Microscopy and Cell Biology Lab 2 Exercises 4 & 5 PDF

Lab 2. Exercises 4 and 5 - Microscopy and Cell Biology ====================================================== **Name:** --------- Learning objectives ------------------- 1. Be familiar with the parts and components of a light microscope. 2. Be familiar with the proper handling, use and care of...

Lab 2. Exercises 4 and 5 - Microscopy and Cell Biology ====================================================== **Name:** --------- Learning objectives ------------------- 1. Be familiar with the parts and components of a light microscope. 2. Be familiar with the proper handling, use and care of a light microscope. 3. Be familiar with the markings on the individual objective lenses and their meanings. 4. Be familiar with the similarities and differences between animal and plant cells. Exercise 4: Microscopy ====================== The microscope is an indispensable tool in the study of cells. Anton van Leeuwenhoek (1632-1723) first observed protozoans using simple microscopes with a single lens to magnify the image. Today, compound microscopes have a two-lens system that achieves much greater magnification with greater resolution. **Figure 1.** The compound microscope. **INTRODUCTION** The microscope has made it possible to study structures too small to be seen with the unaided eye. It is an instrument that magnifies an image, using a series of lenses while maintaining image resolution or visual clarity. This **magnification** (apparent enhancement) and increased resolution allow us to observe and study tiny objects. **Resolution** refers to the ability to distinguish between two points; the greater the resolution or resolving power, the closer two points can be to one another while still being distinguishable as two distinct points. A normal human eye has a resolution of about 0.2 mm, which means that the eye can distinguish between two objects with 0.2 mm or more space between them. The light microscopes that you will use in the lab have a resolution of about a thousand times that, or 0.2 um. As you will see, they can magnify an image up to 1,000 times. An image can be magnified but that does not increase the level of detail that can be observed, as can be seen in the pictures in Figure 2, where the magnified images appear fuzzy. The detail observed when looking under a microscope depends on the **resolving power** of a microscope. **Resolving power** is a measure of image clarity.  **Figure 2.** Resolving Power v. Magnification **MATERIALS** - Compound microscopes - Microscope slides and cover-slips - Letter "e" prepared slide **A. Anatomy of the Compound Microscope** ========================================= The compound microscope that you will use is composed of numerous parts, many of which are adjustable to enable you to change magnification and adjust resolution. Its parts are shown in Figure 1 and 3, and include the following: **Base:** The supportive bottom piece of the microscope. The base includes the following: **Substage light:** A light within the base providing the light source for illumination of the specimen. A switch, usually at the side or front of the base, turns it on and off, and a dial (rheostat) adjusts the light intensity. **Head:** Also called the body tube, it is the upper part of the microscope that contains the viewing pieces (the lenses and rotating nose-piece). **Ocular lenses:** The ocular or eyepieces are two removable lenses that you look through to observe your specimen. Most ocular lenses have a magnification of 10×, increasing the observed size of the specimen by a factor of ten. **Nose-piece:** Located below the ocular lenses, it serves as an attachment for the objective lenses. The viewer can rotate the nose-piece to change from one objective lens to another. **Objective lenses:** Three or four objective lenses are usually attached to the nose- piece, each with a different magnifying power. Most commonly (4× or scanning, 10× or low power, 40× or high power, and if present, 100× or oil immersion.) The magnification levels are written on each objective lens. **Arm:** The narrow, vertical part of the microscope connecting the head and base. **Stage:** The flat platform connected to the arm and suspended beneath the objective lenses, upon which the microscope slide with its specimen is placed. **Coarse Adjustment Knobs:** Two knobs on either side of the arm. They are usually the largest knobs on the arm. These knobs control the focus. **Fine Adjustment Knobs:** Two knobs usually located in the center of each coarse adjustment knob. These knobs are used for precision focusing. **Condenser:** A lens located just below the stage, it focuses or concentrates light on the specimen. In many microscopes, the condenser includes a knob that raises and lowers it to control light intensity. For exercises in this manual, the best position for the condenser is close to the stage. However, you should be sure that it is not so high that your slide scratches the lens when you place it on the stage. **Iris Diaphragm Lever:** A lever located beneath the condenser. It opens and closes the iris diaphragm. The iris diaphragm regulates light passing through the condenser, in the same way that the iris of your eye works. Label the following diagram of the microscope: **B. Basic Care and Setup** =========================== The working parts of a microscope are supported by a strong frame consisting of the head, arm, and base (Figure 4.2). The base and arm are the safe parts of the microscope to hold when carrying it. Grasp the arm with one hand and rest the microscope's base on your other hand. [Before using the microscope]: - If necessary, clean all lenses by carefully wiping them with lens paper. Regular paper towels are too rough and will scratch the lenses. - Plug in the microscope. - Make sure the rheostat is on its minimum position before turning on the light source. [When you are finished working with the microscope]: - Rotate the nosepiece to put the scanning lens in place and turn the coarse adjustment knob so that the stage is in its lowest position. - Turn the rheostat to low before turning off the light source. - Turn off the light source and remove the slide. - Use the mechanical stage adjuster know to make sure the stage arm is not sticking out beyond the stage. - Unplug the power cord and carefully wrap it around the microscope and tuck in the plug so that there are no dangling cords. - Cover microscope (if a cover is available.) - Carefully carry it to its proper storage cabinet. **C. Using the Microscope** =========================== **Determining Magnification** The magnification of any one lens is fixed and given in Table 4.1. But the total amount of magnification you use can be adjusted by changing the combination of lenses you are looking through. Total magnification is calculated by multiplying the fixed magnification of each lens. In other words, since you will always be using the ocular lens (10X), multiple the power of the objective lens currently in place (either 5X, 10X, or 40X) by 10. **Table 1**. Magnification for each combination of lenses.  **Field of View** The field of view is that area of the slide that is visible to you when looking through the microscope. It changes with each objective lens. As seen in Figure 4.3, the greater the magnification of a lens, the closer it is to the slide, so the field of view is smaller and the image appears larger. **Figure 3.** Size of the field of view for each lens. © bluedoor, LLC **Focusing** Bringing an image into clear focus is a series of steps that changes the distance between the lens and the specimen on the slide. The goal is to find the sharpest image so you can study the details of the specimen at appropriate magnification. Figure 4.3 clearly shows why it is important to begin the process with the scanning lens in place. This is the only position that you are sure will not actually push the lens through your slide. Questions --------- Before going on to the next section, answer the following questions. 1. In your own words, explain what is meant by the resolving power of a lens. -- -- 2. **In your own words, explain what is meant by the magnifying power of a lens.** -- -- **Procedure** ============= Familiarization with Microscope Components ========================================== - Familiarize yourself with your microscope by identifying the components labeled in Figure 1. - Note the magnification of each objective and the ocular on your microscope and compute the total magnification with each objective and enter the values in Table 1. **Table 1. Magnification** **Lens Name** **Objective Power** **Ocular lens** **Total magnification** --------------- --------------------- ----------------- ------------------------- low power 10x 10X 100x high dry 10X oil-immersion 10X ### The Letter 'e' Now that you have familiarized yourself with the parts of the microscope, let's try to focus on a slide. Here you will notice the set-up of the lenses on the microscope by viewing the letter 'e'. 1. Select the "Letter E" slide from your instructor 2. Using the Coarse and Fine adjustment knobs on the bottom of the screen, focus on the letter 'e' at [4x magnification]. 3. Draw the letter e at 4x magnification below Figure 1. Letter 'e' at 4X magnification **Post-Lab Questions Exercise 4:** ================================== Q1. When viewing the letter \'e\', what was unusual about its appearance? Q2. Biology labs typically use the \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ microscope. Q3. Another name for rheostat is: Q4. The Field of View is largest when the \_\_\_\_\_\_\_\_ lens is being used. ### **Exercise 5: Viewing Cells under a Microscope** ### **OBJECTIVES** Upon completion of this laboratory, students should be able to - analyze the staining properties of cell organelles; - observe the similarities and differences between animal and plant cells; - recognize the typical structures in plant cells; - recognize the typical structures in animal cells; and - identify the major components of each type of cell and describe the function of each. **INTRODUCTION** One characteristic of living things is that they are made up of cells. A **cell** is the smallest unit capable of sustaining life. While the **Cell Theory** is considered a foundation of biology, cells themselves were not seen until the development of the microscope in the late 1600s, when Robert Hooke built his own microscope and examined a piece of cork from the bark of a tree. The regular structures he saw reminded him of the regular arrangement of rooms, or cells, of monks, so he called these structures cells. We now know that Hooke was actually seeing dead cells, because the cell walls of plants retain their form even after there are no living cells inside. Each cell has three common characteristics: 1. The outer boundary of the cell is the **plasma membrane**. 2. All cells store genetic information in the form of **DNA**. 3. Everything inside the plasma membrane that is not DNA or nucleus is **cytoplasm**. We can distinguish two basic types of cells based on their internal structures: the simpler **prokaryote** cell, and the more complex **eukaryote** cell. **Prokaryotic cells** include organisms that we know as bacteria. They have few internal compartments, their DNA is found in the cytoplasm and not within a nucleus, they do not have any membrane-bound organelles, and they may have rigid cell walls that maintain their characteristic shapes. They are usually very small, just visible with the light microscope. Examples of prokaryotes include bacteria, cyanobacteria, and archaea. **Plants, animals, fungi,** and **protists** are made of eukaryotic cells. **Eukaryotic cells** are usually larger than bacteria and have a variety of internal compartments defined by membranes. They contain membrane-bound organelles such as mitochondria and chloroplasts. The most visible compartment is the nucleus that contains the cell's genetic material in the form of DNA. Other internal structures are difficult to see with the light microscope, but can be seen with special staining or with electron microscopes. Plant Cells Animal Cells ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Plants are unique among the eukaryotes because they can manufacture their own food through a process called photosynthesis. Chlorophyll, which gives plants their green color, enables them to use sunlight to convert water and carbon dioxide into chemicals that cells use for fuel. The basic plant cell does not have centrioles, lysosomes, intermediate filaments, cilia, or flagella. Plant cells do, however, have a number of other specialized structures, including a protective, rigid cell wall structure retained from their prokaryotic ancestors, a central vacuole, and chloroplasts, which store chlorophyll. Animal cells are typical of the eukaryotic cell, enclosed by a plasma membrane and containing a membrane-bound nucleus and organelles. Unlike the eukaryotic cells of plants, animal cells do not have a cell wall, central vacuole, or chloroplasts. The lack of a rigid cell wall allows animals to develop a greater diversity of cell types, tissues, and organs.  Figure 1. Plant Cell Figure 2. Animal Cell **Onion Root Tip** ================== 1. Obtain a compound microscope and an [onion root] prepared slide 2. Using the Coase and Fine adjustment knobs, focus on the slide at 4x magnification. 3. Once you have the slide focused, increase the magnification to 10X, then to 40X 4. Draw what you see at 40X below: **Red Blood Cell** ================== 1. Obtain a compound microscope and a [Red Blood Cell] prepared slide 2. Using the Coase and Fine adjustment knobs, focus on the slide at 4x magnification. 3. Once you have the slide focused, increase the magnification to 10X, then to 40X 4. Draw what you see at 40X below: ### Questions 1. How does using a stain change the visibility of the structures? Why? 2. What features can you see under the microscope which allow you to characterize these cells as eukaryotic cells? 3. Plant cells are surrounded by a plasma membrane and they also have cell walls. What are the major components of plasma membranes? What is the major component of plant cell walls? 4. What differences could you see, using a microscope, between the onion cells and the cheek cells (or alternatively, the blood cells)? Compare structures and size