MICROSCOPY LAB

Lab 6: Introduction to Microscopy Purpose: The purpose of this lab is to use a compound light microscope to examine several specimens and to learn how to make wet mounts for specimen viewing. This lab will help you practice the following skills: 1. Using a light microscope 2. Creating wet mount slides to learn how handle and stain fresh tissues 3. Using a compound light microscope to make observations of fresh tissue and preserved specimens and to learn the appropriate use of the instrument This lab will help you gain the following knowledge: 1. Identifying the differences between plant, animal, and prokaryotic cells under a microscope. Introduction The scientist has a variety of laboratory equipment which allows them to “see” what could not normally be observed with the naked eye. One such instrument is the microscope, which extends the visual range of the scientist. In part 1 of this lab, you will learn the anatomy of the compound light microscope and how to view specimens with it. In part 2, you will use the microscope to observe living specimens which you will prepare for viewing. Finally, in part 3 you will examine the two general types of cells associated with living organisms, prokaryotic and eukaryotic cells, and make some general observations about them. PART 1 – Using a Compound Microscope There are a variety of different kinds of microscopes, each with different merits and uses. In Lab 1, you used the stereoscopic or dissecting microscope. Today you will learn about the compound light microscope . The Compound Light Microscope The compound light microscope (Fig. 1) must be used if you wish to observe smaller and thinner specimens. This type of microscope has much greater powers of magnification than the dissecting scope. The name of this microscope is derived from the fact that it utilizes two (i.e. compound ) optical components and uses light as its source of illumination. Plug in the scope and turn on the light. The optical components are the ocular (eyepiece) and objective lenses. There are usually 3-4 objective lenses projecting from the revolving nosepiece. Each objective has a different power of magnification (indicated on the side of the objective, see Fig. 3). The LOW-POWER OBJECTIVE is the shortest and magnifies objects by 4 times (4X); the MEDIUM-POWER OBJECTIVE magnifies objects by 10 times (10X); the HIGH-POWER OBJECTIVE magnifies objects by 40 times (40X). An OIL-IMMERSION OBJECTIVE may also be present, which magnifies objects by 100 times (100 X). This objective is only used with oil, and won’t be used in this lab. Since the ocular lens is located at the very top of the microscope and the objective lenses are located just above the stage, the object being observed is magnified first by the objective lens and then this image is magnified ten times more by the ocular lens. Thus, if the 10X objective is being used, the total magnification of an object is 10 x 10 or 100X. Magnification powers of 2,000X are possible with more sophisticated compound light microscopes. 1

COMPOUND LIGHT MICROSCOPE Figure 1: Compound Light Microscope oculars revolving nose piece objective 2 arm condenser lens coarse & fine focus knobs iris diaphragm/lever stage adjuster knob condenser adjustment knob base light source

4. When you are finished with an observation, turn off the illuminator and rotate the low power objective into viewing position, lower the stage and set the light intensity to its lowest setting. Practice using the compound light microscope by examining a prepared slide of Spirogyra or Oedogonium . Spirogyra and Oedogonium are species of green algae, though the specimens on the slides you will be using have been stained blue. They are eukaryotes and used to be classified as protists but are now considered to be part of the eukaryotic “supergroup” that includes plants (i.e. they are more closely related to plants than to other protists). For more information, see page 513 of Campbell Biology in Focus by Urry et al., third edition. Figure 2a: Spirogyra http://protist.i.hosei.ac.jp/PDB/Images/Chlorophyta/Spirogyra/group_C/sp_07.html Figure 2b: Oedogonium http://protist.i.hosei.ac.jp/PDB4/PCD2856/htmls/45.html Nucleus

I. (0.2pt) Move the stage/slide towards you. Which way does the specimen move in the field of view? Away from you II. (0.2pt) Move the stage/slide to the right. Which way does the specimen move in the field of view? left III. (0.2pt) What is the relationship between the direction the stage/slide is moved and the direction of movement in the field of view? They are inversely related IV. (0.3pt) Count the number of cells you can see in one strand at 4X, 10X, and 40X without moving the slide: Thousands, hundreds, thousands V. (0.2pt) What is the relationship between magnification and field of view? As magnification increases the field of view decreases VI. (0.2pt) Why do some parts of the specimen appear to be in focus when other parts are not?

PART 2 – Preparing a Wet Mount The material studied with a compound light microscope is usually mounted on a slide. Permanently prepared slides (such as Spirogyra and Oedogonium ) are commercially available, but to study living specimens, a scientist must prepare a temporary slide or wet mount. If the material has natural color, a thin section need only be placed on a microscope slide, a drop of water added to the top of the specimen, and a coverslip slowly lowered onto the specimen (Figure 4). Care should be taken to avoid trapping air bubbles underneath the coverslip. If you examine such a slide for very long some of the water between the slide and coverslip will evaporate. You can avoid letting the specimen dry out by adding a drop of two of water to the edge of the coverslip when needed. Some specimens are colorless and difficult to observe with the microscope. These specimens are usually stained by adding a dye that reacts with one or more molecules contained in the cells of the specimen. Stain adds color to the material being viewed and greatly increases the scientist’s ability to observe detail. Figure 4: Preparing a Wet Mount Slide Make a wet mount of one Elodea leaf using the technique described above. Materials necessary will be available at the front of the room. Figure 5: Elodea leaf. The small green “circles” are chloroplasts. https://commons.wikimedia.org/wiki/File:Turgid_Elodea_Cells_under_400X_Magnification.jpg I. (0.1pt) Examine your prepared slide under the lowest magnification. Will it be necessary to stain these cells? no

II. (0.2pt) Why or why not? Because it is visible (Note: The green color of the leaf comes from the presence of small, green spherical structures known as chloroplasts in the cells.) III. (0.1pt) Can you see the cells of the leaf at the lowest magnification level? no (0.1pt) Switch to the 10x objective lens. Can you see the cells of the leaf at this magnification level? yes IV. (0.1pt) Can you see the individual chloroplasts? yes V. (0.1pt) If so, approximately how many chloroplasts can you see? thousands VI. (0.5pt) Draw an accurate diagram of what you see with the scope at this magnification level. Switch to the x40 objective lens . VII. (0.1pt) Can you see the cells of the leaf under high power? yes VIII. (0.1pt) Can you see individual chloroplasts under high power? yes IX. (0.1pt) If so, approximately how many chloroplasts can you see? thousands X. (0.3pt) Draw an accurate diagram of what you see under high power . (In some cells, the chloroplasts may appear to be moving within the cytoplasm. This phenomenon is called cytoplasmic streaming and is due to the movement of the cytoplasm within the cell.)

Figure 8: Human skin cells https://upload.wikimedia.org/wikipedia/commons/2/25/Human_Cheek_Cells_%28Methylene_Blue_S tain%29.jpg XI. (0.4pt) Draw an accurate diagram of your cells under high power. XII. (0.1pt) Which is bigger – an Elodea leaf cell or your cell? The leaf cell XIII. (0.3pt) What are the differences that you can see between the plant and animal cell? The plant cells are in a pattern and they are more uniform while the animal cells are scattered Once you have finished, place the cover slip in the trash and place the slide in the bleach solution under the fume hood. Part 3 – Examining Prokaryote and Eukaryote cells under a Compound Light Microscope One of the fundamental features of life is that organisms are composed of many cells. As you may know, there are two basic types of cells: prokaryotic and eukaryotic. The distinguishing feature of these two cell types is an intracellular structure called the nucleus. The nucleus is a membrane- bound structure that encloses a cell’s genetic material (DNA). Prokaryotic cells lack a nucleus, and their DNA is only loosely confined to an area within the cell. Eukaryotic cells possess a nucleus. Bacteria are single-celled prokaryotic organisms. Bacteria are extremely small (approximately 1-2 µm in diameter), and many are devoid of natural color. Morphologically, they are either round (cocci), rod-shaped (bacilli), or spiral-shaped (spirilla). They are often found in clusters or in chains. To view bacteria in greatest detail with the compound light microscope, the cells must be stained and one must use an oil-immersion lens (100X). However, for the purposes of this lab, viewing the cells with a 40X objective will give enough detail to be sufficient.

Figure 9: Bacterial types, from left to right — Staphylococcus aureus, Escherichia coli, and Spirillum volutans Obtain slides of Staphylococcus aureus, Escherichia coli, and Spirillum volutans from either your station or the front of the room. I. (1.5pt) Observe a prepared slide of Staphylococcus aureus . Draw and describe the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective. Purple, medium, irregular II. (1.5pt) Observe a prepared slide of Escherichia coli. Draw and describe the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective. Pink, medium, irregular

the DNA is enclosed within a membrane and is visible as a nucleus. Individual chromosomes will be visible in cells undergoing division. Figure 10: Whitefish Blastula http://www.instruction.greenriver.edu/ IV. (1.5pt) Obtain a prepared slide of whitefish blastula. Draw and describe the appearance of these cells (note cell shape, internal structures visible, colors or structures, etc.) using the 40X objective. Record your observations below . Pink, large, round Cell membrane Chromosomes

V. (0.1pt) Which kind of cell is larger, the fish or the prokaryotic cells? The fish VI. (0.1pt) Can you see chromosomes in any cells with this level of magnification? yes VII. (0.1pt) Can you count the number of chromosomes contained in whitefish blastula cells with this level of magnification? yes VIII. (0.1pt) If so, how many chromosomes are present? About 100 IX. (1pt) What are the differences that you can see between the prokaryote and eukaryote cells? They are larger and the nucleus is visible When you are done with your microscope, turn off the illuminator and rotate the low power objective into viewing position, lower the stage and set the light intensity to its lowest setting. CLEANUP: cover slips are placed in the trash and slides are placed in the bleach solution under the fume hood. Gently wipe down the prepared slides and your microscope. WIPE DOWN YOUR EQUIPMENT AND LAB BENCH BEFORE YOU LEAVE.