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Chapter 3 Observing Microorganisms Through a Microscope © 2013 Pearson Education, Inc. Copyright © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case Lectures prepared by Christine L. Case Figure 3.2 Microscopes and Magnification. Unaided eye ≥ 200 m Light microscope 200 nm – 10 mm Tick Actual size Scanning electron microscope 10 nm – 1 mm Red blood cells Transmission electron microscope 10 pm – 100 m E. coli bacteria T-even bacteriophages (viruses) Atomic force microscope 0.1 nm – 10nm DNA double helix © 2013 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 © 2013 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 © 2013 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 © 2013 Pearson Education, Inc. Differential Stains Used to distinguish between bacteria Gram stain Acid-fast stain © 2013 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 © 2013 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 © 2013 Pearson Education, Inc. Figure 3.12 Gram staining. Gram-positive Gram-negative Application of crystal violet (purple dye) Application of iodine (mordant) Rod (gram-negative) Cocci (gram-positive) © 2013 Pearson Education, Inc. Alcohol wash (decolorization) Application of safranin (counterstain) Acid-Fast Stain Stained waxy cell wall is not decolorized by acid-alcohol Mycobacterium Nocardia © 2013 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 © 2013 Pearson Education, Inc. Figure 3.13 Acid-fast bacteria. M. bovis © 2013 Pearson Education, Inc. Special Stains Used to distinguish parts of cells Capsule stain Endospore stain Flagella stain © 2013 Pearson Education, Inc. Negative Staining for Capsules Cells stained Capsule is not stained--negative stain © 2013 Pearson Education, Inc. Figure 3.14a Special staining. Capsules Negative staining © 2013 Pearson Education, Inc. Figure 3.14b Special staining. Endospore Endospore staining © 2013 Pearson Education, Inc. Figure 3.14c Special staining. Flagellum Flagella staining © 2013 Pearson Education, Inc. Table 3.3 A Summary of Various Stains and Their Uses © 2013 Pearson Education, Inc.