gram-positive cells after each step • Compare and contrast Gram stain and the acid fast stain • Explain why each of the following is used: capsule stain, endospore stain, flagella stain Preparation of Specimens for Light Microscopy 23 Preparing Smears for Staining • Live or unstained cells have little contrast with the surrounding medium. • Staining means coloring the microbe with a dye that emphasizes certain structures • A thin film of a solution of microbes on a slide is called a smear. • Before staining, cells must be fixed to a microscope slide. This kills the microbe and attaches them to the slide while preserving the microbe with minimal distortion 24
8/23/2016 Preparing Smears for Staining Differential Stains Stains are salts composed of a positive or negative ion one of which is colored and called the chromophore Dyes create contrast imparting a color to cells or cell parts use a primary stain and a counter stain to distinguish cell types or parts Basic dyes-cationic,with positive charges on the chromophore react differently with different kinds of Acidic dyes-anionic,with negative bacteria and thus can be used to distinguish charges on the chromophore Streptococcus pyog between them Examples of frequently used stains:gram stain and acid-fast stain andcapsuilesaedipt8rtionismimimalsnc 25 Micrococcus luteus Simple Stains Acid-Fast Stain a single basic dye is used Binds only to bacteria that have Pesigtdscolrationwrfalcoho hat primary purpose is to highlight the entire microbe Mycobacterium and Nocardia are reveals shape,size,and arrangement both conain pthogenic Examples:methylene blue,crystal Bacillus obacterium tuberculosis Mycobacterfum bovis stains pin violet,carbolfuchsin,and safranin crystal violet 10anafochsnspedoafad Acid-achd 4.Smear is stained with methylene blue
8/23/2016 7 Stains are salts composed of a positive or negative ion, one of which is colored and called the chromophore Dyes create contrast imparting a color to cells or cell parts • Basic dyes - cationic, with positive charges on the chromophore • Acidic dyes - anionic, with negative charges on the chromophore • Positive staining – surfaces of microbes are negatively charged, attract basic dyes • Negative staining – microbe repels the dye and the dye stains the background – valuable for observing cell sizes, shapes and capsules as distortion is minimal since fixing is not required Preparing Smears for Staining Negative stain Micrococcus luteus Streptococcus pyogenes 25 Simple Stains • a single basic dye is used • primary purpose is to highlight the entire microbe • reveals shape, size, and arrangement • Examples: methylene blue, crystal violet, carbolfuchsin, and safranin Bacillus crystal violet 26 Differential Stains • use a primary stain and a counter stain to distinguish cell types or parts • react differently with different kinds of bacteria and thus can be used to distinguish between them • Examples of frequently used stains: gram stain and acid-fast stain 27 Acid-Fast Stain Binds only to bacteria that have waxy material in their cell walls that resist discoloration with alcohol Mycobacterium and Nocardia are acid-fast, both contain pathogenic strains Mycobacterium tuberculosis Mycobacterium tuberculosis Mycobacterium bovis stains pink (acid-fast) Staphylococcus stain blue (non-acid-fast) 28 1. Carbolfuchsin is applied to a fixed smear 2. Slide is gently heated to drive the stain into the waxy cell walls 3. Acid-alcohol, a decolorizer, removes stain from the non-acid-fast cells. Carbolfuchsin is more soluble in the cell wall lipid than in the alcohol 4. Smear is stained with methylene blue
8/23/2016 Gram Stain Endospore staining An important staining techniques since it classifies bacteria into spore is a special resistant,dormant st formed protects two large groups:gram-positive adverse environmental conditions. and gram-negative Endospores are uncommon but a few genera of bacteria form them Endospores cannot be stained by ordinary methods since Gram staining procedure: the dyes do not penetrate the wall of the endospore. E.coli and Staphylococcus aureus 8018 28eca6 Mature spores stain green 00 Special Stains These stains Negative staining for capsules Many organisms contain a gelatinous covering called a capsule,which plays a role in the virulence of some pathogenic bacteria Capsule staining is more difficult since the capsular material is water soluble Negative staining does not require fixing or washing the cells Due to the biological composition of the capsule,most dyes do not stain the capsule
8/23/2016 8 Gram staining procedure: E. coli and Staphylococcus aureus Gram Stain An important staining techniques since it classifies bacteria into two large groups: gram-positive and gram-negative 29 Special Stains These stains color and isolate specific parts of microbes such as capsule, endospore and flagella stains. Negative staining for capsules Many organisms contain a gelatinous covering called a capsule, which plays a role in the virulence of some pathogenic bacteria Capsule staining is more difficult since the capsular material is water soluble Negative staining does not require fixing or washing the cells Due to the biological composition of the capsule, most dyes do not stain the capsule 30 Endospore staining An endospore is a special resistant, dormant structure formed within a cell that protects the bacteria from adverse environmental conditions. Endospores are uncommon but a few genera of bacteria form them. Endospores cannot be stained by ordinary methods since the dyes do not penetrate the wall of the endospore. Mature spores stain green vegetative cells stain red Bacillus 31