Microbiology An Evolving Science Third Edition Joan L. Slonczewski and John W. Foster Copyright 2014 W. W. Norton & Company, Inc. Permission required for reproduction or display PowerPoint Lecture OutlinesPrepared by Johnny El-Rady, University of South Florida 2 Observing the Microbial Cell 2 Chapter Overview How microorganisms are observed The physics of light The bright-field microscope Staining bacterial cells The fluorescence, dark-field, and phase-contrast microscopes The electron microscope Cutting-edge microscopes How molecules are visualized 3 Figure 1.13 4 Introduction Since Leeuwenhoeks time, powerful microscopes have been devised to search for microbes in unexpected habitats - e.g., the human stomach Microscopy revealedthe presence ofHelicobacter pylori,the cause ofstomach ulcers Figure 2.1 5 Microbes differ in size, over a range of a few orders of magnitude, or powers of ten. - Eukaryotic microbes - Protozoa, algae, fungi - 10100 mm - Prokaryotes - Bacteria, archaea - 0.410 mm Nevertheless, a few bacterial species are large enough to be seen by the unaided eye - Thiomargarita namibiensis Microbial Size 6 Figure 2.4 7 Prokaryotic cell structures are generally simpler than those of eukaryotes Certain shapes of bacteria are common to many taxonomic groups - Bacilli = rods - Cocci = spheres - Spiral forms - Spirochetes - Spirilla Microbial Shape 8 Figure 2.6 9 2.1 Observing Microbes The size at which objects become visible depends on the resolution of the observers eye Resolution is the smallest distance by which two objects can be separated and still be distinguished The resolution of the human retina is about150 mm (1/7 mm) Contrast is the ability to distinguish an object from its surrounding (background) 10 We define what is visible and what is microscopic in terms of the human eye Figure 2.2 11 Different microscopes are required to resolve various cells and subcellular structures. Microscopy for Different-Sized Scales Figure 2.7 12 Detection is the ability to determine the presence of an object Resolution Is Different from Detection Figure 2.3 Magnification means an increase in the apparent size of an image to resolve smaller separations between objects 13 Theoreticalresolution limit = /2 14 Absorption means that the photons energy is acquired by the absorbing object Reflection means that the wavefront bounces off the surface of an object Refraction is the bending of light as it enters a substance that slows its speed Scattering occurs when the wavefront interacts with an object smaller than the wavelength of light Light Interacts with an Object 15 Figure 2.9 16 2.2 Optics and Properties of Light Light is part of the spectrum of electromagnetic radiation.- Wavelength of visible light = 400750 nm For electromagnetic radiation to resolve an object, certain conditions must exist: 1. Contrast between object and its medium 2. Wavelength smaller than the object 3. Magnification 17 Magnification requires the bending of light rays, as in refraction Wavefronts of lightshift direction as theyenter a substance ofhigher refractive index Magnification by a Lens Figure 2.10 18 When light rays enter a parabolic lens, parallel rays each bend at an angle such that all of the rays meet at a certain point, called the focal point Figure 2.11 19 Figure 2.12 20 Figure 2.14 Figure 2.15 R = /2*NA 21 It is resolution, not magnification, that limits the ability of what we can see with a microscope - Indeed, magnification without increasing detail is called empty magnification The resolution of detail in microscopy is limited by the wave nature of light- Light rays actually form wavefronts, which undergo interference - Can be constructive or destructive Resolution of Detail 22 2.3 Bright-Field Microscopy Generates a dark image of an object over a light background To increase resolution: - Use shorter wavelength light - Lessen contrast - Use immersion oil - Use wider lens closer to specimen - Higher numerical aperture (NA) 23 A compound microscope is a system of multiple lenses designed to correct or compensate for aberration - Ocular lens - Objective lens Total magnification = magnification of ocular multiplied by that of the objective The Compound Microscope 24 Figure 2.16 -Objectives may be parfocal25 A simple way to observe microbes is to place them in a drop of water on a slide with a coverslip. - This is called a wet mount preparation. - Advantages: - Observation of cells in natural state - Disadvantages: - Little contrast between cell and background - Sample may dry out quickly Preparing a Specimen for Microscopy 26 Figure 2.17 27 The detection and resolution of cells under a microscope are enhanced by: - Fixation = cells are made to adhere to a slide in a fixed position - Staining = cells are given a distinct color- Most stains have conjugated double bonds or aromatic rings, and one or more positive charges Fixation and Staining 28 A simple stain adds dark color specifically to cells, but not to the external medium or surrounding tissue - Most commonly used stain is methylene blue A differential stain stains one kind of cell but not another- The most famous differential stain is the Gram stain Different Kinds of Stains 29 Figure 2.20 30 The Gram stain was devised in 1884 by the Dutch physician Hans Christian Gram (18531938) - It differentiates between two types of bacteria: - Gram-positive bacteria retain the crystal violet stain because of their thicker cell wall - Gram-negative bacteria do not Figure 2.21 31 Figure 2.22 32 Acid-fast stain = carbolfuchsin used to stain Mycobacterium species Spore stain = malachite green used to detect spores of Bacillus and Clostridium Negative stain = colors the background, which makes capsules more visible Other Differential Stains Figure 2.24 33 2.4 Fluorescence Microscopy In fluorescence microscopy, the specimen absorbs light of a defined wavelength, and then emits light of lower energy, thus longer wavelength; that is, the specimen fluoresces Used to view marine and pathogenic bacteria Figure 2.25 34 Excitation and Emission The specimen absorbs light of a specific wavelength (the excitation wavelength), then emits light at a longer wavelength (the emission wavelength) Figure 2.26 35 The optical system for fluorescence microscopy uses color filters - To limit incidentlight to thewavelength ofexcitation andemitted light tothe wavelengthof emission Figure 2.27 36 A fluorophore is a fluorescent chemical compound - Its cell specificity can be determined in three ways: - Chemical affinity - Labeled antibodies - DNA hybridization Fluorophores for Labeling Figure 2.29 37 2.5 Dark-Field Microscopy Dark-field optics enables microbes to be visualized as halos of bright light against darkness Light shines at oblique angle - Only light scattered by sample reaches objective - Makes visible objects below resolution limit - Flagella - Very thin bacteria 38 Figure 2.33 Figure 2.32 Figure 2.31 39 2.5 Phase-Contrast Microscopy Superimposes refracted light and transmitted light shifted out of phase- Reveals differences in refractive index as patterns of light and dark - Can be used to view live cells and cellular organelles Figure 2.34 40 Figure 2.35 41 2.6 Electron Microscopy Electrons behave like light waves - Very high frequency - Allow very great resolution - A few nanometers Sample must absorb electrons - Coated with heavy metal Electron beam and sample are in a vacuum - Lenses are magnetic fields 42 Figure 2.36 43 2.6 Electron Microscopy Two major types - Transmission electron microscopy (TEM) - Electrons pass through the specimen - Reveals internal structures - Scanning electron microscopy (SEM) - Electrons scan the specimen surface - Reveals external features in 3D 44 Figure 2.37 The TEM closely parallels the design of the bright-field microscope 45 The SEM is arranged somewhat differently from the TEM Figure 2.38 46 The specimens for electron microscopy can be prepared in several ways - Embedded in a polymer for thin sections - Microtome is used to cut slices - Sprayed onto a copper grid The specimen is then treated with a heavy-metal salt such as uranyl acetate Note: For SEM, specimen is coated with heavy metal and it is not sliced Sample Preparation 47 Figure 2.39 Figure 2.40 48 2.6 Tomography In cryo-EM, or electron cryomicroscopy, the specimen is flash-frozen - Suspended in water and frozen rapidly in a refrigerant Cryo-electron tomography, or electron cryotomography, avoids the need to physically slice the sample for thin-section TEM - The images are combined digitally to visualize the entire object in 3D - Generates high-resolution models of virus particles 49 Figure 2.42 50 Figure 2.43 51 Figure 2.44 52 Scanning probe microscopy (SPM) enables nanoscale observation of cell surfaces The atomic force microscope (AFM) is an example of an SPM It measures the van der Waals forces between electron shells of adjacent atoms of the cell surface and the sharp tip It can be used to observe live bacteria in water or exposed to air (unlike electron microscopy) Emerging Methods of Microscopy 53 Figure 2.45 54 2.7 Visualizing Molecules X-ray diffraction analysis - For samples that can be crystallized, X-ray diffraction makes it possible to fix the position of individual atoms in a molecule - A beam of X-rays is shot at a crystallized sample - Many molecules in identical conformation - X-rays diffract according to position of atoms - Compute position of atoms from pattern of scattered X-rays 55 Figure 2.46 56 Today, X-ray data undergo digital analysis to generate sophisticated molecular models - Example: the anthrax lethal factor - A toxin producedby Bacillus anthracis - Note: The model wasencoded in a proteindata bank (PDB)text file Figure 2.48 57 Chapter Summary When observing microbes, resolution and magnification are paramount Different kinds of microscopes are required to resolve cells and subcellular structures: - Bright-field: employs various stains - Fluorescence: employs fluorophores for labeling - Dark-field: detects unresolved objects - Phase-contrast: exploits differences in refractive indices 58 Chapter Summary Electron microscopes use beam of electrons instead of light rays - TEM: provides internal details in 2D - SEM: provides external details in 3D Scanning probe microscopes (SPMs) include the atomic force microscope (AFM) - Allow observation of living cells in water or in air Molecules can be visualized by X-ray crystallography 59 Concept CheckSection 2.1 Which of the following statements about the size of microbes is FALSE? a) Eukaryotic microbes tend to have a size of10100 mm. b) Prokaryotic microbes tend to have a size that is less than 10 mm. c) A few bacterial species are large enough to be seen by the unaided eye. d) Choose this answer if all the above are true. 60 Concept CheckSection 2.2 Which of the following properties is most important for a lens to magnify an image? a)Absorption b)Reflection c)Scattering d)Refraction 61 Concept CheckSection 2.3 You are observing a bacterium using a 10 ocular lens and a 45 objective lens. What would the total magnification be? a) 10 b) 45 c)145 d)450 e)Need more information 62 Concept CheckSection 2.3 What is the correct order of reagents in the Gram stain? a) Iodine, crystal violet, ethanol, safranin b) Crystal violet, iodine, ethanol, safranin c)Crystal violet, ethanol, iodine, safranin d) Iodine, ethanol, safranin, crystal violet e)Safranin, ethanol, iodine, crystal violet63 Concept CheckSection 2.4 In fluorescence microscopy, the specimen absorbs incident light and then re-emits it at a _______ energy and thus, a _______ wavelength. a)lower; shorter b)lower; longer c)higher; shorter d)higher; longer 64 Concept CheckSection 2.5 Which of the following microscopes allows the best view of bacterial flagella during motility? a)Bright-field microscope b)Dark-field microscope c)Fluorescence microscope d)Transmission electron microscope e) Scanning electron microscope 65 Concept CheckSection 2.5 Which of the following statements aboutphase-contrast microscopy is true? a)It exploits differences in refractive indices between cell parts and surrounding media. b)It can be used to view live cells. c)Both A and B d)Neither A nor B 66 Concept CheckSection 2.6 All of the following statements apply to scanning electron microscopy EXCEPT a)The specimen is usually fixed and embedded b)The embedded specimen is cut into thin sections with a microtome c)It cannot be used to view live specimens d)It provides 3D images of the specimen 67 Concept CheckSection 2.6 Which of the following statements about theatomic force microscope is true? a)It is an example of a scanning-probe microscope b)It measures van der Waals forces c)Both A and B d)Neither A nor B 68 Concept CheckSection 2.7 What is the best technique for examining the presence of a chemical structure with a diameter of 3 nm? a)Light microscopy b)Electron microscopy c)Ultracentrifugation d)Tomography e)X-ray crystallography