Microscopy Lecture Notes PDF

MICROBIOLOGY WITH DISEASES BY TAXONOMY, THIRD EDITION Chapter 4 Microscopy, Staining, and Classification Lecture prepared by Mindy Miller-Kittrell, University o...

MICROBIOLOGY WITH DISEASES BY TAXONOMY, THIRD EDITION Chapter 4 Microscopy, Staining, and Classification Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville Copyright © 2011 Pearson Education Inc. Microscopy and Staining Tortora Play Button Animation: Microscopy and Staining: Overview Copyright © 2011 Pearson Education Inc. Units of Measurement Copyright © 2011 Pearson Education Inc. Table 4.1 Microscopy General Principles of Microscopy –Wavelength of radiation –Magnification –Resolution –Contrast Copyright © 2011 Pearson Education Inc. The electromagnetic spectrum Copyright © 2011 Pearson Education Inc. Figure 4.1 Light refraction and image magnification Copyright © 2011 Pearson Education Inc. Figure 4.2 The limits of resolution Copyright © 2011 Pearson Education Inc. Figure 4.3 Microscopy General Principles of Microscopy – Contrast –Differences in intensity between two objects, or between an object and background –Important in determining resolution –Staining increases contrast –Use of light that is in phase increases contrast Copyright © 2011 Pearson Education Inc. Microscopy Light Microscopy – Bright-field microscopes –Simple –Contain a single magnifying lens –Similar to magnifying glass –Leeuwenhoek used simple microscope to observe microorganisms Copyright © 2011 Pearson Education Inc. Microscopy Light Microscopy – Bright-field microscopes – Compound – Series of lenses for magnification – Light passes through specimen into objective lens – Oil immersion lens increases resolution – Have one or two ocular lenses – Total magnification = magnification of objective lens X magnification of ocular lens – Most have condenser lens (direct light through specimen) Copyright © 2011 Pearson Education Inc. A bright-field, compound light microscope Copyright © 2011 Pearson Education Inc. Figure 4.4 The effects of immersion oil on resolution Copyright © 2011 Pearson Education Inc. Figure 4.5 Microscopy Light Microscopy – Dark-field microscopes – Best for observing pale objects – Only light rays scattered by specimen enter objective lens – Specimen appears light against dark background – Increases contrast and enables observation of more details Copyright © 2011 Pearson Education Inc. The light path in a dark-field microscope Copyright © 2011 Pearson Education Inc. Figure 4.6 Microscopy Light Microscopy – Phase microscopes – Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining – Light rays in phase produce brighter image, while light rays out of phase produce darker image – Contrast is created because light waves are out of phase – Two types – Phase-contrast microscope – Differential interference contrast microscope Copyright © 2011 Pearson Education Inc. Principles of phase microscopy Copyright © 2011 Pearson Education Inc. Figure 4.7 Four kinds of light microscopy Copyright © 2011 Pearson Education Inc. Figure 4.8 Copyright © 2011 Pearson Education Inc. Microscopy Light Microscopy – Fluorescent microscopes – Direct UV light source at specimen – Specimen radiates energy back as a longer, visible wavelength – UV light increases resolution and contrast – Some cells are naturally fluorescent; others must be stained – Used in immunofluorescence to identify pathogens and to make visible a variety of proteins Copyright © 2011 Pearson Education Inc. Fluorescent microscopy Copyright © 2011 Pearson Education Inc. Figure 4.9 Immunofluorescence Copyright © 2011 Pearson Education Inc. Figure 4.10 Microscopy Light Microscopy – Confocal microscopes – Use fluorescent dyes – Use UV lasers to illuminate fluorescent chemicals in a single plane – Resolution increased because emitted light passes through pinhole aperture – Computer constructs 3-D image from digitized images Copyright © 2011 Pearson Education Inc. Microscopy Electron Microscopy – Light microscopes cannot resolve structures closer than 200 nm – Electron microscopes have greater resolving power and magnification – Magnifies objects 10,000X to 100,000X – Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms – Two types – Transmission electron microscopes – Scanning electron microscopes Copyright © 2011 Pearson Education Inc. A transmission electron microscope (TEM) Copyright © 2011 Pearson Education Inc. Figure 4.11 SEM images Copyright © 2011 Pearson Education Inc. Figure 4.13 Microscopy Tortora Play Button Animation: Electron Microscopy Copyright © 2011 Pearson Education Inc. Microscopy Probe Microscopy – Magnifies more than 100,000,000 times – Two types –Scanning tunneling microscopes –Atomic force microscopes Copyright © 2011 Pearson Education Inc. Probe microscopy Copyright © 2011 Pearson Education Inc. Figure 4.14 Staining Increases contrast and resolution by coloring specimens with stains/dyes Smear of microorganisms (thin film) made prior to staining Microbiological stains contain chromophore Acidic dyes stain alkaline structures; more commonly, basic dyes stain acidic structures Copyright © 2011 Pearson Education Inc. Preparation of the Biological Specimen The preparation of the biological specimen to be analyzed under a microscope depends on the type of staining. Given below are some procedures that are carried out. 1. Wet Mounting: Living biological specimens are mounted on a glass slide with water and specific stains. Copyright © 2011 Pearson Education Inc. Copyright © 2011 Pearson Education Inc. Preparation of the Biological Specimen 2. Fixation: It is a multi-step process which is done to preserve the shape of cells and tissues. ✓Heat fixation is done to kill and adhere the specimens. ✓Chemical fixation is done to generate strong bonds and increase the rigidity of the samples. Common chemical fixatives used are formaldehyde, picric acid, methanol and ethanol. Copyright © 2011 Pearson Education Inc. Preparation of the Biological Specimen 3. Mordant: Mordants are chemical agents that are used along with dye to make the specimen stainable, which otherwise is unstainable. Mordants are of two types: ✓Basic Mordants: They react with acidic dyes. ✓Acidic Mordants: They react with basic dyes. Copyright © 2011 Pearson Education Inc. Preparing a specimen for staining Copyright © 2011 Pearson Education Inc. Figure 4.15 Copyright © 2011 Pearson Education Inc. Staining Simple stains Differential stains – Gram stain – Acid-fast stain – Endospore stain Special stains – Negative (capsule) stain – Flagellar stain Copyright © 2011 Pearson Education Inc. Simple stains Copyright © 2011 Pearson Education Inc. Figure 4.16 The Gram staining procedure Copyright © 2011 Pearson Education Inc. Figure 4.17 Schaeffer-Fulton endospore stain Copyright © 2011 Pearson Education Inc. Figure 4.19 Negative (capsule) stain Copyright © 2011 Pearson Education Inc. Figure 4.20 Flagellar stain Copyright © 2011 Pearson Education Inc. Figure 4.21 Copyright © 2011 Pearson Education Inc.