Observing Microorganisms Through a Microscope Chapter 3 Observing Microorganisms Through a Microscope Lectures prepared by Christine L. Case © 2013 Pearson Education, Inc. 1

Tick Actual size Red blood cells 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

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

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

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

Differential Stains Used to distinguish between bacteria Gram stain Acid-fast stain

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

Gram Stain Color of Gram-Positive Cells Gram-Negative Cells Primary Stain: Crystal Violet Purple Mordant: Iodine Decolorizing Agent: Alcohol-Acetone Colorless Counterstain: Safranin Red

Application of crystal violet (purple dye) Figure 3.12 Gram staining. Gram-positive Gram-negative Application of crystal violet (purple dye) Application of iodine (mordant) Alcohol wash (decolorization) Application of safranin (counterstain) Rod (gram-negative) Cocci (gram-positive)

Acid-Fast Stain Stained waxy cell wall is not decolorized by acid-alcohol Mycobacterium Nocardia

Acid-Fast Stain Color of Acid-Fast Non–Acid-Fast Primary Stain: Carbolfuchsin Red Decolorizing Agent: Acid-alcohol Colorless Counterstain: Methylene Blue Blue

Figure 3.13 Acid-fast bacteria. M. bovis

Special Stains Used to distinguish parts of cells Capsule stain Endospore stain Flagella stain

Negative Staining for Capsules Cells stained Capsule is not stained--negative stain

Figure 3.14a Special staining. Capsules Negative staining

Figure 3.14b Special staining. Endospore Endospore staining

Figure 3.14c Special staining. Flagellum Flagella staining

Table 3.3 A Summary of Various Stains and Their Uses