Microbiology: An Introduction (Thirteenth Edition) Chapter 3 PDF

Microbiology an Introduction Thirteenth Edition Chapter 3 Observing Microorganisms through a Microscope Copyright © 2019 Pearson Education, Inc. All Rights Reserved Helicobacter Pylori Bacteria Copyright © 2019 Pearson Education, Inc. All Rights Reserved Foundation Figure 3.2 Microscopes and Magnifi...

Microbiology an Introduction Thirteenth Edition Chapter 3 Observing Microorganisms through a Microscope Copyright © 2019 Pearson Education, Inc. All Rights Reserved Helicobacter Pylori Bacteria Copyright © 2019 Pearson Education, Inc. All Rights Reserved Foundation Figure 3.2 Microscopes and Magnification Copyright © 2019 Pearson Education, Inc. All Rights Reserved Units of Measurement (1 of 2) Learning Objective 3-1 List the units used to measure microorganisms. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Units of Measurement (2 of 2) Microorganisms are measured in micrometers μm and nanometers (nm) 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 Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-1 Check Your Understanding ü 3-1 How many nanometers is 10 μm? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Microscopy: The Instruments (1 of 2) Learning Objectives 3-2 Diagram the path of light through a compound microscope. 3-3 Define total magnification and resolution. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Microscopy: The Instruments (2 of 2) A simple microscope has only one lens Similar to a magnifying glass, but with a much better (higher magnification) lens Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 1.3b Anton van Leeuwenhoek's Microscopic Observations Lens Location of specimen on pin Specimenpositioning screw Focusing control Stagepositioning screw Microscope replica Copyright © 2019 Pearson Education, Inc. All Rights Reserved Light Microscopy Any kind of microscope that uses visible light to observe specimens Types of light microscopy – Compound light microscopy – Darkfield microscopy – Phase-contrast microscopy – Differential interference contrast (DIC) microscopy – Fluorescence microscopy – Confocal microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.1a The Compound Light Microscope Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Body tube Transmits the image from the objective lens to the ocular lens Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Coarse focusing knob Base Fine focusing knob Principal parts and functions Copyright © 2019 Pearson Education, Inc. All Rights Reserved Compound Light Microscopy (1 of 4) In a compound microscope, the image from the objective lens is magnified again by the ocular lens Total magnification = objective lens × ocular lens Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.1b The Compound Light Microscope Line of vision Ocular lens Path of light Prism Body tube Objective lenses Specimen Condenser lenses Illuminator Base with source of illumination The path of light (bottom to top) Copyright © 2019 Pearson Education, Inc. All Rights Reserved Compound Light Microscopy (2 of 4) Resolution is the ability of the lenses to distinguish two points A microscope with a resolving power of 0.4 nm can distinguish between two points at least 0.4 nm apart Shorter wavelengths of light provide greater resolution Copyright © 2019 Pearson Education, Inc. All Rights Reserved Compound Light Microscopy (3 of 4) The refractive index is a measure of the lightbending ability of a medium Light may refract after passing through a specimen to an extent that it does not pass through the objective lens Immersion oil is used to keep light from refracting Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.3 Refraction in the Compound Microscope Using an Oil Immersion Objective Lens Oil immersion objective lens Unrefracted light Without immersion oil, most light is refracted and lost Immersion oil Air Glass slide Condenser lenses Condenser Light source Iris diaphragm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Compound Light Microscopy (4 of 4) Brightfield illumination – Dark objects are visible against a bright background – Light reflected off the specimen does not enter the objective lens Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.4a Brightfield, Darkfield, and Phase-contrast Microscopy (a). Brightfield. (Top) The path of light in brightfield microscopy, the type of illumination produced by regular compound light microscopes. (Bottom) Brightfield illumination shows internal structures and the outline of the transparent pellicle (external covering). Copyright © 2019 Pearson Education, Inc. All Rights Reserved Animation: Light Microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-2 Check Your Understanding ü 3-2 Through what lenses does light pass in a compound microscope? ü 3-3 What does it mean when a microscope has a resolution of 0.2 nm? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Microscopy: The Instruments Learning Objectives 3-4 Identify a use for darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, two-photon, and scanning acoustic microscopy, and compare each with brightfield illumination. 3-5 Explain how electron microscopy differs from light microscopy. 3-6 Identify uses for the transmission electron microscope (TEM), scanning electron microscope (SEM), and scanned-probe microscopes. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Darkfield Microscopy Light objects are visible against a dark background Opaque disk placed in condenser Only light reflected off the specimen enters the objective lens Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.4b Brightfield, Darkfield, and Phase-Contrast Microscopy (b). Darkfield. (Top) The darkfield microscope uses a special condenser with an opaque disk that eliminates all light in the center of the beam. The only light that reaches the specimen comes in at an angle; thus, only light reflected by the specimen (blue lines) reaches the objective lens. (Bottom) Against the black background seen with darkfield microscopy, edges of the cell are bright, some internal structures seem to sparkle, and the pellicle is almost visible. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Phase-Contrast Microscopy Allows examination of living organisms and internal cell structures Brings together two sets of light rays, direct rays, and diffracted rays to form an image Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.4c Brightfield, Darkfield, and Phase-Contrast Microscopy (c). Phase-contrast. (Top) In phasecontrast microscopy, the specimen is illuminated by light passing through an annular (ring-shaped) diaphragm. Direct light rays (unaltered by the specimen) travel a different path from light rays that are reflected or diffracted as they pass through the specimen. These two sets of rays are combined at the eye. Reflected or diffracted light rays are indicated in blue; direct rays are red. (Bottom) Phase-contrast microscopy shows greater differentiation of internal structures and clearly shows the pellicle. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Differential Interference Contrast (DIC) Microscopy Similar to phase-contrast Uses two light beams and prisms to split light beams, giving more contrast and color to the specimen Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.5 Differential Interference Contrast (DIC) Microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Fluorescence Microscopy Uses UV (short wavelength) light Fluorescent substances absorb UV light and emit longer wavelength (visible) light Cells may be stained with fluorescent dyes (fluorochromes) if they do not naturally fluoresce Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.6b The Principle of Immunofluorescence Copyright © 2019 Pearson Education, Inc. All Rights Reserved Confocal Microscopy Cells are stained with fluorochrome dyes Short-wavelength (blue) light is used to excite a single plane of a specimen Each plane in a specimen is illuminated and a threedimensional image is constructed with a computer Can examine layers of cells to a depth of 100 μm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.7 Confocal Microscopy Nucleus Copyright © 2019 Pearson Education, Inc. All Rights Reserved Two-Photon Microscopy Cells are stained with fluorochrome dyes Two photons of long-wavelength (red) light are used to excite the dyes Can study living cells up to 1 mm deep Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.8 Two-Photon Microscopy (TPM) Nucleus Copyright © 2019 Pearson Education, Inc. All Rights Reserved Super-Resolution Light Microscopy Uses two laser beams – One wavelength stimulates fluorescent molecules to glow – Second wavelength cancels out all fluorescence except for that in one nm A computer scans the specimen nm by nm, then puts the images together Copyright © 2019 Pearson Education, Inc. All Rights Reserved Fig. 3.9 Super-Resolution Light Microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Scanning Acoustic Microscopy Measures sound waves that are reflected back from a specimen Used to study cells attached to surfaces Resolution of 1 μm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.10 Scanning Acoustic Microscopy (SAM) of a Bacterial Biofilm on Glass Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-3 Check Your Understanding ü 3-4 How are brightfield, darkfield, phase-contrast, and fluorescence microscopy similar? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Electron Microscopy Uses electrons instead of light The shorter wavelength of electrons gives greater resolution Used for images too small to be seen with light microscopes, such as viruses Copyright © 2019 Pearson Education, Inc. All Rights Reserved Transmission Electron Microscopy (1 of 2) A beam of electrons passes through ultrathin sections of a specimen, then through an electromagnetic lens, then focused on a projector lens Specimens may be stained with heavy-metal salts for contrast Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.11a Transmission and Scanning Electron Microscopy Electron gun Electron beam Electromagnetic condenser lens Specimen Electromagnetic objective lens Electromagnetic projector lens Fluorescent screen or photographic plate Viewing eyepiece Transmission. (Left) In a transmission electron microscope, electrons pass through the specimen and are scattered. Magnetic lenses focus the image onto a fluorescent screen or photographic plate. (Right) This colorized transmission electron micrograph (TEM) shows a thin slice of Paramecium. In this type of microscopy, the internal structures present in the slice can be seen. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Transmission Electron Microscopy (2 of 2) Magnifies objects 10,000 to 10,000,000x; resolution of 10 pm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Scanning Electron Microscopy (1 of 2) An electron gun produces a beam of electrons that scans the surface of an entire specimen Secondary electrons emitted from the specimen produce a three-dimensional image Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.11b Transmission and Scanning Electron Microscopy Electron gun Primary electron beam Electromagnetic lenses Viewing screen Electron collector Secondary electrons Specimen Amplifier Scanning. (Left) In a scanning electron microscope, primary electrons sweep across the specimen and knock electrons from its surface. These secondary electrons are picked up by a collector, amplified, and transmitted onto a viewing screen or photographic plate. (Right) In this colorized scanning electron micrograph (SEM), the surface structures of Paramecium can be seen. Note the three-dimensional appearance of this cell, in contrast to the two-dimensional appearance of the transmission electron micrograph in part (a). Copyright © 2019 Pearson Education, Inc. All Rights Reserved Scanning Electron Microscopy (2 of 2) Magnifies objects 1,000 to 500,000x; resolution of 10 nm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Animation: Electron Microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-4 Check Your Understanding ü 3-5 Why do electron microscopes have greater resolution than light microscopes? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Scanning Tunneling Microscopy Uses a tungsten probe to scan a specimen and reveal details of its surface Resolution of 1/100 of an atom Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.12a Scanned-Probe Microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Atomic Force Microscopy Uses a metal-and-diamond probe placed onto a specimen; movements are recorded Produces three-dimensional images at near atomic detail Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.12b Scanned-Probe Microscopy Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-5 Check Your Understanding ü 3-6 For what is TEM used? SEM? Scanned-probe microscopy? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Preparation of Specimens for Light Microscopy Learning Objectives 3-7 Differentiate an acidic dye from a basic dye. 3-8 Explain the purpose of simple staining. 3-9 List Gram stain steps, and describe the appearance of gram-positive and gram-negative cells after each step. 3-10 Compare and contrast the Gram stain and the acid-fast stain. 3-11 Explain why each of the following is used: capsule stain, endospore stain, flagella stain. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Preparing Smears for Staining (1 of 3) Staining: coloring microorganisms with a dye that emphasizes certain structures Smear: a thin film of a material containing microorganisms spread over a slide Microorganisms are fixed (attached) to the slide, which kills the microorganisms Copyright © 2019 Pearson Education, Inc. All Rights Reserved Preparing Smears for Staining (2 of 3) Live and/or unstained specimens have little contrast with the surrounding medium. Live specimens are used to study cell behavior. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Animation: Microscopy and Staining: Overview Copyright © 2019 Pearson Education, Inc. All Rights Reserved Preparing Smears for Staining (3 of 3) Stains consist of a positive and negative ion, one of which is colored (chromophore) In a basic dye, the chromophore is a cation In an acidic dye, the chromophore is an anion Staining the background instead of the cell is called negative staining Copyright © 2019 Pearson Education, Inc. All Rights Reserved Simple Stains Simple stain: use of a single basic dye Highlights the entire microorganism to visualize cell shapes and structures A mordant may be used to hold the stain or coat the specimen to enlarge it Copyright © 2019 Pearson Education, Inc. All Rights Reserved Animation: Staining Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-6 Check Your Understanding ü 3-7 Why doesn't a negative stain color a cell? ü 3-8 Why is fixing necessary for most staining procedures? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Differential Stains Used to distinguish between bacteria – Gram stain – Acid-fast stain Copyright © 2019 Pearson Education, Inc. All Rights Reserved The First Synthetic Drugs Classifies bacteria into gram-positive or gramnegative – Gram-positive bacteria have thick peptidoglycan cell walls – Gram-negative bacteria have thin peptidoglycan cell walls and a layer of lipopolysaccharides Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.13a Gram Staining KEY Crystal violet Iodine Alcohol Safranin Gram-positive Gram-negative Application of crystal violet (purple dye) Application of iodine (mordant) Alcohol wash (decolorization) Application of safranin (counterstain) Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.13b Gram Staining Coccus (gram-positive) Rod (gram-negative) Copyright © 2019 Pearson Education, Inc. All Rights Reserved Acid-Fast Stain (1 of 2) Binds only to bacteria that have a waxy material in their cell walls, which is not decolorized by acidalcohol Used for the identification of – Mycobacterium – Nocardia Copyright © 2019 Pearson Education, Inc. All Rights Reserved Acid-Fast Stain (2 of 2) Blank Primary Stain: Carbolfuchsin Decolorizing Agent: Acid-Alcohol Counterstain: Methylene Blue Color of Acid-Fast Color of Non–Acid-Fast Red Red Red Colorless Red Blue Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.14 Acid-Fast Bacteria M. tuberculosis Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-7 Check Your Understanding ü 3-9 Why is the Gram stain so useful? ü 3-10 Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Special Stains Used to distinguish parts of microorganisms – Capsule stain – Endospore stain – Flagella stain Copyright © 2019 Pearson Education, Inc. All Rights Reserved Negative Staining for Capsules Capsules are a gelatinous covering that do not accept most dyes Suspension of India ink or nigrosin contrasts the background with the capsule, which appears as a halo around the cell Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.15a Special Staining Capsules Negative staining Copyright © 2019 Pearson Education, Inc. All Rights Reserved Endospore Staining Endospores are resistant, dormant structures inside some cells that cannot be stained by ordinary methods Primary stain: malachite green, usually with heat Decolorize cells: water Counterstain: safranin Spores appear green within red or pink cells Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.15b Special Staining Endospore Endospore staining Copyright © 2019 Pearson Education, Inc. All Rights Reserved Flagella Staining Flagella are structures of locomotion Uses a mordant and carbolfuchsin to thicken appearance of flagella, making them visible under the light microscope Copyright © 2019 Pearson Education, Inc. All Rights Reserved Figure 3.15c Special Staining Flagellum Flagella staining Copyright © 2019 Pearson Education, Inc. All Rights Reserved Check Your Understanding-8 Check Your Understanding ü 3-11 How do unstained endospores appear? Stained endospores? Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright Copyright © 2019 Pearson Education, Inc. All Rights Reserved