Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
CHAPTER 2: Observing the Microbial Cell CONCEPT MAP I. Section 2.1: Observing Microbes A. Resolution of objects i. Resolution—smallest distance by which two objects can be separated and still be distinguished B. Resolution differs from detection i. Detection—the ability to determine the presence of an object. a. The eye can detect the presence of mold but can’t resolve individual cells ii. Magnification—increase in the apparent size of an object C. Microbial size and shape i. Prokaryotes are generally between 0.4–10 m ii. Three common prokaryotic shapes a. Bacilli—rod shaped b. Cocci—spherical shaped c. Spiral—corkscrew shaped D. Microscopy for different size scales i. Different types of microscopy are used to view structures of different sizes II. Section 2.2: Optics and Properties of Light A. Light carries information i. Electromagnetic radiation interacts with objects and acquires information that can be used to detect the objects ii. Conditions necessary for electromagnetic radiation to resolve and object a. Contrast between object and surroundings b. Wavelength smaller than object c. Detector with enough resolution for that wavelength B. Light interacts with objects i. Particles of light called photons interact with objects in many ways a. Absorption—light energy is acquired by object b. Reflection—wave front bounces off of object at angle equal to its incident angle c. Refraction—bending of light when it enters a substance that slows its speed d. Scattering—wave front interacts with object of smaller dimension than the wavelength C. Refraction enables magnification i. Magnification requires refraction of light through medium of high refractive index D. Magnification and resolution i. Empty magnification—magnification without increase in resolution 277 278 | Chapter 2 III. Section 2.3: Bright-Field Microscopy A. Increasing resolution i. Resolution depends on a. Wavelength of light b. Contrast c. Lens quality and magnifying power d. Position of the focal plane B. The compound microscope i. Uses a system of lenses to achieve magnification and resolution a. Condenser lens—concentrates light from light source to slide b. Objective lenses—closest to specimen; typically magnify 10×, 40×, or 100× c. Ocular lens—closest to eye; typically magnifies 10× ii. Steps for observing specimen a. Position specimen in middle of field of view b. Optimize light—need more at higher powers c. Focus objective lens C. Is the object in focus? i. Object is in focal plane of lens when edges of object appear sharp and distinct D. Fixing and staining improve resolution and contrast i. Wet mount—water and specimen on slide with cover slip a. Can observe living cells in natural state b. Most cells are transparent so show little contrast ii. Fixation—cells are treated with alcohol or heat to make them adhere to slide iii. Stains—adhere to bacteria to increase contrast E. Different kinds of stains i. Simple stain—uses one stain to color all cells ii. Differential stain—distinguishes between different types of bacteria by using different stains a. Gram stain—bacteria are classified as Gram positive or Gram negative based on whether they retain crystal violet stain b. Acid-fast stain—carbolfuchsin is used to stain Mycobacterium species c. Spore stain—malachite green and heat are used to stain endospores d. Negative stain—stains background but not cells; used to view capsules e. Antibody stain—antibody proteins linked to fluorophores bind specific components of cells IV. Section 2.4: Dark-Field, Phase-Contrast, and Interference Microscopy A. Dark-field microscopy i. Uses scattered light so cells appear bright on dark background ii. Can be used to view very small microbes and motility iii. Disadvantage—particulates will scatter light; can be difficult to distinguish from microbes from particulates B. Phase-contrast microscopy i. Enhances differences in refractive index so live cells can be observed without staining C. Interference microscopy i. Superimposes interference bands on an image, accenting small differences in refractive index Observing the Microbial Cell | 279 V. Section 2.5: Fluorescence Microscopy A. Fluorescence requires excitation and emission at different wavelengths B. Fluorophores can label specific parts of cells by i. Chemical affinity ii. Labeled antibodies iii. Gene fusion iv. DNA hybridization C. Laser confocal microscopy i. Fluorescence is used along with laser optics to produce 3D images VI. Section 2.6: Electron Microscopy A. The electron microscope focuses beams of electrons and achieves resolution 1000× resolution of light microscopy i. Transmission electron microscopy a. Electrons are transmitted through specimen ii. Scanning electron microscopy a. Electrons scan surface of specimen and are reflected to produce 3D image B. Electron microscopy requires specialized sample preparation i. Specimen can be embedded in polymer and cut into thins sections using microtome then coated with heavy metal ii. Specimen can be sprayed onto copper grid then coated with heavy metal iii. Specimen can be flash frozen for cryo-EM C. Microscopy results require careful interpretation i. Artifact—microscopic structure that is interpreted incorrectly D. Emerging methods of microscopy i. Cryo-EM a. Samples are flash frozen in water solution; very high resolution ii. Atomic force microscopy a. Measure van der Waals forces between atoms on cell surface and a sharp tip; very high resolution VII. Section 2.7: Visualizing Molecules A. X-ray diffraction i. Uses X-ray interference patterns to generate computational model of crystallized macromolecules ii. Cryocrystallography—uses frozen crystals to determine structures of macromolecular complexes