Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Chapter 3 Observing Microorganisms Through A Microscope Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Q&A Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis? Copyright © 2010 Pearson Education, Inc. Look for the answer in the chapter . Observing Microorganisms Copyright © 2010 Pearson Education, Inc. Figure 3.2 Units of Measurement Learning Objectives 3-1 List the metric units of measurement that are used for microorganisms. Copyright © 2010 Pearson Education, Inc. Units of Measurement 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 © 2010 Pearson Education, Inc. Figure 3.2 Check Your Understanding If a microbe measures 10 μm in length, how long is it in nanometers? 3-1 Copyright © 2010 Pearson Education, Inc. Microscopy: The Instruments Learning Objectives 3-2 Diagram the path of light through a compound microscope. 3-3 Define total magnification and resolution. Copyright © 2010 Pearson Education, Inc. Microscopy: The Instruments A simple microscope has only one lens Copyright © 2010 Pearson Education, Inc. Figure 1.2b Light Microscopy Use of 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 microscopy Fluorescence microscopy Confocal microscopy Copyright © 2010 Pearson Education, Inc. The Compound Light Microscope Copyright © 2010 Pearson Education, Inc. Figure 3.1a Compound Light Microscopy In a compound microscope, the image from the objective lens is magnified again by the ocular lens Total magnification = objective lens ocular lens Copyright © 2010 Pearson Education, Inc. Figure 3.1b Compound Light Microscopy 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 ≥ 0.4 nm Shorter wavelengths of light provide greater resolution Copyright © 2010 Pearson Education, Inc. Compound Light Microscopy The refractive index is a measure of the lightbending ability of a medium The light may bend in air so much that it misses the small high-magnification lens Immersion oil is used to keep light from bending Copyright © 2010 Pearson Education, Inc. Refraction in the Compound Microscope Copyright © 2010 Pearson Education, Inc. Figure 3.3 Check Your Understanding Through what lenses does light pass in a compound microscope? 3-2 What does it mean when a microscope has a resolution of 0.2 nm? 3-3 Copyright © 2010 Pearson Education, Inc. 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 one use for the TEM, SEM, and scannedprobe microscopes. Copyright © 2010 Pearson Education, Inc. Brightfield Illumination Dark objects are visible against a bright background Light reflected off the specimen does not enter the objective lens ANIMATION Light Microscopy Copyright © 2010 Pearson Education, Inc. Figure 3.4a Darkfield Illumination Light objects are visible against a dark background Light reflected off the specimen enters the objective lens Copyright © 2010 Pearson Education, Inc. Figure 3.4b Phase-Contrast Microscopy Accentuates diffraction of the light that passes through a specimen Copyright © 2010 Pearson Education, Inc. Figure 3.4c Differential Interference Contrast Microscopy Accentuates diffraction of the light that passes through a specimen; uses two beams of light Copyright © 2010 Pearson Education, Inc. Figure 3.5 Fluorescence Microscopy Uses UV light Fluorescent substances absorb UV light and emit visible light Cells may be stained with fluorescent dyes (fluorochromes) Copyright © 2010 Pearson Education, Inc. Figure 3.6b Confocal Microscopy Cells stained with fluorochrome dyes Short wavelength (blue) light used to excite the dyes The light illuminates each plane in a specimen to produce a three-dimensional image Up to 100 µm deep Copyright © 2010 Pearson Education, Inc. Figure 3.7 Two-Photon Microscopy Cells stained with fluorochrome dyes Two photons of longwavelength (red) light used to excite the dyes Used to study cells attached to a surface Up to 1 mm deep Copyright © 2010 Pearson Education, Inc. Figure 3.8 Scanning Acoustic Microscopy (SAM) Measures sound waves that are reflected back from an object Used to study cells attached to a surface Resolution 1 µm Copyright © 2010 Pearson Education, Inc. Figure 3.9 Electron Microscopy Uses electrons instead of light The shorter wavelength of electrons gives greater resolution ANIMATION Electron Microscopy Copyright © 2010 Pearson Education, Inc. Transmission Electron Microscopy (TEM) Ultrathin sections of specimens Light passes through specimen, then an electromagnetic lens, to a screen or film Specimens may be stained with heavy metal salts Copyright © 2010 Pearson Education, Inc. Figure 3.10a Transmission Electron Microscopy (TEM) 10,000–100,000; resolution 2.5 nm Copyright © 2010 Pearson Education, Inc. Figure 3.10a Scanning Electron Microscopy (SEM) An electron gun produces a beam of electrons that scans the surface of a whole specimen Secondary electrons emitted from the specimen produce the image Copyright © 2010 Pearson Education, Inc. Figure 3.10b Scanning Electron Microscopy (SEM) 1,000–10,000; resolution 20 nm Copyright © 2010 Pearson Education, Inc. Figure 3.10b Scanned-Probe Microscopy Scanning tunneling microscopy (STM) uses a metal probe to scan a specimen Resolution 1/100 of an atom Copyright © 2010 Pearson Education, Inc. Figure 3.11a Scanned-Probe Microscopy Atomic force microscopy (AFM) uses a metaland-diamond probe inserted into the specimen. Produces three-dimensional images. Copyright © 2010 Pearson Education, Inc. Figure 3.11b Check Your Understanding How are brightfield, darkfield, phase-contrast, and fluorescence microscopy similar? 3-4 Why do electron microscopes have greater resolution than light microscopes? 3-5 For what is TEM used? SEM? Scanned-probe microscopy? 3-6 Copyright © 2010 Pearson Education, Inc. 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 the steps in preparing a Gram stain, 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 © 2010 Pearson Education, Inc. Preparing Smears for Staining Staining: Coloring the microbe with a dye that emphasizes certain structures Smear: A thin film of a solution of microbes on a slide A smear is usually fixed to attach the microbes to the slide and to kill the microbes Copyright © 2010 Pearson Education, Inc. Preparing Smears for Staining Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens. ANIMATION Microscopy and Staining: Overview Copyright © 2010 Pearson Education, Inc. Figures B and C Preparing Smears for Staining Stains consist of a positive and negative ion 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 © 2010 Pearson Education, Inc. Simple Stains Simple stain: Use of a single basic dye A mordant may be used to hold the stain or coat the specimen to enlarge it ANIMATION Staining Copyright © 2010 Pearson Education, Inc. Differential Stains Used to distinguish between bacteria Gram stain Acid-fast stain Copyright © 2010 Pearson Education, Inc. Gram Stain Classifies bacteria into gram-positive or gram-negative Gram-positive bacteria tend to be killed by penicillin and detergents Gram-negative bacteria are more resistant to antibiotics Copyright © 2010 Pearson Education, Inc. Gram Stain Color of Gram-positive cells Color of Gram-negative cells Primary stain: Crystal violet Purple Purple Mordant: Iodine Purple Purple Decolorizing agent: Alcohol-acetone Purple Colorless Counterstain: Safranin Purple Red Copyright © 2010 Pearson Education, Inc. Micrograph of Gram-Stained Bacteria Copyright © 2010 Pearson Education, Inc. Figure 3.12b Check Your Understanding Why doesn’t a negative stain color a cell? 3-7 Why is fixing necessary for most staining procedures? 3-8 Why is the Gram stain so useful? 3-9 Copyright © 2010 Pearson Education, Inc. Acid-Fast Stain Stained waxy cell wall is not decolorized by acidalcohol Mycobacterium Nocardia Copyright © 2010 Pearson Education, Inc. Acid-Fast Stain Color of Acid-fast Color of Non–Acid-fast Primary stain: Carbolfuchsin Red Red Decolorizing agent: Acid-alcohol Red Colorless Counterstain: Methylene blue Red Blue Copyright © 2010 Pearson Education, Inc. Acid-Fast Bacteria Copyright © 2010 Pearson Education, Inc. Figure 3.13 Q&A Acid-fast staining of a patient’s sputum is a rapid, reliable, and inexpensive method to diagnose tuberculosis. What color would bacterial cells appear if the patient has tuberculosis? Copyright © 2010 Pearson Education, Inc. Special Stains Used to distinguish parts of cells Capsule stain Endospore stain Flagella stain Copyright © 2010 Pearson Education, Inc. Negative Staining for Capsules Cells stained Negative stain Copyright © 2010 Pearson Education, Inc. Figure 3.14a Endospore Staining Primary stain: Malachite green, usually with heat Decolorize cells: Water Counterstain: Safranin Copyright © 2010 Pearson Education, Inc. Figure 3.14b Flagella Staining Mordant on flagella Carbolfuchsin simple stain Copyright © 2010 Pearson Education, Inc. Figure 3.14c Check Your Understanding Which stain would be used to identify microbes in the genera Mycobacterium and Nocardia? 3-10 How do unstained endospores appear? Stained endospores? 3-11 Copyright © 2010 Pearson Education, Inc.