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CH 3 Observing Microorganisms through a Microscope

by: Madeleine Reinstein

CH 3 Observing Microorganisms through a Microscope BIOL 210

Marketplace > Montgomery County Community College > Biology > BIOL 210 > CH 3 Observing Microorganisms through a Microscope
Madeleine Reinstein
Montgomery County Community College
GPA 3.73
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Types of Microscopes Definition and Process of Gram Stain, Acid-Fast Stain, Differential Stains, Capsule Stain, Endospore Stain
Ijeoma N. Otigbuo
Class Notes
Microbiology, gram staining, Microscopes





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This 3 page Class Notes was uploaded by Madeleine Reinstein on Wednesday September 21, 2016. The Class Notes belongs to BIOL 210 at Montgomery County Community College taught by Ijeoma N. Otigbuo in Fall 2016. Since its upload, it has received 4 views. For similar materials see MICROBIOLOGY in Biology at Montgomery County Community College.

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Date Created: 09/21/16
CH 3 Observing Microorganisms through a Microscope Microscopy: The Instruments Light Microscopy  Light Microscopy: the use of any kind of microscope that uses visible light to observe specimens Compound Light Microscope  Compound Light Microscope: series of lenses and uses visible light as a source of illumination Illuminator The light source Condenser Lenses that direct the light rays through the specimen Objective lenses Lenses closest to the specimen; Ocular lens Eyepiece; further magnifies the image Total Objective lens magnification X ocular lens magnification magnification Resolution Ability of the lenses to distinguish final detail and structure Refractive index Measure of the light-bending ability of a medium o Bright Field: dark objects are visible against a bright background o Dark Field: translucent objects are visible against a dark background Phase-Contrast Microscopy  Phase-Contract Microscopy: permits detailed examinations of internal structures in living microorganisms; Accentuates diffraction of the light that passes through a specimen Fluorescence Microscopy  Fluorescence Microscopy: uses UV light; organisms are dyed o Ex. FAT Fluorescence-Antibody Technique; used to diagnose HIV and typhoid fever Scanning Acoustic Microscopy  Scanning Acoustic Microscopy (SAM): interpreting the action of a sound wave being sent through a specimen; measures sound waves Electron Microscopy  Electron Microscopy: uses electrons instead of light to view objects smaller than about .2µm o Transmission Electron Microscopy (TEM): need to stain ultrathin slices of specimens with heavy metal salts o Scanning Electron Microscopy (SEM): electron gun produces beam of electrons, gives 3D configuration Preparation of Specimens for Light Microscopy Preparing Smears for Staining  Smear: thin film of microorganisms spread over slide and allowed to air dry  Staining: coloring the organism with a dye that emphasizes certain structures  Fixing: attaching organism to the slide o Heat; passed through Bunsen burner, smear side up, several times o Non Heat; cover the slide with methyl alcohol Basic Stains: have positive ions; stain organism directly examples: Crystal Violet, methylene blue, malachite green, and safranin Acidic Stains Have negative ions; valuable for observing overall cell shapes, sizes and capsules Examples: eosin, acid fuchsin, and nigrosin Simple Stains  Simple Stain: aqueous or alcohol solution of a single basic dye o Primary purpose is to highlight the entire microorganism so that the cellular shapes and basic structures are visible  Mordant: chemical that is added to the solution to intensify the stain and coat the structure, making it easier to see  Steps for Basic/ Acidic Staining Steps: Basic Acidic Smear organism onto slide; let air Smear organism onto slide; let air dry dry Fix organisms with heat No heat fixing Primary Stain: Methylene Blue Primary Stain: Nigrosin Differential Stains  Differential Stains: Employ different stains to identify bacteria Gram Stain  Gram Stain: differentiates bacteria into gram-positive and gram-negative based on the chemical content of the cell wall  Gram Positive/Gram Negative Comparison Gram Positive Gram Negative Many thick layers of peptidoglycan in Single thin layer of peptidoglycan in cell cell wall wall Teichoic acid in cell wall Liposaccharides in cell wall Less virulent; sensitive to antibiotics More virulent; resistant to antibiotics Have no periplasmic spaces to store Have periplasmic spaces poison Retains color of primary stain crystal Loses color of primary stain crystal violet at the end of gram stain violet, retains pink or red color of secondary stain safranin  Steps for Gram Staining Steps: Gram + Gram - Primary Stain Crystal Violet Purple purple Mordant Iodine Purple Purple Decolorizing Agent Alcohol/ ethyl Purple Colorless alcohol Counter Stain Safranin Purple Red/Pink Acid-Fast Stain  Acid-Fast Stain: binds strongly to bacteria that have a waxy substance in their cell walls o Used to identify bacteria in the Mycobacterium family o Examples; Mycobacterium tuberculosis and Mycobacterium leprae  Steps for Acid-Fast Staining Steps: Acid Fast Non-Acid Fast Primary Stain Carbolfuchsin Red Red Decolorizing Agent Acid alcohol Red Colorless Counter Stain Methylene Blue Red blue Special Stains  Special Stains: used to color and isolate specific parts of microorganisms, such as endospores and flagella, and to reveal the presence of capsules Capsule Stain  Capsule Stain: makes organisms pathogenic  Steps for Capsule Staining Steps: Primary Stain Congo Red Fixing Agent Acid Alcohol Counter Stain Acid Fuschin Endospore Stain  Endospore Stain: Bacillus and Clostridium makes organisms heat and acid resistant; forms in adverse conditions; Autoclave and ethylene oxide gas will kill endospore  Steps for Endospore Staining Steps: Primary Stain Malachite Green Fixing Agent Heat Counter Stain Spore Safranin Flagella Stain  Flagella Stain: has motility to infect multiple places


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