Week 1 Lab Handout1 (1)

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Purdue University *

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135

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Biology

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Feb 20, 2024

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Week 1: First Week in Lab Learning Goal: 1. I can demonstrate the proper technique of a compound light microscope by correctly setting up and focusing on a specimen. (Essential skill for future courses like microbiology and future biology-related careers) Overview: (1) Get to know your teaching team and peers. Complete the Padlet introducing yourself if you have yet to. (2) Introduction to lab safety and equipment tour. (3) Activity: See below material. Comparison of stomata across leaf areas and then use bar graphs of your data of stomatal counts and a compilation of the classes’ data to analyze the differences. Lab Prep: To prepare for the lab: (1) Read this handout in full before lab. (2) Complete the Padlet activity. (3) Download Excel to your computer from: https://www.itap.purdue.edu/services/microsoft- office-365.html You will need the full version of Excel downloaded to your laptop computer, unfortunately Microsoft 365 online does not support some of the graph design settings that we will use. Background: Information needed on how to use your microscope: Ocular Eyepiece Nosepiece Objective Lens Stage Clip Stage Stage Control Knob Light Intensity Knob Power Switch Course Focus Knob Fine Focus Knob Iris Diaphragm Digital Camera Switch between Digital Camera and Ocular Eyepiece Light Source Figure 1. Labelled diagrams of a common compound light microscope that we will be using in lab. Familiarize yourself with the parts, their names, and their locations on the microscope. In the biological sciences, the compound light microscope (Figure 1) is a useful tool for studying small specimens (object under the microscope) that are not visible to the naked eye. The microscope uses bright light to illuminate through the specimen and provides an inverted image at high magnification and resolution. There are two lenses that magnify the image of the specimen – the objective lens on the 1

nosepiece and the ocular eyepiece . To determine the total magnification of the specimen, you must multiply the objective lens magnification with the ocular lens magnification. The ocular lens has a magnification of 10X, and the two objective lens have magnifications of 4X and 10X respectively. Therefore, if the ocular lens has a magnification of 10X and the 4X objective lens is in place in the light path, then the specimen is being magnified 40X (or appears 40 times larger than without magnification). Question: If you have the 10X objective lens in the light path, then the specimen is being magnified _____________ the ability of your eyes. Fill in the blank with what you believe is the total magnification before continuing. Total Magnification: 100X Compound light microscopes are valuable tools in the lab. They magnify our ability to see in detail by up to 1,000 times (depending upon the objective lens in the light path), allowing us to study things as small as the nucleus of a cell. With them, we can determine the shape and structure of cells, observe the movements of microorganisms, and examine the smallest parts of plants, animals and fungi. Question: Before continuing, practice with your lab peers by pointing to a part of the microscope and asking the team to identify the name and what it does to the microscope. Try to complete 3 parts for each peer. It is okay if you do not know these or get this correct, we are just practicing. We want you to become familiar with the parts of the microscope so that you are better able to talk about using the microscope. The locations of the parts of the microscope are listed in Figure 1 and the tasks of some key parts are outlined below. Part 1 Identified: Course Focus Knob Part 1 Function: To adjust the focus for the slide being viewed Part 2 Identified: Ocular eyepiece Part 2 Function: the part of the microscope which allow you to see the specimen, it has a 10X magnification Part 3 Identified: stage clip Part 3 Function: secures the slide to the stage for viewing Digital Camera – see separate handout on how to connect your phone or laptop to this camera to take images via Bluetooth (optional). Ocular Eyepiece – provides 10X magnification and allows you to adjust for your eyes so that you can see the specimen; adjustable to fit each person’s unique distance between their eyes; needs to be adjusted prior to use. Switch between Digital Camera and Ocular Eyepiece - switches the light path between the two viewers (push in or pull out to change between the two). Nosepiece – provides a place to hold and rotate from one objective lens to the next to change the magnification level. Objective Lens – when rotating the nosepiece, the objective lens that is in the light path (facing down) will determine the total magnification (ocular eyepiece 10X magnification multiplied by the objective lens magnification = total magnification). Used to magnify very small things. There are two objective lenses (one is 4X and is small with a red stripe, the other is 10X and is slightly larger with a yellow stripe). Stage Clip – secures the slide with your specimen to the stage so that the slide does not move. Stage – the flat surface that can be adjusted to move the specimen closer or further from the objective lens to help focus the specimen. 2

Course Focus Knob – a knob that can be turned to adjust the stage closer or further from the objective lens to help focus the specimen (careful: only use this when the 4X objective lens is in the light path). Fine Focus Knob – a knob that can be turned to adjust the stage closer or further from the objective lens to help focus the specimen in fine detail. Stage Control Knob – a bidirectional knob that can move the stage clip and thus the slide and specimen to line up better in the light path so that you can see the specimen, can move the specimen in all directions (North/South/East/West). Iris Diaphragm – can be used to adjust the contrast of some specimen. Light Source – where the light will shine up through the microscope through the objective lens and to the ocular eyepiece, the light intensity can be adjusted on the base via the Light Intensity Knob. Technique: Learning to use the microscope: Before we get our leaf sample today, please follow directions from your TA and the video on how to use the microscope and then practice using the microscope with a “letter e slide” as your first specimen. (If the “letter e slide” sounds too simplistic for you, feel free to ask your TA if you could have an onion root tip slide or various anatomy slides.) Check out this hyperlinked video walking you through how to these microscopes in BIOL 135. How to use the Biol 135 microscope Note: Throughout this course, the column of boxes on the left in all the technique protocols is for you to check off the steps as you do them. This will help you keep track of where you are in your experiments. The middle column is the step-by-step protocol. The column to the right is where you can add observations, notes, and answer questions that are being asked. Check (X) Protocol Questions to Answer and Notes (very important section for observations and notes) x 1. Plug the microscope in. x 2. Turn on the power switch on the right side of the base so that the light on the microscope turns on. (If it looks too dark or too bright, you can adjust the amount of light at the light source by adjusting the light intensity knob on the right side of the base.) x 3. Align the 4X objective lens so that it is directly over the middle of the stage in the light path by rotating the nosepiece. You should hear a clicking sound once it is aligned. Why must you use only the smallest (4X) objective lens before adding your slide? So there is enough space between the stage and the objective lens. It also aids in allowing the 10X to be better focused to the slide. What could happen if there is not enough space between the objective lens and the slide? If there is not enough space the objective lens could shatter the slide. x 4. Move the stage to lowest setting using the course focus 3

knob (larger inner knob on the left side). x 5. Place your slide (with the coverslip and/or specimen face up) on stage and secure with slide clip (careful slides can crack). x 6. Adjust the ocular eyepiece so that your eyes are comfortable. x 7. Adjust focus with coarse focus knob by bringing the stage to the highest position (without touching the slide), and then slowly move it down until the sample is visible. x 8. Further sharpen focus with fine focus knob (outer knob on left side) until the image is clear, if the image is not getting clear, go back a few steps and double check these steps. x 9. The specimen should be in focus at 40X total magnification (4X objective lens X 10X ocular eyepiece). Write down your observations and/or draw an image of what you see under the microscope. Observations: The image is upside down and it is spotty Drawing: (TA said description will suffice) Compare the image under the microscope to the letter without the microscope. What do you see? Has anything happened unexpected? The image is right side up again and much more clear. x 10. Increase magnification to 10X objective lens by rotating the nosepiece so that the 10X objective lens is in the light path. x 11. Now only use the fine focus knob to adjust until the image is clear. Careful: Often when you zoom in on a specimen by increasing the magnification you might zoom in on a place where there is no specimen. It is not your eyes that cannot see, it is the fact that you zoomed in on a specific location where there is no specimen. In this case, use the stage control knob to adjust so that the specimen comes into view. Then adjust ever so little the fine focus knob. x 12. Write down your observations and/or draw an image of what you see with the 10X objective lens. Observations: The image is much closer and is super spotty Drawing: x 13. Once finished, adjust the nosepiece again so that the 4

4X objective lens is in the light path. Then use the course focus knob to lower the stage. x 14. Remove your slide carefully by releasing the stage clip carefully. x 15. When finished, turn off the power switch (careful the light bulb can get hot when on for a long time) Tip: Only increase in magnification once you have located and focused your specimen: if you can’t find it at 4X, you won’t be able to find it at 10X. We won’t be using higher powered objective lens in this course, but you will in future courses. Background: Information on plants (the specimen under the microscope today will be plant leaves) All life requires energy to survive and grow. Plants are photosynthetic autotrophs, meaning they synthesize molecules to produce and store their own energy, a process that produces glucose. Glucose is an important biological molecule because of its ability to store large amounts of energy. Through a series of chemical reactions, carbon dioxide, water, and light are brought together to produce glucose and oxygen. Below is the overall equation for photosynthesis: 6 CO 2 + 6 H 2 O -----> C 6 H 12 O 6 + 6 O 2 Formula showing carbon dioxide and water yields glucose and oxygen in the presence of light. There are many elements required for this chemical reaction to take place successfully. In lab this week, we are going to explore the specialized cells that manage the movement of carbon dioxide and water in the plant. Stomata and Guard Cells Two necessary ingredients for photosynthesis are carbon dioxide and water. Plants acquire water through a passive process called osmosis, after which it is transported to the leaves for photosynthesis. The transportation process occurs through a combination of cohesion (when water molecules cling to each other through hydrogen bonds) and adhesion (where water molecules are also attracted the walls of the vessel that is holding them). Water molecules climb up, against gravity, through the xylem tubes and into the leaves. Finally, the water molecules not used in photosynthesis exit the plant through transpiration, or the exhalation of water vapour from the plants. The cohesions between the water molecules continues to pull more water molecules up from the roots and into the leaves. The rate of water movement in a plant depends on how much water is available in the environment. In environments with plenty of water, the movement of water through the plant can occur almost continuously. In contrast, when limited amounts of water are available, plants must ensure that water is available in leaves for photosynthesis and not lost through transpiration. It is therefore very important for plants to control the amount of transpiration that occurs. 5

Figure 2. A leaf cross section, showing the movement of carbon dioxide and water through the stoma found at the bottom of the image. It is important to note that a quick internet or AI search would tell you that stoma are found on both sides of a leaf but not always in equal numbers. The interior of leaves is in communication with the atmosphere through pores called stomata. A single pore, a stoma, is created by the gap between two specialized epithelial cells called guard cells (Figure 2). The stomata allow the evaporated water to escape and allows carbon dioxide to enter, which is also necessary for photosynthesis. The amount of water loss is regulated by changing the size of the guard cells (Figure 3). Stomatal opening is also related to the amount of atmospheric carbon dioxide. While plant leaves contain many air spaces (the space between the cells in the spongy mesophyll layer, Figure 2), the opening and closing also relates to the concentration of carbon dioxide inside the leaf. As both atmospheric carbon dioxide and temperature increase, as expected with climate change, plants must strike a balance between water loss and carbon dioxide entry. Figure 3. A comparison of open and closed stomata based on the shape of the guard cells. Activity: Estimating stomatal numbers across leaf surfaces In this activity, you will be formulating and testing a hypothesis regarding the number of stomata on leaves. In general, you form a hypothesis when you use previous data or information to make predictions about how the new experimental data will turn out. In this case, you will examine the epidermal layer of a leaf under the microscope and count the number of stomata. The goal of this experiment is to address the question: Will there be more stomata on the top or bottom of a leaf? Consider what you know about the function of stomata (in background information above). There is no 6

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