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

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Presentation on theme: "Copyright © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 3 Observing Microorganisms Through a Microscope © 2013 Pearson."— Presentation transcript:

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

2 © 2013 Pearson Education, Inc.

3 Figure 3.2 Microscopes and Magnification. Tick Actual size Red blood cells E. coli bacteria T-even bacteriophages (viruses) DNA double helix Unaided eye ≥ 200  m Light microscope 200 nm – 10 mm Scanning electron microscope 10 nm – 1 mm Transmission electron microscope 10 pm – 100  m Atomic force microscope 0.1 nm – 10nm

4 © 2013 Pearson Education, Inc.  1 µm = 10 –6 m = 10 –3 mm  1 nm = 10 –9 m = 10 –6 mm  1000 nm = 1 µm  µm = 1 nm Units of Measurement

5 © 2013 Pearson Education, Inc.  A simple microscope has only one lens Microscopy: The Instruments

6 © 2013 Pearson Education, Inc. Figure 1.2b Anton van Leeuwenhoek’s microscopic observations. Microscope replica Lens Location of specimen on pin Specimen-positioning screw Focusing control Stage-positioning screw

7 © 2013 Pearson Education, Inc. Light Microscopy  The use of any kind of microscope that uses visible light to observe specimens  Types of light microscopy  Compound light microscopy  Darkfield microscopy  Phase-contrast microscopy  Differential interference contrast microscopy  Fluorescence microscopy  Confocal microscopy

8 © 2013 Pearson Education, Inc. Ocular lens (eyepiece) Remagnifies the image formed by the objective lens Body tube Transmits the image from the objective lens to the ocular lens Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Coarse focusing knob Base Fine focusing knob Principal parts and functions Figure 3.1a The compound light microscope.

9 © 2013 Pearson Education, Inc. Compound Light Microscopy  In a compound microscope, the image from the objective lens is magnified again by the ocular lens  Total magnification = objective lens  ocular lens

10 © 2013 Pearson Education, Inc. The path of light (bottom to top) Line of vision Ocular lens Path of light Prism Body tube Objective lenses Specimen Condenser lenses Illuminator Base with source of illumination Figure 3.1b The compound light microscope.

11 © 2013 Pearson Education, Inc. Compound Light Microscopy  Resolution is the ability of the lenses to distinguish two points  A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm  Shorter wavelengths of light provide greater resolution

12 © 2013 Pearson Education, Inc. Compound Light Microscopy  The refractive index is a measure of the light-bending ability of a medium  The light may bend in air so much that it misses the small high-magnification lens  Immersion oil is used to keep light from bending

13 © 2013 Pearson Education, Inc. Figure 3.3 Refraction in the compound microscope using an oil immersion objective lens. Unrefracted light Immersion oil Condenser Light source Iris diaphragm Condenser lenses Air Without immersion oil most light is refracted and lost Oil immersion objective lens Glass slide

14 © 2013 Pearson Education, Inc. Brightfield Illumination  Dark objects are visible against a bright background  Light reflected off the specimen does not enter the objective lens ANIMATION Light Microscopy

15 © 2013 Pearson Education, Inc. Figure 3.4a Brightfield, darkfield, and phase-contrast microscopy. Eye Ocular lens Objective lens Specimen Condenser lens Light Opaque disk Unreflected light Annular diaphragm Only light reflected by the specimen is captured by the objective lens Condenser lens Refracted or diffracted light (altered by specimen) Objective lens Diffraction plates Undiffracted light (unaltered by specimen) Brightfield Specimen

16 © 2013 Pearson Education, Inc. Darkfield Illumination  Light objects are visible against a dark background  Light reflected off the specimen enters the objective lens

17 © 2013 Pearson Education, Inc. Figure 3.4b Brightfield, darkfield, and phase-contrast microscopy. Eye Ocular lens Objective lens Specimen Condenser lens Light Opaque disk Unreflected light Annular diaphragm Only light reflected by the specimen is captured by the objective lens Condenser lens Refracted or diffracted light (altered by specimen) Objective lens Diffraction plates Undiffracted light (unaltered by specimen) Darkfield Specimen

18 © 2013 Pearson Education, Inc. Phase-Contrast Microscopy  Accentuates diffraction of the light that passes through a specimen

19 © 2013 Pearson Education, Inc. Figure 3.4c Brightfield, darkfield, and phase-contrast microscopy. Eye Ocular lens Objective lens Specimen Condenser lens Light Opaque disk Unreflected light Annular diaphragm Only light reflected by the specimen is captured by the objective lens Condenser lens Refracted or diffracted light (altered by specimen) Objective lens Diffraction plates Undiffracted light (unaltered by specimen) Phase-contrast Specimen

20 © 2013 Pearson Education, Inc.  Accentuates diffraction of the light that passes through a specimen; uses two beams of light Differential Interference Contrast Microscopy

21 © 2013 Pearson Education, Inc. Figure 3.5 Differential interference contrast (DIC) microscopy.

22 © 2013 Pearson Education, Inc. Fluorescence Microscopy  Uses UV light  Fluorescent substances absorb UV light and emit visible light  Cells may be stained with fluorescent dyes (fluorochromes)

23 © 2013 Pearson Education, Inc. Figure 3.6b The principle of immunofluorescence.

24 © 2013 Pearson Education, Inc. Confocal Microscopy  Cells are stained with fluorochrome dyes  Short-wavelength (blue) light is used to excite the dyes  The light illuminates each plane in a specimen to produce a three-dimensional image  Up to 100 µm deep

25 © 2013 Pearson Education, Inc. Figure 3.7 Confocal microscopy. Nucleus

26 © 2013 Pearson Education, Inc. Two-Photon Microscopy  Cells are stained with fluorochrome dyes  Two photons of long-wavelength (red) light are used to excite the dyes  Used to study cells attached to a surface  Up to 1 mm deep

27 © 2013 Pearson Education, Inc. Figure 3.8 Two-photon microscopy (TPM). Food vacuoles © 2013 Pearson Education, Inc.

28 Scanning Acoustic Microscopy (SAM)  Measures sound waves that are reflected back from an object  Used to study cells attached to a surface  Resolution 1 µm

29 © 2013 Pearson Education, Inc. Figure 3.9 Scanning acoustic microscopy (SAM) of a bacterial biofilm on glass.

30 © 2013 Pearson Education, Inc.  Uses electrons instead of light  The shorter wavelength of electrons gives greater resolution ANIMATION Electron Microscopy Electron Microscopy

31 © 2013 Pearson Education, Inc. Transmission Electron Microscopy (TEM)  Ultrathin sections of specimens  Light passes through specimen, then an electromagnetic lens, to a screen or film  Specimens may be stained with heavy-metal salts

32 © 2013 Pearson Education, Inc. Figure 3.10a Transmission and scanning electron microscopy. Electron beam Electromagnetic condenser lens Specimen Electromagnetic objective lens Electromagnetic projector lens Fluorescent screen or photographic plate Viewing eyepiece Electron gun Specimen Secondary electrons Primary electron beam Electromagnetic lenses Viewing screen Electron collector Amplifier Transmission

33 © 2013 Pearson Education, Inc.  10,000–100,000  ; resolution 2.5 nm Transmission Electron Microscopy (TEM)

34 © 2013 Pearson Education, Inc. Scanning Electron Microscopy (SEM)  An electron gun produces a beam of electrons that scans the surface of a whole specimen  Secondary electrons emitted from the specimen produce the image

35 © 2013 Pearson Education, Inc. Figure 3.10b Transmission and scanning electron microscopy. Electron beam Electromagnetic condenser lens Specimen Electromagnetic objective lens Electromagnetic projector lens Fluorescent screen or photographic plate Viewing eyepiece Electron gun Specimen Secondary electrons Primary electron beam Electromagnetic lenses Viewing screen Electron collector Amplifier Scanning

36 © 2013 Pearson Education, Inc.  1,000–10,000  ; resolution 20 nm Scanning Electron Microscopy (SEM)

37 © 2013 Pearson Education, Inc.  Scanning tunneling microscopy (STM) uses a metal probe to scan a specimen  Resolution 1/100 of an atom Scanned-Probe Microscopy

38 © 2013 Pearson Education, Inc. Figure 3.11a Scanned-probe microscopy.

39 © 2013 Pearson Education, Inc.  Atomic force microscopy (AFM) uses a metal-and-diamond probe inserted into the specimen  Produces three-dimensional images Scanned-Probe Microscopy

40 © 2013 Pearson Education, Inc. Figure 3.11b Scanned-probe microscopy.

41 © 2013 Pearson Education, Inc.  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 Preparing Smears for Staining

42 © 2013 Pearson Education, Inc.  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

43 © 2013 Pearson Education, Inc. Preparing Smears for Staining  Stains consist of a positive and negative ion  In a basic dye, the chromophore is a cation  In an acidic dye, the chromophore is an anion  Staining the background instead of the cell is called negative staining

44 © 2013 Pearson Education, Inc.  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 Simple Stains

45 © 2013 Pearson Education, Inc. Differential Stains  Used to distinguish between bacteria  Gram stain  Acid-fast stain

46 © 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

47 © 2013 Pearson Education, Inc. Color of Gram-Positive Cells Color of Gram-Negative Cells Primary Stain: Crystal Violet Purple Mordant: Iodine Purple Decolorizing Agent: Alcohol-Acetone PurpleColorless Counterstain: Safranin PurpleRed Gram Stain

48 © 2013 Pearson Education, Inc. Figure 3.12b Gram staining. Cocci (gram-positive) Rod (gram-negative)

49 © 2013 Pearson Education, Inc. Acid-Fast Stain  Stained waxy cell wall is not decolorized by acid-alcohol  Mycobacterium  Nocardia

50 © 2013 Pearson Education, Inc. Color of Acid-Fast Color of Non–Acid-Fast Primary Stain: Carbolfuchsin Red Decolorizing Agent: Acid-alcohol RedColorless Counterstain: Methylene Blue RedBlue Acid-Fast Stain

51 © 2013 Pearson Education, Inc. Figure 3.13 Acid-fast bacteria. M. bovis

52 © 2013 Pearson Education, Inc. Special Stains  Used to distinguish parts of cells  Capsule stain  Endospore stain  Flagella stain

53 © 2013 Pearson Education, Inc.  Cells stained  Negative stain Negative Staining for Capsules

54 © 2013 Pearson Education, Inc. Figure 3.14a Special staining. Capsules Negative staining

55 © 2013 Pearson Education, Inc.  Primary stain: malachite green, usually with heat  Decolorize cells: water  Counterstain: safranin Endospore Staining

56 © 2013 Pearson Education, Inc. Figure 3.14b Special staining. Endospore Endospore staining

57 © 2013 Pearson Education, Inc.  Mordant on flagella  Carbolfuchsin simple stain Flagella Staining

58 © 2013 Pearson Education, Inc. Figure 3.14c Special staining. Flagellum Flagella staining


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