Unformatted text preview: 2/2/2010 Chapter 3:
Learning Objectives: Students should be able to…
1. Convert between metric size units of meters, Convert metric of centimeters, millimeters, microns, and nanometers. centimeters, 2. Describe the basic differences between Light Describe Microscopy (bright field, dark field, phase-contrast, and Microscopy contrast, fluorescent) and Electron Microscopy (Transmission and Electron (Transmission Scanning EM). Scanning 3. Describe the method and utility of several different Describe specimen staining procedures. staining Chapter 3
Observing Microorganisms Observing Through a Microscope Through 1 2/2/2010 3.1) Units of Measurement
NANOMETERS & MICRONS: • 1 µm = 10-6 m = 10-3 mm • 1 nm = 10-9 m = 10-6 mm • 1000 nm = 1 µm 1000 • 0.001 µm = 1 nm 0.001 Figure 3.2 3.2) Microscopy: The Instruments • A simple microscope simple has only one lens. Figure 1.2b 2 2/2/2010 Microscopy: The Instruments
• In a compound microscope In compound the image from the the objective lens is magnified again by the ocular lens. again • Total magnification = objective lens (10, 40, objective 100X) ocular lens (10X)
Figure 3.1b Microscopy: The Instruments
• Resolution iis the ability s of the lenses to distinguish two points. distinguish
– A microscope with a microscope resolving power of 0.4 nm 0.4 – can distinguish between can two points ≥ 0.4 nm apart. • Shorter wavelengths of Shorter light provide greater resolution
– (eg: UV).
Figure 3.2 3 2/2/2010 Microscopy: The Instruments
• Refractive index is the light-bending bending ability of a medium. ability
– The light may bend The in air so much that it misses the small high-magn’n lens. high • Immersion oil is Immersion used to keep light from bending. from
Figure 3.3 A. Brightfield Illumination A. Brightfield
• Dark objects are Dark visible against a bright background. bright
– Light reflected off Light the specimen does not enter the not enter objective lens. objective
• Creates contrast / Creates positive image positive Figure 3.4a, b 4 2/2/2010 B. Darkfield Illumination B. Darkfield
• Light objects are Light visible against a dark background. dark
– Light reflected off Light the specimen enters the objective lens. the
• Creates contrast / Creates negative image negative Figure 3.4a, b C. Phase-Contrast Microscopy
• Accentuates diffraction of Accentuates diffraction of the light that passes through a specimen. specimen.
– Peaks and valleys of incoming Peaks light waves accentuate bright and dark regions of the specimen specimen Figure 3.4c 5 2/2/2010 D. Fluorescence Microscopy
• Uses UV (to red) light. • Fluorescent substances Fluorescent
– absorb UV light (or other absorb wavelengths) and – emit visible light (at a emit different wavelength). • Cells may be stained Cells with fluorescent dyes (“fluorochromes”).
Figure 3.6b E. Confocal Microscopy Confocal
• Uses fluorochromes and a laser light. Uses fluorochromes • The laser illuminates each plane in a The specimen to produce a 3-D iimage. mage. specimen Figure 3.7 6 2/2/2010 T ransgenic, green GFP-expressing Transgenic, GFP expressing bacteria invading plant root tissues bacteria • Symbiotic plant cell wall Symbiotic plant proteins labeled Red proteins Red • Allows visualization of LIVE Allows biological processes!! biological 3.3) Electron Microscopy
• Uses electrons instead of light. • The shorter wavelength of The electrons gives greater resolution. electrons 7 2/2/2010 F. Transmission Electron F. Microscopy (TEM) Microscopy
• Ultrathin sections of specimens. • Light passes through specimen, then an Light electromagnetic lens, to a screen or film. electromagnetic • Specimens may be stained with heavy metal salts. Specimens heavy • 10,000-100,000; resolution = 2.5 nm Figure 3.10a • An electron gun produces a beam of electrons that An scans the surface of a whole specimen. scans • Secondary electrons emitted from the specimen Secondary produce the image. produce • 1000-10,000; resolution = 20 nm G. Scanning Electron G. Microscopy (SEM) Microscopy Fig 3.10b 8 2/2/2010 3.4) Preparation of Specimens 3.4) for Light Microscopy for
• A thin film of a solution of microbes on a thin slide is a smear. smear • A smear is usually fixed to attach the smear fixed to microbes to the slide and to kill the microbes. Preparing Smears for Staining
• Live or unstained cells Live have little contrast with the surrounding medium. • However, researchers However, do make discoveries about cell behavior looking at live specimens. specimens. 9 2/2/2010 Preparing Smears for Staining
• Stains consist of a positive and negative ion.
– In a basic dye, the chromophore (colored In basic the chromophore (colored molecule) is a cation. cation
• Carbol fuschin, methylene blue, crystal violet, safranin methylene blue, safranin – In an acidic dye, the chromophore iis an anion. In acidic the chromophore s anion • Staining the background instead of the cell is Staining called negative staining. negative
– Use acidic dyes – India ink, nigrosin. Use India nigrosin A. Simple Stains
• Use of a single basic dye is called a simple Use stain. stain • A mordant may be used to hold the stain, or mordant may coat the specimen to enlarge it. coat 10 2/2/2010 B. Differential Stains: Gram Stain Differential Gram
• The Gram stain classifies bacteria into grampositive and gram-negative.
– Gram-positive bacteria tend to be killed by penicillin and bacteria detergents. detergents. – Gram-negative bacteria are more resistant to antibiotics. Figure 3.12b Gram Stain
Color of Color Gram + cells Gram Primary stain: Crystal violet Primary Mordant: Iodine Mordant: Decolorizing agent: Alcohol Decolorizing Counterstain: Safranin Purple Purple Purple Purple Color of Gram – cells Gram Purple Purple Colorless Red/Pink Figure 3.12a 11 2/2/2010 C. Differential Stains: C. Acid-Fast Stain Acid
• Waxy cells that retain a basic stain in the Waxy presence of acid-alcohol are called acid-fast. presence alcohol acid
– Mycobacterium; Nocardia • Non–acid-fast cells lose the basic stain when fast rinsed with acid-alcohol rinsed
– usually counterstained (with a different color usually basic stain; eg: methylene blue) to see them. eg methylene Figure 3.13 D. Special Stains
• Negative staining is useful for capsules. • Differential: Endospore Stain – Heat is Differential: Endospore Heat required to drive a stain into endospores. endospores
– Phenol solvent sometimes added. Figure 3.14 12 ...
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This note was uploaded on 03/18/2010 for the course BIOL 240 taught by Professor Staples during the Spring '09 term at Canada College.
- Spring '09