EXERCISE 1 MICROSCOPY BLOOD SMEAR Procedure: 1. Prepare 3 glass slides. Swab your finger (to be punctuated) with alcohol. Puncture your fingertip with a sterile lancet. 2. Let a drop of blood fall on one end of the slide. Let the end of the other slide come in contact with the droplet. Draw the second slide across the first slide at a 30-40 angle to make a thin smear of blood. The smear should cover about half of the slide. Allow air to dry. 3. Fix the smear by putting 95% ethyl or methyl alcohol on the smear. Let it stand for 3-5 minutes. Let dry. 4. Repeat steps 1-3. 5. Examine one of the blood smears under the low power objective, then switch to the high power objective. Note you observations. 6. Stain one of the smears following the procedure below. a) Add a few drops of Wright-Giemsa stain on the smear until it is fully covered ( take note of the stain: specimen volume ratio). Stain for about 16 minutes, renewing the stain about four times. b) Rinse the slide with distilled water at room temperature. Drain off the water and leave the slide to dry. c) Perform steps a) and b) to the other smear using Mayer’s Stain. 7. Examine each slide under the low power and high power objectives. Compare the smears with and without stains. What are the most notable differences among the three preparations? WET MOUNTS Procedure: 1. Gently scrape the inside of your cheek with the blunt end of a toothpick. Bathe the scrapings with a drop of Ringer’s solution on a glass slide and gently lower a cover slip unto the droplet using forceps and avoiding bubbles. Remove excess water on or around the cover slip with absorbent paper. 2. Place a drop of aceto-carmine or methylene blue stain at the edge of the cover slip and allow it to diffuse into the preparation. Let it stand for 3-5 minutes. Observe under low and high power objectives. 3. Obtain a protozoan culture from the hay infusion. Place a drop of the culture on a cavity slide and another on an ordinary slide. 4. Gently lower a cover slip on each side. Remove excess water. Observe movement of organisms in each preparation. What other differences can you observe in the two preparations? 5. Gently apply pressure on the cover slip on the ordinary slide. Observe what happens to the specimen. B. ESTIMATING MAGNIFICATION AND MEASURING MICROSCOPIC OBJECTS Procedure: 1. Mount a square millimeter of graph paper on a slide under a cover slip. Move the slide such that the paper is on the center of the field and the two sides of the square are parallel to the edge of the microscope stage. 2. Place a metric ruler on the right edge of your microscope stage so that you can see the ruler with one eye while you view the slide with the other eye. 3. Keep both eyes open and focus your eyes on both images with the square of graph paper in one eye and the scale of the ruler in the other eye. 4. When you see the two superimposed images, measure the apparent size of the superimposed millimeter square as it appears on the ruler. Be sure that you measure from the upper edge of the one line to the upper edge of the next line to include the thickness of the line in your measurement of the square. Record your measurement. 5. Calculate the magnification under the low power by dividing the apparent size of the millimeter square by its actual size (1mm). E.g. 100 mm (apparent size)/ 1mm (known size) = 100 X. 6. Using the stained blood smear, determine the size of the red blood cells under the low and high power objectives. EXERCISE 2 Procedure: 1. Examine under the high power objective the prepared slide of the bacterium Bacillus sp. Focus on one cell and draw all the visible structures (Fig. 2.1) Label accordingly and indicate the magnification. 2. Examine under low or high power objective the prepared slide of the protozoan Paramecium sp. Draw and label structures identified (Fig. 2.2). What are the functions of each structure? 3. Examine the prepared slide of a kidney cross-section. Focus on one cell and identify the visible structures (Fig. 2.3). Label properly. Compare and contrast the three cells based on the structures visible in each. Indicate which is a eukaryotic and a prokaryotic cell. Are all single celled individuals considered prokaryotic? Explain. Are all eukaryotes multi-cellular? Explain. 4. Examine the remaining slides. Draw each cell type studied. Compare the different cell types studied based on the following characteristics: a) basic shape, b) relative size, c) location of the cell in the body, d) function, and e) location of nucleus. PARAMECIUM SP. OVARY(cross-section) FROG SKIN(cross-section) STOMACH(cross-section) FROG SKIN(cross-seciton) KIDNEY(cross-section) TESTIS(cross-section) Schuch, Raymond, Daniel Nelson & Vincent A. Fischetti. 2002. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature, Vol. 418: 884-889 YEAST University of Texas Medical Branch: Bacillus University of Wisconsin-Madison: Bacillus anthracis and anthrax Paramecium sp Sources: Kidney(cross-section) parts http://legacy.owensboro.kctcs.edu/gcaplan/anat2/h istology/histo%20e%20urinary%20system.htm Bacillus sp. http://microbewiki.kenyon.edu/index.php/Bacillus Graumann, P. 2007. Bacillus subtilis: Cellular and Molecular Biology. Caister Academic Press. ISBN 9781-904455-12-7 Innovation in Europe: The decoding of the Bacillus subtilis genome Ivanova, Natalia et al. 2003. Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature, Vol. 423: 87-91. Kunst, F. 1997. The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature, 390: 249-256. Read, T. D. et al. 2003. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature, Vol. 423: 23-25. Rosovitz, M. J. & Stephen H. Leppla. 2002. Medicine: Virus deals anthrax a killer blow. Nature, Vol. 418: 825-826. Pellicle - a membrane covering that protects the paramecium like skin Cilia - hair like appendages that help the paramecium move food into the oral groove Oral Groove - collects and directs food into the cell mouth Cell Mouth - opening for food Anal Pore - disposes of waste Contractile Vacuole - contracts and forces extra water out of the cell Radiating Canals - paths to the contractile vacuole Cytoplasm - intercellular fluid needed to contain vital cell parts Trichocyst - used for defense Gullet - forms food vacuoles Food Vacuole - storage pocket for food Macronucleus - larger nucleus which performs normal cell functions Micronucleus - smaller nucleus which is responsible for cell division. BioMedia. "The Classics of Biology: Paramecium. Coleman, A.W. "Paramecium aurelia Revisited." The Journal of Eukaryotic Microbiology. Fujishima, Masahiro, Miki Kawai, and Ryu Yamamoto. "Paramecium caudatum acquires heatshock resistance in ciliary movement by infection with the endonuclear symbiotic bacterium Holosporaobtusa." FEMS Microbiology Letters 243 Gerritsen, Vivienne Baillie. "The Arsenal of Paramecium." Protien Spotlight. Haselton, Aaron. "Paramecium putrinum." The Connecticut River Homepage. Kawano T, Kadono T, Kosaka T, Hosoya H. "Green paramecia as an evolutionary winner of oxidative symbiosis: a hypothesis and supportive data." Z Naturforsch (C). Kimball, John W. Ciliated Protozoans. 14 June 2003. Samworth, Mike. "Paramecium." Microscopy UK. 1999. FROG SKIN http://science.tjc.edu/Course/BIOLOGY/1413/frog %20skin%20high.jpg nucleus- at center, hexagonal in shape TESTIS Sperling, Linda. Paramecium Genomics. 17 April 2005. EUKARYOTIC & PROKARYOTIC CELLS http://grauhall.com/catalog/product_info.php?prod ucts_id=1057&osCsid=7139a9bc9cf1274711d3c3b0f 36f9327 http://www.diffen.com/difference/Eukaryotic_Cell _vs_Prokaryotic_Cell Nucleus- center, small spaces between cells, circular in shape Prokaryotes (pro-KAR-ee-ot-es) (from Old Greek probefore + karyon nut or kernel, referring to the cell nucleus, + suffix -otos, pl. -otes; also spelled "procaryotes") are organisms without a cell nucleus (= karyon), or any other membrane-bound organelles. Most are unicellular, but some prokaryotes are multicellular). OVARY Eukaryotes (IPA: [juːˈkæɹɪɒt]) are organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane bound structure is the nucleus. This feature gives them their name, (also spelled "eucaryote,") which comes from the Greek ευ, meaning good/true, and κάρυον, meaning nut, refering to the nucleus. Animals, plants, fungi, and protists are eukaryotes. YEAST SMOOTH MUSCLE http://www.promocell.com/products/humanprimary-cells/smooth-muscle-cells/ Smooth muscle cells, which form non-striated muscles, are structurally and functionally different from skeletal and cardiac muscle cells. They are located in the walls of blood vessels and hollow organs like the bladder, the uterus, and the gastrointestinal tract and are responsible for involuntary movements like peristaltic contractions, which propel food along the gastrointestinal tract. Nucleus- at the middle of strands http://www.sciencedirect.com/science/article/pii/ S0016648007001062 Nucleus- center, circular in shape, spaces like kidney cells http://www.sciencedirect.com/science/article/pii/ S0016648007001062 Nucleus- apparently near the side, semi-oblong in shape