Lab 5 - Cells

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Texas A&M University, San Antonio *

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Biology

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Apr 3, 2024

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Microscopes and the Cell (Adapted from Biology Laboratory Manual , 10th Edition; Darrell S. Vodopich and Randy Moore. 2013 and Northwest Vista College stock laboratories.) Microscopes We will use the light microscope to see objects that cannot be seen with the naked eye. The light microscope works by shining light from the bottom of the microscope through a specimen and then refracting (bending) the light rays with lenses. This will be discussed in more depth once the parts of the microscope have been covered. Most people are aware that microscopes are used to magnify , which means to make something appear larger. Just making things larger is NOT sufficient, however. When you make a photograph larger (magnify), eventually the picture will get fuzzy and you may not see the details in the picture any better. Observing the details of a specimen is called resolution . Resolution allows us to see the parts within a cell. A fuzzy picture has lost resolution. Thus, magnification and resolution are both important factors when viewing a specimen with a microscope. It is also important to be able to distinguish the specimen from the background; this is called contrast . Sometimes a specimen is treated with chemical stains in order enhance contrast. Black letters against white background has a high degree of contrast and makes the letter easier to see. White letters on a white background have no contrast and are difficult to see. The Proper Care of a Microscope When carrying a microscope, one hand should be placed on the bottom (base) of the microscope and the other should grasp the handle (arm). The microscope should be carried close to the body, like a baby. Once the instructor has demonstrated the proper manner for carrying, students can obtain a microscope. Other important considerations include:  Only using lens paper (kimwipes) to clean lenses  Turning off the light source when no longer viewing a specimen  Always using a fastener or tie to secure the electrical cord (Never wrap the cord around the base of the microscope.)  Avoid water around the electrical outlet Parts of the Light Microscope 1

Base : This is the bottom portion of the microscope that is grasped when carrying the microscope. Arm : This is the handle of the microscope that is used when carrying. Eyepiece : This is the area where the eye is placed when viewing specimens. Body tube : This cylinder joins the eyepiece to the arm. It contains a lens that magnifies 10X. Stage : This is a flat area where slide is placed when viewing a specimen. There is an opening on the stage that allows light to pass through the specimen. Stage bracket : This is a contraption that holds the slide on the stage when viewing specimens. Stage movement knobs : Some microscopes have knobs that can move the slide around on the stage. Light source : This supplies light for viewing. It is turned on and off by the power switch located on the base. Condenser : This is a lens beneath the stage that bends light rays on to the specimen. Diaphragm : This part controls the amount of light passing through the specimen through the opening on the stage. It is adjusted by a lever that sticks out from beneath the stage. Revolving nosepiece : This is a textured knob that allows the user to switch objectives so that different magnifications can be obtained. Scanning objective : This is a cylinder that contains a lens that magnifies 4X; in fact, the number 4 is etched on the objective. This is the shortest objective. Low power objective : This is a cylinder that contains a lens that magnifies 10X; in fact, the number 10 is etched on the objective. High power objective : This is a cylinder that contains a lens that magnifies 40X; in fact, the number 40 is etched on the objective. Oil immersion objective : This is a cylinder that contains a lens that magnifies 100X; in fact, the number 100 is etched on the objective. We have no need to use this lens for our activities. This is the longest of the objectives. Coarse adjustment knob : This is a knob on the arm of the microscope that is used to focus an image when the scanning or low power objective is being used. Fine adjustment knob : This is a knob on the arm of the microscope that is used to focus an image when the high power or oil immersion lens objective is being used. 4. Label the following parts on the figure below : Base, Arm, Eyepiece, Body tube, Stage, Stage bracket , Light source, Condenser, Diaphragm lever, Revolving nosepiece, Objectives, Coarse adjustment knob, Fine adjustment knob, and, if present, the Stage movement knobs. 2

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How a microscope works Now that you are somewhat familiar the parts, let’s examine how the light microscope works. Light from the base of the microscope is projected upward. The condenser lens refracts (bends) the light on to the specimen. As light travels upward through the objectives and body tube to the eyepiece, it is bent by additional lenses (a lens in the objective and a lens in the eyepiece), until an image forms on the retina of the eye. Magnification The magnification power (how many times an object is magnified) is determined by the specific magnification of the objective lens, as written on the objective, and by the magnification of the eyepiece found in the body tube. Total magnification is the mathematical product of magnification of the eyepiece (10X) multiplied by the magnification of the objective that is being used. Fill in the following table: The first row has been completed for you. Complete the remaining portions. Remember the magnification of an objective appears on the side of the objective. Lens Eyepiece Magnificatio n Lens Magnification Calculate Total Magnification Which adjustment knob is used with this lens? Scanning 10X 4X 10X * 4X= 40X Coarse adjustment knob Low power 10X High power 10X Oil immersion 10X Steps for Getting a Focused Image 4

2. The scanning power objective should be clicked into place and the light turned on LOW. 3. The microscope should be plugged in and turned on. 4. The slide is placed on the stage in the stage bracket. 5. While looking from the side of the microscope, move the stage up, as close as possible to the objective by manipulating the coarse adjustment knob. BE SURE THE SCANNING POWER OBJECTIVE IS IN PLACE. 6. Line the specimen up with the light coming through the stage (center the specimen in the light). 7. While looking through the eyepiece, use the coarse adjustment knob to slowly move the stage AWAY from the objective until the specimen comes into focus (though the focus may NOT be clear). 8. Use the fine adjustment knob to sharpen the focus on the specimen. 9. Move the specimen until what you want to see is in the center of the field. 10.When the specimen is in focus at scanning power, the low power objective may be clicked into place. Since a microscope is parfocal , it is designed to be more or less in focus when switching from one objective to another. Adjust the focus using the fine adjustment knob. Though the coarse adjustment knob may still be used at low power, its use should NOT be necessary. NEVER USE THE COARSE ADJUSTMENT KNOB WHEN THE MEDIUM OR OIL OBJECTIVES ARE IN PLACE. 11.You may want to adjust the light by manipulating the diaphragm lever and/or turning the knob on the base to make the light source brighter or darker to better see the specimen. In general, use the least amount of light to see the specimen clearly. 12.If a higher magnification is needed, the high power objective is clicked into place. 13.The image is focused ONLY with the fine adjustment knob. The parfocal nature of the microscope still applies when switching from the low power to the high power objective. 14.The light should be adjusted as necessary. Generally, use the least amount of light that still allows you to see the specimen clearly. 15.The last objective is an oil immersion objective. The instructor will provide directions for using the oil immersion lens, when it is needed. Activities with Prepared Slides To get accustomed to focusing an image, the following prepared slides should be viewed using the steps above.  The letter “e”  Micrometer slide and “Field of View” The Letter “e” Let’s first look at the letter “e” using the scanning objective; total magnification of 40X. A. Obtain a slide with the letter “e” on it and follow the steps above to get the “e” into focus. B. Place the letter “e” so that you can read it, right side up, when viewed without the microscope. C. Look from the side of the microscope and move each stage adjustment knob and observe what happens to the stage. a. When you move the top knob, which direction does the stage move? 5

b. When you move the bottom knob, which direction does the stage move? D. Now look under the microscope. a. When the stage moves to the right, which direction does the “e” move? b. When the stage moves up, which direction does the “e” move? E. How do you think the stage movement knobs will help when viewing specimens? F. View the letter “e” at a total magnification of 40X, 100X, & 400X. Total magnification: ________ Total magnification: _______ Total magnification: _______ Objective used: ____________ Objective used: ___________ Objective used: ___________ Which objective allows you to see the “e” best? What happens to the field of view (how much of the “e” you get to see), when you move to higher powers? When viewing the letter “e,” how does the orientation of the image compare to its orientation with the naked eye? Look back at the figure explaining how a microscope works (above). Note how the light moves as the light passes through the lens? How does this explain what you saw when you looked at the “e” under the microscope? Micrometer slide and “Field of View” 6

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Earlier, when we looked at the letter “e, did you notice that less of this letter was visible when you moved to a higher power? This change was due to a decrease in the field of view. Now, we will measure that difference. A. The instructor will have prepared slides with a small straight edge (ruler). Place this slide on the stage. As always, start at scanning power and adjust the focus as described above. B. Looking through the objective, move the stage so that one of the large markings on the ruler is at the far left of the image and the straight edge of the ruler is running left to right at the widest part of the circle of light in the objective (see figure at right). Note the diameter of the field of view and record it in the chart below. C. Move progressively up to higher powers and repeat this procedure, each time noting the diameter of the field of view. Diameter of the field Scanning (40X) Low Power (100X) High Power (400X) As you progressed from low to high power what happened to the field of view? Why do you think we ALWAYS start to look at a specimen under scanning power before proceeding to a higher power? (Hint: the depth of focus is very small at high magnification). The Cell 7

Microscopes provide us with the ability to see what is invisible to the naked eye, although crucially important for life. Early scientists who first discovered cells with the aid of the light microscope formulated observations which led to the cell theory . Cell Theory : 1. All organisms are composed of one or more cells 2. The cell is the basic structural and functional unit of all living organisms 3. Cells arise only from the division of preexisting cells Whether a cell falls under the categorization of a eukaryotic cell (true nucleus) or a prokaryotic cell (void of a true nucleus, contains a nucleoid region) the cell theory applies. There are many differences between eukaryotic and prokaryotic cells, mainly eukaryotic cells containing a true-membrane bound nucleus, other membrane bound organelles (e.g. chloroplasts, mitochondria, Golgi, etc), size (eukaryotes are typically larger), and complexity (eukaryotes more complex). There are similarities between eukaryotic and prokaryotic cells (some of which relate back to the cell theory). The following are structures that are shared characteristics: Plasma membrane : phospholipid bilayer embedded with proteins that surround cells and creates intracellular environments that are unique compared to the extracellular environment. Cytoplasm : aqueous/gel-like internal region of the cell which contains all the cellular organelles DNA : the genetic material of life. While eukaryotic cells contain DNA in a true nucleus and prokaryotes in a nucleoid region, they both have DNA. Ribosomes : a non-membrane bound organelle that processes genetic information from mRNA to synthesize proteins. This is the only organelle that is shared between eukaryotes and prokaryotes. While discussing the differences between prokaryotic and eukaryotic cells, it is important to take note that there are many different classes of these cells. For example, not all eukaryotic cells are alike. A cell could be a eukaryotic plant cell or eukaryotic animal cell. Both are eukaryotes meaning that they have true nuclei, but they both have differences that make them distinct. The following outline the structural/cellular differences between plant and animal cells: Plants : Animals : Cell walls lysosomes (rare in plants) Plasmodesmata centrioles Chloroplasts cilia are also very rare for plants Central vacuole Fixed Shape/Rectangular Irregular Shape/Round Cell Organelles 8

In the following diagrams, identify each type as prokaryotic or eukaryotic, then label each of the following cell organelles (not all cells will have all of these parts: nucleus , nucleoid , ribosomes , golgi apparatus , smooth endoplasmic reticulum , rough endoplasmic reticulum , vacuole , chloroplasts , mitochondria , lysosomes , cell wall , cell membrane , vesicle, cilia, flagella Activities with slides We will be using prepared slides to view a prokaryotic cell A slide is prepared by making a wet mount of a specimen. Two wet mount specimens will be prepared:  Elodea, a freshwater plant  Human cheek cells Bacteria prepared slides 9

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A. Obtain a prepared bacterial slide that may contain different morphological types – you may see Bacillus , Streptococcus , Staphylococcus , etc. B. Observe two different strains of bacteria. C. Make a drawing of each morphological type under high power. Elodea wet mount The steps below should be followed to prepare a wet mount of Elodea . No stain is required because the organism contains a pigment that makes it easy to observe. A. In the center of a clean slide, a drop of water is placed using a plastic pipette. The water puddle will spread out slightly on the slide forming a “little puddle.” B. A single leaf of Elodea is placed in the little puddle of water. C. A cover slip is placed over the specimen by gently lowering one edge of a cover slip at an angle touching the slide at one edge of the water drop. Draw the cover slip slightly toward the leaf and the held edge of the cover slip will be lowered, so that the air is pushed out from under the cover slip. The instructor can demonstrate the technique. D. Follow the steps for getting an image in focus (above) and increase the magnification to 400X. E. Draw and label what you see of the Elodea leaf at 100X and 400X. Use your text book / online references to answer these questions. 10 Organism Viewed: _______________ Magnification: __________________ Organism Viewed: _______________ Magnification: __________________ Organism Viewed: _______________ Magnification: __________________ Organism Viewed: _______________ Magnification: __________________

What are the big rectangles edged by a clear (like cellophane) border? What are the green circles within the big rectangles? Can you see a nucleus in any of the cells? What do they look like? Cheek cells wet mount Human Epithelial cells are sloughed off from the inner surface of your mouth. They are flat cells with a readily visible nucleus. A. Gently scrape the inside of your cheek with the broad end of a clean toothpick. B. Spread the cheek cells onto a clean microscope slide and place a cover slip on top of the cell spread. Add one drop of methylene blue to one side of the cover slip and watch the blue dye wick across the slide to stain your cells blue. Major organelles that you may see include the nucleus and cell membrane. C. Draw what you see at two magnifications. Use the dimensions of the Field of View diameter calculated earlier to approximate the size of a human epithelial cells. What organelles could you see in each specimen? Elodea:____________________________ Cheek cells: ________________________ 11 Organism Viewed:_______________ Magnification: __________________ Organism Viewed:_______________ Magnification: __________________

Write a summary paragraph comparing and contrasting what you saw in each of the three cell types (bacterial, animal, and plant). Comment on things like size, shapes, what magnification could you see the organisms at, etc. 12

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Lab 5 - Cells

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