Microbiology An Introduction Chapter 3 PDF

Microbiology an Introduction Thirteenth Edition Chapter 3 Observing Microorganisms through a Microscope Copyright © 2019 Pearson Education, Inc. All Righ...

Microbiology an Introduction Thirteenth Edition Chapter 3 Observing Microorganisms through a Microscope Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 1 2 Units of Measurement Microscopy: The Instruments (2 of 2) Microorganisms are measured in micrometers μm A simple microscope has only one lens and nanometers (nm) 1 μm = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm 1000 nm = 1 μm 0.001 μm = 1 nm micro is bigger than nano but smaller than milli Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 3 4 1  Figure 1.3b Anton van Leeuwenhoek's Microscopic Observations Light Microscopy Any kind of microscope that uses visible light to Lens observe specimens Location of specimen on pin Types of light microscopy read the textbook for descriptions Specimen- – Compound light microscopy positioning screw – Darkfield microscopy Focusing control – Phase-contrast microscopy Stage- positioning screw – Differential interference contrast (DIC) microscopy – Fluorescence microscopy – Confocal microscopy Microscope replica Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 5 6 Compound Light Microscopy Compound Light Microscopy In a compound microscope, the image from the Resolution is the ability of the lenses to distinguish objective lens is magnified again by the ocular lens two points Total magnification = objective lens (4x, 10x, 40x, 100x) x ocular lens (10x) A microscope with a resolving power of 0.4 nm can distinguish between two points at least 0.4 nm apart Shorter wavelengths of light provide greater resolution Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 7 8 2  Compound Light Microscopy Compound Light Microscopy The refractive index is a measure of the light- Brightfield illumination bending ability of a medium – Dark objects are visible against a bright Light may refract after passing through a specimen to background an extent that it does not pass through the objective lens Immersion oil is used to keep light from refracting (has the same refraction index as glass) Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 9 10 Figure 3.4a Brightfield, Darkfield, and Phase-contrast Microscopy Darkfield Microscopy (a). Brightfield. (Top) The Light objects are visible against a dark path of light in brightfield background microscopy, the type of Opaque disk placed in condenser illumination produced by regular compound light Only light reflected off the specimen enter the objective lens microscopes. (Bottom) Brightfield illumination shows internal structures and the outline of the transparent pellicle (external covering). Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 11 12 3  Figure 3.4b Brightfield, Darkfield, and Phase-Contrast Microscopy Phase-Contrast Microscopy (b). Darkfield. (Top) The darkfield Allows examination of living organisms and internal microscope uses a special condenser with an opaque disk that cell structures eliminates all light in the center of the beam. The only light that Brings together 2 sets of light rays, direct rays, & diffracted rays to form an image reaches the specimen comes in at an angle; thus, only light reflected by the specimen (blue lines) reaches the objective lens. (Bottom) Against the black background seen with darkfield microscopy, edges of the cell are bright, some internal structures seem to sparkle, and the pellicle is almost visible. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 13 14 Figure 3.4c Brightfield, Darkfield, and Differential Interference Contrast Phase-Contrast Microscopy (DIC) Microscopy (c). Phase-contrast. (Top) In phase- Similar to phase-contrast contrast microscopy, the specimen is illuminated by light passing through an Uses two light beams and prisms to split light beams, annular (ring-shaped) diaphragm. Direct light rays (unaltered by the specimen) giving more contrast and color to the specimen travel a different path from light rays that are reflected or diffracted as they pass through the specimen. These two sets of rays are combined at the eye. Reflected or diffracted light rays are indicated in blue; direct rays are red. (Bottom) Phase-contrast microscopy shows greater differentiation of internal structures and clearly shows the pellicle. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 15 16 4  Fluorescence Microscopy Confocal Microscopy Uses UV (short wavelength) light Cells are stained with fluorochrome dyes Fluorescent substances absorb UV light and emit Short-wavelength (blue) light is used to excite a single longer wavelength (visible) light plane of a specimen Cells may be stained with fluorescent dyes (fluorochromes) if Each plane in a specimen is illuminated and a three- they do not naturally fluoresce dimensional image is constructed with a computer Can examine layers of cells to a depth of 100 μm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 17 18 Transmission Electron Microscopy Electron Microscopy Uses electrons instead of light A beam of electrons passes through ultrathin sections of a specimen, then through an electromagnetic lens, The shorter wavelength of electrons gives greater resolution then focused on a projector lens Specimens may be stained with heavy-metal salts for Used for images too small to be seen with light contrast microscopes, such as viruses Magnifies objects 10,000 to 10,000,000x; resolution of 10 pm Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 19 20 5  Figure 3.11a Transmission and Scanning Scanning Electron Microscopy Electron Microscopy (1 of 2) Electron gun Electron beam An electron gun produces a beam of electrons that Electromagnetic scans the surface of an entire specimen condenser lens Specimen Secondary electrons emitted from the specimen Electromagnetic objective lens produce a three-dimensional image Electromagnetic Viewing projector lens eyepiece Magnifies object 1,000 to 500,000x; resolution of 10 nm Fluorescent screen or photographic plate Transmission. (Left) In a transmission electron microscope, electrons pass through the specimen and are scattered. Magnetic lenses focus the image onto a fluorescent screen or photographic plate. (Right) This colorized transmission electron micrograph (TEM) shows a thin slice of Paramecium. In this type of microscopy, the internal structures present in the slice can be seen. Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 21 22 Figure 3.11b Transmission and Scanning Electron Microscopy Preparing Smears for Staining Electron gun Primary electron beam Staining: coloring microorganisms with a dye that Electromagnetic lenses emphasizes certain structures Viewing Smear: a thin film of a material containing screen Electron microorganisms spread over a slide collector Secondary electrons Microorganisms are fixed (attached) to the slide, Specimen which kills the microorganisms Amplifier Scanning. (Left) In a scanning electron microscope, primary electrons sweep Live and/or unstained specimens have little contrast with the across the specimen and knock electrons from its surface. These secondary electrons surrounding medium. Live specimens are used to study cell are picked up by a collector, amplified, and transmitted onto a viewing screen or photographic plate. (Right) In this colorized scanning electron micrograph (SEM), the behavior surface structures of Paramecium can be seen. Note the three-dimensional appearance of this cell, in contrast to the two-dimensional appearance of the transmission electron micrograph in part (a). Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 23 24 6  Preparing Smears for Staining Simple Stains Stains consist of a positive and negative ion, one of Simple stain: use of a single basic dye which is colored (chromophore) Highlights the entire microorganism to visualize cell In a basic dye, the chromophore is a cation gets color shapes and structures In an acidic dye, the chromophore is an anion A mordant may be used to hold the stain or coat the specimen to enlarge it - Staining the background instead of the cell is called negative staining Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 25 26 Differential Stains Differential Stains Used to distinguish between bacteria Classifies bacteria into gram-positive or gram- - Gram stain negative - Acid-fast stain – Gram-positive bacteria have thick peptidoglycan cell walls Purple – Gram-negative bacteria have thin peptidoglycan cell walls and a layer of lipopolysaccharides Pink Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 27 28 7  Acid-Fast Stain Binds only to bacteria that have a waxy material in their cell walls, which is not decolorized by acid-alcohol Acid-Fast Stain Used for the identification of Color of Color of Blank – Mycobacterium Acid-Fast Non–Acid-Fast – Nocardia Primary Stain: Red Red Carbolfuchsin is applied and slide is heated to enhance Carbolfuchsin dye penetration and retention Decolorizing Agent: Red Colorless Treated with acid-alcohol Acid-Alcohol Acid-fast cells will not decolorize because the Counterstain: carbolfuchsin is more soluble in the wall lipids than in acid Methylene Red Blue alcohol Blue Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 29 30 Special Stains Negative Staining for Capsules Used to distinguish parts of microorganisms Capsules are a gelatinous covering that do not – Capsule stain accept most dyes – Endospore stain Suspension of India ink or nigrosin contrasts the – Flagella stain background with the capsule, which appears as a halo around the cell Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 31 32 8  Endospore Staining Flagella Staining Endospores are resistant, dormant structures inside Flagella are structures of locomotion some cells that cannot be stained by ordinary Uses a mordant and carbolfuchsin to thicken methods appearance of flagella, making them visible under the Schaeffer-Fulton Endospore stain light microscope Primary stain: malachite green, usually with heat Decolorize cells: water Counterstain: safranin Spores appear green within red or pink cells Flagellum Copyright © 2019 Pearson Education, Inc. All Rights Reserved Copyright © 2019 Pearson Education, Inc. All Rights Reserved 33 34 9