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INTRODUCTION TO THE MICRSCOPE Introduction to microscopy Severall ti S times during d i the th semester t you will ill be b required i d to t utilize tili the light microscope to observe cells and tissues. To be successful you will need to have a working knowledge of how to use the microscope. Improper use of the microscope is not only frustrating to the student, but may result in damage to the microscope or the specimen being observed. Goals By the end of this laboratory, laboratory you will have the ability to: 1) Name the parts of the microscope and their functions. 2) S Successfully f ll use the th microscope i to t observe b biological bi l i l specimens i . MICROSCOPY AND CELL STRUCTURE Experimental procedure for basic microscopy A A. I Important t t parts t off the th microscope. i 1. The microscopes used in lab are called compound microscopes because they have two magnifying lens. lens a. The ocular lens is the lens that you look through. bb. The h objective bj i lens l i the is h lens l that h is i immediately i di l above the specimen. There are three objective objecti e lens. lens - the scanning lens with the 4X label - the low power lens with the 10X label - the high power lens with the 43X or 45X label. MICROSCOPY AND CELL STRUCTURE Experimental procedure for basic microscopy (cont.) A A. I Important t t parts t off the th microsocpe i (cont.). ( t) c. To calculate the total magnification, the magnifying power of the ocular lens is multiplied by the magnifying power of the objective lens. Example: When using the scanning power lens (4X) the total magnification is 40X. Ocular lens = 10X X X Objective lens = 4X Total magnification 40X MICROSCOPY AND CELL STRUCTURE Experimental procedure for basic microscopy (cont.) A A. I Important t t parts t off the th microsocpe i (cont.). ( t) 2. There are two different focusing knobs that will be used. a. The course focus knob which is the outer larger portion of the focus knob allows for dramatic or gross movement of the objective lens.This lens This focus knob should not be used when focusing the high power objective lens. b. The fine focus knob which is the inner small portion of the focus knob allows for very slight movement off the h objective bj i lens. l This Thi lens l should h ld only l be b used for focusing the low and high power lens. MICROSCOPY AND CELL STRUCTURE Experimental procedure for basic microscopy (cont.) A A. I Important t t parts t off the th microsocpe i (cont.). ( t) 3. The stage has two metal clips to hold the microscope slide in place. place A grease interface between the stage and the stage platform allows for slow precise movement of the stage. 4. Below the stage is a condenser with an iris diaphragm that allows light to be focused onto the specimen through a hole in the stage. a. As the magnification is increased, you may have to open the iris diaphragm to allow more light onto the specimen. specimen MICROSCOPY AND CELL STRUCTURE Experimental procedure for basic microscopy B B. Ob Observation ti off the th letter l tt “e”. “ ” 1. You will place a slide with a typed letter “e” on the stage of your microscope. microscope 2. You will then observe the letter “e” under the scanning, low power, and finally high power objective lens. Remember: If you cannot find the specimen/object you are looking for j lens. As the magnification g f increases, the do not ggo to the next objective depth of field and the diameter of the field decrease making it harder, not easier to find what you are looking for. 3. Rotate the objective lens back to scanning, raise the lens with the coarse adjustment knob, and carefully remove the slide. MICROSCOPY AND CELL STRUCTURE Experimental procedure basic microscopy (cont.) C C. Di Diameter t off fi field. ld 1. You will place a measuring stick on the stage of the microscope to measure the diameter of the field. field 2. You will align the measuring stick so that it passes through the middle of the circular field and count the number of millimeters. (See Figure 4.2 in the lab manual). 3. Since microbes are usuallyy measured in units of micrometer rather than millimeters, you will need to convert your millimeter measurement into micrometers. There are 1,000 micrometers in 1 millimeter. millimeter MICROSCOPY AND CELL STRUCTURE Experimental procedure basic microscopy (cont.) D. Focal plane/depth of field 1. A slide with three colored overlapping threads will be used to illustrate the difference in the depth p of field between the three objective lens. 2. By focusing carefully, it may be determined which of the three threads is on the top, top which is in the middle, middle and which is on the bottom. MICROSCOPY AND CELL STRUCTURE Introduction to cell structure The cell Th ll is i the th smallest ll t unit it off life lif andd all ll living li i organisms i are composed of cells or cell products. There are two basic types of cells found on our planet, the eukaryotic cells and the prokaryotic cells. cells The names of these two cell types indicate the condition of their nuclear material. The eukaryotic cells have a true (eu eu) nucleus (karyo). The h prokaryotic k i cells ll on the h other h hand h d are though h h to be b more primitive i ii (pro pro or pre pre) nucleus (karyo). All living li ing cells whether hether eukaryotic e kar otic or prokaryotic prokar otic have ha e several se eral common characteristics. This list includes a cell membrane, cytoplasm, ribosomes, and DNA. MICROSCOPY AND CELL STRUCTURE Experimental procedure observing cells A. Observation of prokaryotic cells. 1. Bacteria will observed on a prepared slide containing three different morphological types. bacillus - rod spirillium - spiral coccus - round MICROSCOPY AND CELL STRUCTURE Experimental procedure observing cells (cont.) A. Observation of prokaryotic cells (cont.). 2. Cyanobacteria (Blue-green algae) will be available as live specimens. MICROSCOPY AND CELL STRUCTURE Experimental procedure observing cells (cont.) B. Observation of eukaryotic cells. 1. Amoeba will be available as live organisms. A wet mount will be prepared from a culture dish. For best results take your sample l from f the th bottom b tt off the th dish. di h The Th amoeba b will ill be b observed as a gray granular non-distinct object. After a few minutes of adaptation to the microscope slide the amoeba will begin to move using its pseudopods. This projection is a pseudopod. MICROSCOPY AND CELL STRUCTURE Experimental procedure for observing cells (cont.) B. Observation of eukaryotic cells (cont.). 2. Elodea will be available as live organisms. A wet mount of a leaf will be prepared. The individual cells will appear rectangular t l with ith distinct di ti t cell ll walls ll andd internal i t l chloroplasts. hl l t The small green spheres are the chloroplasts ==> This distinct Thi di i line is a cell wall. MICROSCOPY AND CELL STRUCTURE Experimental procedure observing cells (cont.) B. Observation of eukaryotic cells (cont.). 3. Onion cells will be observed from the skin taken from a piece of onion which has been prepared with Janus green and sucrose. The Th cells ll will ill be b observed b d as irregular i l cells ll that th t remind i d me of chicken wire. MICROSCOPY AND CELL STRUCTURE Experimental procedure observing cells (cont.) B. Observation of eukaryotic cells (cont.). 4. Cheek epithelial cells will be acquired from the lining of your own cheek. The blunt end of a toothpick will be used to scrape cell ll from f the th inside i id off the th cheek. h k The Th cells ll will ill be b dispersed in iodine on the microscope slide to make the nucleus more apparent. The distinct structure in i the h center is the nucleus. The outer edge d off the h cell ll is the cell membrane. MICROSCOPY AND CELL STRUCTURE Introduction electron microscopy An electron A l t microscope i has h the th capability bilit off magnifying if i a specimen many thousands of times thus allowing observation of organelles that cannot be seen with light microscopy. An electron micrograph may be use to determine the true size of a cell or organelle. Experimental procedure for electron microscopy A. Calculation of actual size from a micrograph. Step1: p Measure the cell or organelle g and convert the units to micrometers Step2: Divide the measurement in micrometers by the magnification. MICROSCOPY AND CELL STRUCTURE Experimental procedure for electron microscopy (cont.) B B. Ob Observation ti off organelles. ll You should be able to observe the following organelles in the electron micrographs provided: nucleus, nucleus mitochondria mitochondria, endoplasmic reticulum, ribosomes, golgi complex, cell membrane, and in addition to these structures cell wall, central vacuole, and chloroplasts in plant cells.