Brief Overview of Different Types of Microscopes Commonly Used | PLP 3343, Lab Reports of Plant Taxonomy and Evolution

Download Brief Overview of Different Types of Microscopes Commonly Used | PLP 3343 and more Lab Reports Plant Taxonomy and Evolution in PDF only on Docsity! LAB PERIOD #1 Wednesday August 20, 2008 I. Objectives A. Brief overview of different types of microscopes commonly used in plant pathology B. Learn how they work C. Proper use and care of microscopes D. Practice using both stereomicroscopes and compound microscopes E. Learn about sterile technique and pouring solid medium II. Information Covered A. Storage and proper handling of microscopes in PLP 3343 B. Magnification versus Resolution (value and purpose of each). i. Calculate magnification of your field of view on each type of light microscope. C. Microscopes used in PLP 3343 i. Stereomicroscope (= “dissecting microscope”) 1. reflected light, most common 2. three-dimensional, lower resolution image produced 3. common uses: whole or pieces of specimens ii. Compound Microscope 1. transmitted light 2. flat, two-dimensional, higher resolution and higher magnification image produced 3. common uses: minute (sectioned) specimens mounted on slides D. Electron microscopes not used in PLP 3343 but important to plant pathology research, especially minute pathogens such as bacteria and viruses. i. Scanning Electron Microscope (SEM) three-dimensional image produced. 1. Whole-mounted, fixed specimen coated in gold to reflect electrons ii. Transmission Electron Microscope (TEM) two-dimensional image produced, very high resolution and very high magnification. 1. Fixed specimens sectioned to ultra thin thicknesses, stained with heavy metals (U or Pb) to reflect electrons Angle 12° | ‘Ocular Focusing knob Objectives Stage Fig. 1-6. The dissecting microscope. Ocular lens Fig. 1-2. Carrying a Turret microscope Objective lens Specimen on slide Stage Condenser diaphragm lever Condenser Condenser adjustment knob _) (not on all micrascopes) Fine focus Coarse focus Light switch Base LAB #1 Magnifier/Microscoper Information Magnification Range Maximum Resolution1 Price Range Human Eye 1x 100 – 200 μm Free Hand lens/magnifier 4 – 20x 100 – 200 μm $5-50 Stereomicroscope2 10 – 250x 100 – 200 μm $1,000-9,000 Compound microscope2,3 50 – 1,000x 0.2 μm $3000-50,000 Scanning Electron Microscope (SEM) 100 – 50,000x 0.01 $200,000-1,000,000 Transmission Electron Microscope (TEM) 1,000 – 500,000x 0.0002 μm $200,000-1,000,000 1 “Resolution” or “resolving power” depends on: (a) lens quality (expressed as the numerical aperture [N.A.]; e.g. low=0.1, medium 0.7, highest=1.4 (oil immersion)), (b) refractive index of the medium (air, water, immersion oil) between the specimen and the objective lens, and (c) wavelength of incident illumination (the shorter, the higher the resolution); visible light beams of the compound light microscope range from 300 - 600 nm in wavelength and can resolve objects 0.3 – 0.6 μm apart,accordingly, electron beams in an EM resolve objects with accelerated electrons (radius 0.000000002 μm) and have much higher resolution. Microscopy unit of measure one “micron” = 1 μ = 1 μm = 0.001 mm = 0.000001 m = 1000 nm 2 “light microscopes” 3 Magnification = power of objective lens x power of ocular lens: (10x ocular x 2x zoom) = 20x (10x ocular x 10x objective) = 100x (10x ocular x 40x objective) = 400x “Resolution” and “Magnification” are totally different things: Resolution – the ability to resolve closely spaced objects as distinct, without blurring together. Magnification – simple enlargement of an image. Resolution is NOT enhanced unless a lens with a greater resolving power is used. Stereomicroscope (or “dissecting microscopes”): has two sets of compound (stacked) lenses; ~15° off parallel, providing 3-D, “stereoscopic” image. Stereomicroscopes have lower magnification and resolution than the compound microscope, but have greater working distances and can be used with both transmitted and reflected illumination. Compound microscope: has one set of compound (stacked) lenses, often split apart at the top (yet always as parallel beams) to allow for binocular viewing. Produces a flat, 2-D image, but with greater magnification and resolution than a stereomicroscope; usually only transmitted illumination. Fluorescent compound microscopes use reflected fluorescent illumination of specifically filtered wavelengths. Examples of Types of Microscopy Budding Yeast Cells (Saccharomyces cerevisiae) ELECTRON MICROSCOPY TRANSMITTED LIGHT SCANNING TRANSMITTED DIC coupled with fluorescent microscopy nuclei stained blue and spindle pole body labeled green Filamentous algal cells (Zygnema spp.) (a) Bright Field (b) Phase Contrast (c) Differential Interference Contrast (DIC) or Nomarski DIC optics SEM TEM halo shadow light Making Sterile Nutrient Media for isolation of pure cultures of microbes 1. Pour solid media into Petri Plates – two plates each of • Potato Dextrose Agar (PDA) = a rich medium • Water Agar (WA) = a weak medium – label with your initials, date and media type 2. Observe an autoclave “The Story of Agar”: Koch's Lab releases a method to pure culture microoganisms (1881) MICROBIAL SERENDIPITY: Until recently, few women have played a significant role in microbiology, however this young science owes the development of a crucial technique to a woman. Fanny Angelina Eilshemius was born in 1850 in New York of a wealthy Dutch immigrant family. As a young girl, Angelina toured Europe where she met a young German doctor, Walther Hesse, whom she married in 1874. Angelina Hesse settled down to being the wife of a busy country physician. W. Hesse, became interested in the new science of Microbiology and joined Koch's lab in 1881. Dr. Hesse studied many aspects of bacterial public health and bacterial metabolism. His wife, nicknamed Lina, assisted him, much like my wife does me, as a laboratory technician. She was a talented artist who drew illustrations for his publications. One hot summer when Walther was attempting to do counts on bacterial air contaminates he was having trouble with his GELATIN (what we call Jell-O) plates melting in the heat and being digested by many of the bacteria he tried to grow on them. In frustration, he asked his wife "Why do your jellies and puddings stay solid in the warm weather?". She explained to him that she used AGAR-AGAR, a complex polysaccharide extracted from seaweed, to keep them solid in hot weather. AGAR-AGAR had been used as a gelling agent in ASIA for centuries. She had learned of it as a youngster in New York from a Dutch neighbor who had immigrated from Java. Presumably Dr. & Mrs. Hesse discussed the possibility of using agar-agar to prepare microbial media and Dr. Hesse subsequently found that it worked beautifully. The following characteristics of AGAR-AGAR make it almost perfect for the growth of microbes on solid medium: (a) non-toxic to most microbes. (b) only melts at 100°C, but solidifies at about 45°C (a temperature most bacteria can survive). (c) nontoxic to other forms of life. (d) stable to sterilization temperatures. (e) physiologically inert as very few bacteria have the #enzymes for digesting it. This kitchen ingredient revolutionized the science of microbiology as it made what had been an arduous task of separating and growing microbes on solid surfaces a routine procedure. Dr. Hesse went on to make other important bacteriological discoveries and advances, such as bringing the pasteurization of milk to Germany; which prevented the death of children from TB- and milk contaminated with intestinal pathogens.