Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 The Dark-Field Microscope Image is formed by light reflected or refracted by specimen produces a bright image of the object against a dark background used to observe living, unstained preparations –For eucaryotes has been used to observe internal structures –For procaryotes has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 Figure 2.7 (b)

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 The Phase-Contrast Microscope enhances the contrast between intracellular structures having slight differences in refractive index excellent way to observe living cells –Especially useful for detecting bacterial components such as endospores and inclusion bodies that have refractive indices different from that of water

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4 Figure 2.8

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 The Differential Interference Contrast Microscope (DIC) creates image by detecting differences in refractive indices and thickness of different parts of specimen excellent way to observe living cells –Live, unstained cells appear brightly colored and three-dimensional

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6 Figure 2.11

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7 The Fluorescence Microscope exposes specimen to ultraviolet, violet, or blue light specimens usually stained with fluorochromes shows a bright image of the object resulting from the fluorescent light emitted by the specimen Has applications in medical microbiology and microbial ecology studies

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 8 Figure 2.12

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 9 Table 2.3

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 10 Figure 2.13

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 11 Preparation and Staining of Specimens increases visibility of specimen accentuates specific morphological features preserves specimens

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12 Staining Specific Structures negative staining –e.g., capsule stain used to visualize capsules surrounding bacteria –capsules are colorless against a stained background

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 13 Staining Specific Structures endospore staining –double staining technique –bacterial endospore is one color and vegetative cell is a different color flagella staining –mordant applied to increase thickness of flagella

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 14 Figure 2.14

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 15 Electron Microscopy beams of electrons are used to produce images wavelength of electron beam is much shorter than light, resulting in much higher resolution Figure 2.16

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 16 Figure 2.17

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17 The Transmission Electron Microscope (TEM) electrons scatter when they pass through thin sections of a specimen transmitted electrons (those that do not scatter) are used to produce image denser regions in specimen, scatter more electrons and appear darker

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18 Figure 2.18

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19 Figure 2.19

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 20 Table 2.4

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 21 Specimen Preparation analogous to procedures used for light microscopy for transmission electron microscopy, specimens must be cut very thin specimens are chemically fixed and stained with electron dense material

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 22 Other preparation methods shadowing –coating specimen with a thin film of a heavy metal freeze-etching –freeze specimen then fracture along lines of greatest weakness (e.g., membranes)

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 23 Figure 2.20

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24 Figure 2.21

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 25 Figure 2.22

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 26 The Scanning Electron Microscope uses electrons reflected from the surface of a specimen to create image produces a 3-dimensional image of specimen’s surface features

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 27 Figure 2.23

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 28 Figure 2.24

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 29 Confocal Microscopy confocal scanning laser microscope laser beam used to illuminate a variety of planes in the specimen computer compiles images created from each point to generate a 3- dimensional image used extensively to observe biofilms

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 30 Figure 2.25