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Biology 1001 Laboratory 4

MICROSCOPES, MEASUREMENTS AND CELLS PREPARATION - Read this exercise before you come to the laboratory. - Search the internet for images of Elodea cells, cyclosis in Elodea cells and human cheek cells. OBJECTIVES At the end of this lab you should be able to: 1. Give the name and function of parts of the compound microscope. 2. Align the light source of a compound microscope to give optimum illumination of a specimen at all magnifications. 3. Find a specific part of a specimen under low power and change to high power without disrupting the field of view. 4. Determine the three-dimensional shape of objects viewed with a microscope. 5. Accurately determine the size of a microscopic specimen. 6. Prepare a temporary whole mount. 7. Prepare a stained whole mount. 8. Make a proper biological drawing of a microscopic specimen. LABORATORY ASSIGNMENTS 1. Set up a compound microscope for use. 2. Answer the questions on the lab assignment sheet. 3. Fully label a drawing of a cell from an Elodea leaf. 4. Make a fully labeled drawing of epithelial cheek cells. (you must pass in this drawing for marking) 5. Upon completion of the lab, have a demonstrator check that you have properly cleaned your microscope and set it up for storage.

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THE LIGHT MICROSCOPE INTRODUCTION The light microscope is an indispensable tool of a working biologist. Its proper care and use is essential for success in Biology 1001. Even if you have used a microscope before, do not omit this lesson. This will be a good opportunity to review, and at the same time, to become familiar with the workings and quirks of the microscopes in this lab. Most people, even those who use a microscope daily, tend to become negligent and forgetful of the best procedures. IF AT ANY TIME YOUR MICROSCOPE DOES NOT SEEM TO BE WORKING PROPERLY, CALL AN INSTRUCTOR. DO NOT ATTEMPT TO REPAIR IT YOURSELF. This lab is divided into two parts. First, you will learn about the parts of the compound microscope and their functions, and you will learn how to properly set up and care for these delicate and expensive instruments. In the second part of this lab, you will use your new microscope skills to examine living cells. You will be guided through this lab by doing the TASKS and LAB ASSIGNMENTS in order. There are 8 TASKS and 11 ASSIGNMENTS to be completed. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - PART I A. THE PARTS OF THE COMPOUND MICROSCOPE There is a microscope that corresponds to your seat number, either on the bench top or stored in the under-bench cupboard. USE BOTH HANDS TO HOLD AND CARRY IT - one hand under the base, the other holding the arm. Put the microscope on your bench directly in front of you, so that when you are seated, you can look into it without twisting or straining. Read the following descriptions of the parts of the microscope and find these parts on your own scope. Refer to Figure 4.1. Compound microscopes were designed to observe fine details of specimens. Most compound microscopes use transmitted light. The light usually originates in a source in the base of the microscope. The light must be concentrated and organized into coherent, parallel beams before it can be used. A condenser below the specimen stage of the microscope does this job. These beams must be focused on the specimen. Thus the condenser is attached to the microscope by a rack and pinion device, so it can be moved up and down for proper focus. As the light reaches the specimen, some of it is absorbed by dense or coloured areas; some of it may pass undisturbed through clear areas of the specimen and some of it may be refracted (bent) by dense areas of the specimen. Thus we can perceive differences in a specimen.

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Eventually the light after passing through the specimen enters the objective lens and the image is magnified. The image is magnified again by the ocular lens at the top of the body tube. (See Figure 4.1). Make sure you can find and name the following parts of the microscope and can give their functions: Ocular (lens) - Often called the eyepiece. It is the topmost series of lenses through which an object is viewed. Its magnification is ten times (10x). This lens is unattached and will slip out of the body tube unless the microscope is kept upright. Body tube - Joins the nosepiece to the ocular lens. Arm - Supports the body tube and provides a carrying handle.

Figure 4.1 The Olympus compound microscope. Nose piece - The revolving part to which the objective lenses are attached. It must be firmly clicked into position when the objective lenses are changed. Objective lenses - There are four objective lenses on the nose piece of your microscope. The magnifying power of each is marked on side of the lens. Notice that when an object is in focus under the low-power objective it is also in focus under the high-power objective as well and vice-versa. In other words, these objectives are said to be parfocal.

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Scanning Objective - This objective has a wide field of view at relatively low magnification, and is used to get a general view of a specimen on a slide, or to examine at low magnification an object too large to be seen in its entirety with the other objectives. The magnification given by the scanning objective is 4x.

Low-Power Objective - The low power objective is the normal working objective of the

microscopes. Its magnifying power is usually 10x.

High Power Objective - The high-power objective is to be used when the magnification of the low-power objective is not adequate. When preparing to use the high-power objective, always bring the specimen into focus with the low-power objective first, then turn the nose piece to bring the high-power objective into line with the body tube of the microscope. The magnifying power of the high-power objective is usually 40x. USE ONLY THE FINE ADJUSTMENT KNOB FOR FOCUSING WHEN THE HIGH-POWER OBJECTIVE IS IN THIS POSITION: DO NOT TOUCH THE COARSE ADJUSTMENT KNOB which might cause you to CRUSH THE MICROSCOPE SLIDE.

Oil Immersion Lens - The magnifying power of this lens is usually 100x. NEVER, UNDER ANY CIRCUMSTANCES, USE THIS LENS WITHOUT THE APPROPRIATE IMMERSION OIL.

Slide holder - Holds the slide firmly in place. Control knobs on this mechanical stage allow you to move the slide back and forth, and from side to side. Stage - Supports the slide that is held onto it by the slide holder. Has a hole so the light can shine up through the specimen. Condenser - A lens located under the stage which focuses and concentrates the light before it passes through the specimen. Its position is controlled by a knob. Iris Diaphragm - Controls how much light and lamp heat go through a specimen. It is controlled by a lever on the side of the condenser. Coarse Focus Adjustment - Moves the stage up and down to bring the object into approximate focus; used only with scanning and low-power objectives. Fine Focus Adjustment - Moves the stage up and down very slightly to bring the object into precise focus. The only adjustment used with high-power and oil immersion objective. Light source - An attached lamp that directs a beam of light up through the object. Base - Gives the microscope a firm, steady support. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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B. CARE OF A MICROSCOPE - ALWAYS carry a microscope in BOTH HANDS. One hand holds the arm securely while the other hand is under the base to support it. Do not tilt the microscope - carry it in an UPRIGHT position. - Check over your microscope each time you use it. You share this scope with other students. If something is wrong, please report it. Call an instructor. DO NOT ATTEMPT TO FIX IT YOURSELF. - If you must wear mascara, please clean the oculars when you are finished. - Don't touch the lenses with your fingers. Oily fingerprints attract dust and are hard to clean off. - The lenses should ONLY be cleaned with FRESH CLEANING TISSUE and LENS CLEANING SOLUTION or water. These are found in each student bench. Use gentle pressure when cleaning a lens; the glass is soft and easily scratched. For this reason, also, you should never use a dry tissue on the lens, there should always be a liquid with the tissue. WHEN YOU ARE FINISHED with the microscope, BE SURE that: 1. The shortest objective lens is in line with the body tube. 2. No slide is left on the stage.

3. The microscope is clean - no water or dust on the stage or lenses. Make a routine habit of cleaning both the lenses and the stage of your scope after each use.

4. The cord is coiled around the base to avoid entanglement in the cabinet. Make sure the cord is not pinched under the condenser gears.

5. Replace the dust cover and return the microscope to where you found it. Your microscope will be checked after each lab session to ensure that it has been put away properly. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

* * * * * TASK 1 * * * * ** * * * * TASK 1 * * * * * C. SETTING UP THE OLYMPUS MICROSCOPE This microscope (Figure 4.2) enables you to observe fine details of very small or thinly sectioned subjects in transmitted light. The specimen must be properly illuminated in order to see . Here is the procedure for setting up the illumination of the microscope. It must be followed each time you begin to use the microscope. 1. Plug the microscope into the electrical outlet on your lab bench. 2. Turn on the microscope light using the on/off switch on the base.

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3. Put the scanning objective lens (4x) into working position. 4. Using the light intensity lever, increase the light intensity until a reasonable amount of light is visible when you are looking in the oculars. 5. Place slide 81 (cross section of corn stem) on the stage and use the arms of the slide holder to grip the slide. Use the slide holder controls to centre the specimen over the hole in the stage. (This must be done before any further adjustments are made to insure the correct position of the light at the level of the specimen. 6. Look through the right ocular and use the coarse focus knob to carefully move the stage up until the specimen can be seen. 7. While looking through the eyepieces with both eyes, hold the right and left eyepiece tubes

with both hands and push the tubes together, or pull them apart laterally, whichever is required, until the two circles of light merge into one and you obtain binocular vision. You should be able to see down both tubes at the same time.

8. Look at the image through the right eyepiece with your right eye, and focus on the specimen with the focus adjustment knobs. 9. Next, look at the specimen through the left eyepiece (with your left eye) and rotate the ring on the left eyepiece to focus the specimen without touching the coarse and fine focus knobs. 10. Using the condenser focus knob, put the substage condenser all the way up. 12. Remove one of the ocular lenses and look down the body tube to the circle of light. Use the iris diaphragm lever to adjust the circle of light until it fills about 3/4 of the field of view. Put the ocular lens back in. 13. The microscope is now ready for use. Each time you use the microscope, you should repeat this procedure. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 1 Have a demonstrator check your microscope to insure that you have set it up properly. If you have, the demonstrator will sign on the assignment page. If you haven't succeeded, try this exercise again. You will need to properly set up your microscope each time that you start to use it. With practice this will become second nature to you. Trying to use an improperly set up microscope is an exercise in frustration - not only will you be unable to see what you should - you may also develop headaches and eyestrain.

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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HINTS AND RULES FOR EXAMINING A MICROSCOPIC SPECIMEN 1. Examine only specimens on prepared slides, or on slides after mounting in liquid and covered by a cover slip. Before using a prepared slide, clean it with lens tissue and lens cleaner, if necessary. 2. Try to keep both eyes open; it lessens eye strain. 3. Always study the specimen first under the 10x objective lens. Centre the part you are interested in before switching to the 40x objective. 4. Use only the fine focus with the 40x objective.

5. Adjust the fine focus almost continuously when viewing a specimen in order to get a three-dimensional appreciation of the specimen. Even microscopic tissues have depth!

6. Don’t forget that you can change the intensity of illumination of the specimen by

adjusting the light intensity knob or the iris diaphragm lever. Hidden structures can be observed by examining a specimen under different light intensities!

7. If the image is weak or blurred, check the adjustment of the condenser, then look for dirt on eyepiece or objective. If this does not correct the trouble, call an instructor. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

* * * * * TASK 2 * * * * ** * * * * TASK 2 * * * * * D. USING THE COMPOUND MICROSCOPE Focusing - Low Power

1. Obtain slide #102B of the letter e from your slide box. Look at it with the slide label on the left and the cover slip uppermost. 2. Put the letter e slide on your microscope stage centering it over the hole in the stage. Make sure the scanning objective is in line with the body tube. Using the coarse focus knob put the stage all the way up. Now look in the microscope and, using the coarse focus knob, slowly increase the distance between the objective lens and the stage until the object, the letter e, comes into focus. 3. Move the slide away from you. In which direction did the e in the microscope move?

4. Now move the slide to your right. In which direction did the e in the microscope move?

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What you have just observed is called inversion. The image in the microscope is inverted, that is, it is upside down to the actual specimen. Also, the image in the microscope is reversed meaning that the actual right side of a specimen is viewed as the left side in the microscope. Keep in mind the fact that microscope images are inverted and reversed as you study various microscopic specimens during the year. Focusing - High Power The lenses in compound microscopes are parfocal, that is, if an object is in focus under low power, it should also be in focus under high power. To switch to higher power objective use the following procedure. 1. Make sure the object, in this case the letter e, is in the centre of the field and is in focus.

2. Rotate the nose piece so that the 10x objective, the low power objective, clicks into place in line with the body tube. If the objective has not clicked into place, you will

not be able to see down the body tube.

3. Locate the object in the field of view. Put the part you are interested in, in the centre of the field of view. 4. To switch to high power, the 40x objective, these steps are repeated. It is very important to remember that once you put the 40x objective in line with the body tube, USE ONLY THE FINE ADJUSTMENT KNOB. The distance between the slide and the objective lens is so small that you must be very careful or you will crack the slide and possibly damage the lens.

5. Removing a slide - When you are finished with your observations of this slide (or any slide), rotate nose piece back until the 4x objective clicks into place and only then remove the slide from the stage. If you try to remove a slide while the microscope is still on high power, you may scratch or damage to objective lens.

You should observe these steps each time you put a slide on the microscope, switch to high power, then switch back to low power and then remove the slide. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 2 Based on what you have done in Part D answer questions 2, 3 and 4 on the assignment pages. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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E. MAGNIFICATION The magnification of the image that you see through the microscope is given by the formula:

i.e. Using the low power objective, the total magnification is 100x = 10x (eyepiece) x 10x (low power objective). What is the total magnification of your microscope when the 40x objective is in use? Please note that this magnification will not be the magnification of a drawing that you make of an observed specimen. This will have to be calculated in another way, as explained in a later section of this lab. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * * * * * TASK 3 * * * * * * * * * * TASK 3 * * * * * F. DIAMETER OF FIELD OF VIEW 1. Obtain slide #85b, a stage micrometer slide containing part of a millimeter ruler from your slide box and put it on the stage so that the ruler is visible as a line along the diameter of the scanning power field. Estimate the number of millimeters that you can see in the diameter of the field when you are using the 4x lens. 2. Carefully change objectives to the 10x objective and again calculate the diameter of the field for low power. Is it the same as scanning power? 3. Carefully try to do the same thing for the 40x objective lens. Record these diameters below. Diameter of field for scanning power (4x) = _____________ mm Diameter of field for low power (10x) = ______________ mm Diameter of field for high power (40x) = _____________ mm - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 3 Based on what you have done in Part F answer questions 5 and 6 on the assignment pages.

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* * * * * TASK 4 * * * * ** * * * * TASK 4 * * * * * G. DEPTH OF FOCUS Lenses have a plane of focus. This is the thickness of the plane in which an object appears to be in focus. This is true for the lens of your eye as well as for the lens in the microscope. To illustrate this principle, extend your fist at arms length in front of you and hold your thumb up. Look at your thumb continuously while noticing that the objects past your thumb on the other side of the room are not clearly seen. Do not look at these objects past your thumb, but just concentrate on your thumb. You have focused the lens of your eye on your thumb and in order to see objects farther away it would be necessary to look away from your thumb - to refocus. We are so used to refocusing the lens of our eyes that we don't even think about it. Similarly with a microscope, when it is focused on one surface, the surfaces lower or higher will be out of focus and not seen so clearly. You will notice that the plane within which objects can be seen clearly will become narrower as the magnification of the microscope is increased. The distance within which objects can be seen clearly is called the depth of field or depth of focus. 1. From your slide box obtain slide #102A with three coloured threads mounted together. With the 4x objective, find a point where the threads cross. Slowly focus up and down. Notice that all three threads seem to be in focus. 2. Change to the 10x objective and use the fine focus to slowly focus up and down. Now when one thread is in focus, the others seem blurred. This is because the 10x objective lens has a smaller depth of field then the 4x lens. 3. If you are very, very careful, you could switch to the 40x objective. Remember to only use fine focus adjustment. The vertical distance that remains in focus at one time is called the depth of focus. Which appears to have a greater depth of focus, the 4x, 10x, or 40x objective lens? 4. Determine the order of the threads and decide which thread is on top and which is on the bottom. If you make constant use of the fine adjustment knob when viewing a slide with the 40x lens, you will get an idea of the specimen's three-dimensional form ____________________________________________________________ LABORATORY ASSIGNMENT 4 Answer question 7 on the assignment pages. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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* * * * * TASK 5 * * * * ** * * * * TASK 5 * * * * * H. MEASURING MICROSCOPIC SPECIMENS Since you know the diameter of each of the fields of view, you can make an educated guess about the size of an object you view in the microscope. (Is it half the size of the 10X field or does it fill the whole 40X field, etc.). However, it is much better to be able to measure the size of an object accurately. To measure microscopic objects, microscopists use a device called an ocular micrometer. You may already have noticed that when you look in your microscope, you can see what looks like a tiny ruler. This is the ocular micrometer that is in the ocular lens. When you rotate your eyepiece, this scale will also rotate. Notice that it is divided into 100 units and some of these are marked - 0, 10, 20, 30, etc. The whole scale is 100 ocular units. To be useful, the ocular scale has to be calibrated - that is, the exact size of one ocular unit must be measured at each different magnification. You will notice that as you change magnifications, the diameter of the field of view changes, but the ocular scale, because it is in the eyepiece, does not. This means that the length of one ocular unit is different for each of the magnifications. Here are the approximate values for your microscopes. (You will learn to calibrate your microscope in Biology 1501, next term) Please note that 1 micron = 1/1000mm. CALIBRATION FOR OLYMPUS SCOPES: Objective Calibration 4X 1 ocular unit (o.u.) = 0.025 mm = 25 µm 10X 1 o.u. = 0.010 mm = 10 µm 40X 1 o.u. = 0.0025 mm = 2.5 µm 100x 1 o.u. = 0.001 mm = 1 m 1 micron = 1/1000mm (the micron is very useful for measuring very small things like cells) Each time you want to know the size of a microscopic specimen, you can calculate it by measuring how many ocular units it is and knowing what objective you are using. For example, if your specimen measured 22 ocular units with the 10X objective you can calculate its size to be 22 o.u. x 0.01 mm = 0.22 mm or 220 m.

Mark this page of measurements in your lab manual. You will need to quickly refer to it each time you draw microscopic specimens.

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Depth can be measured if the fine focus knob on your microscope has a micrometer etched on it. If so, by focusing from one level to another, depth can be determined directly from the focus micrometer. LABORATORY ASSIGNMENT 5 Measure the width (diameter) of one of the threads on the crossed thread slide using the scanning objective and the low power objective lenses. Answer question 8 on the assignment pages. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I. PREPARATION OF MICROSCOPE SLIDES Slides may be either temporary (usually made with water), or permanent (made with a mounting medium that lasts for many years). Preparation of Temporary Wet Mounts In the Biology l001 laboratory, washed glass microscope slides are provided for you. They may be found in the glass jar on the front or side benches. Make sure the slide is perfectly clean. If the slide is badly scratched, throw it out in the SHARPS container and take another. Wipe carefully with paper towel. Make sure the slide is perfectly dry. When you are finished with this slide, you must clean it and return it to the jar. You will also need a cover slip. Boxes of new cover slips may be found in each lab bench cupboard. Cover slips are only used once, then discarded in the SHARPS container for safety (never in the general garbage containers). - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

First, one edge of the coverslip contacts the drop of water, then the coverslip is gently lowered to expel the air. Figure 4.2 Preparation of a temporary wet mount slide.

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* * * * * TASK 6 * * * * ** * * * * TASK 6 * * * * * J. UNSTAINED WHOLE MOUNT – Elodea (pond weed) 1. Put a drop of water in the centre of a clean slide. 2. Transfer one whole Elodea leaf to the drop of water, making sure that it is wetted in the drop. 3. Apply a second drop of water over the specimen. 4. Holding the cover slip by its edges, lower it so that one edge contacts the drop of water first, then lower the rest of the cover slip carefully so that all he air is expelled. It is often convenient to support the upper edge of the cover slip on the point of your tweezers or a pin as it is lowered into place. See Figure 4.2. The amount of water you use is critical. Too little water will not support and float the cover slip. Too much water means that your specimen will not stay in your field of view and you will get the microscope stage wet. Do not squash down on the cover slip, it needs to float on the drop of water. If you got large air bubbles under the cover slip that obscure the specimen, make a new preparation. 5. Place your slide of the Elodea leaf on the microscope stage. Centre the specimen over the light source. Bring it into focus with the scanning objective (4x objective).

6. Examine the specimen carefully by moving the slide around to see what is there. This is called scanning the slide. Can you see the individual cells? Using what you know about the diameter of your field of view, estimate the length and width of your leaf. 7. Now choose one area of the specimen (the leaf) and increase the magnification by

changing to the 10x objective. Look carefully over all of the leaf, both at the edges and in the centre. You should be able to see the cells more clearly. If it seems too dark, you can increase the light to the specimen by opening the iris diaphragm a bit more.

Can you figure out how many layers of cells there are? (Hint, use the fine focus knob to focus up and down through the leaf, a new cell layer comes into view when the cell boundaries seem to suddenly shift). You might want to increase the magnification even more by going up to the 40X objective lens. Do this carefully; as long as the cells are in focus with the 10X objective lens, then you will be able to swing the high power objective into place without trouble. Remember, the distance between the slide and the 40x objective lens is very small - use the fine focus only!

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Are all the cells of the leaf the same shape or are there differences? Locate the cell walls. Inside the cells will be cytoplasm, a granular material containing cell organelles such as chloroplasts. Cytoplasm in mature plant cells is often displaced to the perimeter of the cell by the large central vacuole. The vacuole itself is hard to see in these live cells, it looks like a large empty space. Each of the cells will also have a nucleus, seen as a clear rounded area in the cell, although many of these may be hard to see. Just inside the cell will also be a cell membrane (also called the plasma membrane), although this is difficult to see. Try looking closely at the corner of the cell. [Demonstrations - Cell membranes may be difficult to see, especially in live whole mount preparations. To help, we have prepared a slide of onion skin cells that are in a saline solution. A saline solution draws water out of the cell, causing the cell membranes to pull away from the cell walls. This should make the membranes more visible. Also, a slide showing stained Elodea nuclei may also be seen on the demonstration bench] Do all the cells of the Elodea leaf contain green chloroplasts? Which ones do and which ones don't? Can you see any movement of the chloroplasts within any of the cells? Be patient; you should be able to. Such movement of chloroplasts around the interior of the cell gives evidence that the cytoplasm within a cell circulates. This cytoplasmic movement is termed cyclosis. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 6 Answer questions 9 and 10 on the assignment pages - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 7 Now that you know how to measure cells in the microscope, fill in the “Cell Measures” portion of the assignment pages by measuring the length and width of one cell from the middle rib of the leaf. Measure the length and width of one cell from the blade (i.e. neither the edge cells nor the midrib cells). Which type of cell is longer? Which type of cell is wider? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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LABORATORY ASSIGNMENT 8 Label the drawing of an Elodea cell, Figure 4.3. Since this is a drawing of a plant cell, double lines are used to demonstrate the cell wall. Also, it is not a solitary cell existing by itself, it is attached to neighbouring cells. These attachments are indicated in the drawing. Label cell wall, cytoplasm, nucleus, cell membrane, chloroplasts, vacuole. Review the instructions for biological drawings, items 8 through 13 on pages A-8 and 9. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

* * * * * TASK 7 * * * * * * * * * * TASK 7 * * * * * K. STAINED WHOLE MOUNT - Human Epithelial Cells Staining The cells from the Elodea leaf are easy to see in a microscope for two reasons. First, they contain many small green chloroplasts which are very visible. Secondly, being plant cells they have a thick, rigid cell wall made of cellulose. It is not so easy to look at animal cells since they do not have cell walls nor do they contain coloured, visible organelles like chloroplasts. In fact, because many animal cells are so transparent and colourless, it is the usual practice to stain them in order to increase the contrast between the specimen and the background.

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One commonly used stain is methylene blue. When it is prepared with water, methylene blue is a vital stain, that is, it will stain cells without killing them. You will find a bottle of methylene blue in the stain tray at each lab bench. There are two ways in which you can add stain to a temporary whole mount. One, you can add a drop of the stain to the water used in making slide before putting on the cover slip. Or you can add the stain afterward to one edge of the cover slip and draw it through underneath the cover slip as is shown in Figure 4.4. Epithelial cells are the covering or lining cells in animals. The cells making up the lining of the inside of your mouth are epithelial cells which are easily rubbed off. Using your own epithelial cells, make a slide in the following manner. 1. On a clean microscope slide place drop of water and a small drop of methylene blue. 2. Using the blunt end of a toothpick, gently scrape off some of the epithelial tissue that covers the inside of your cheek and stir the scraping in the drop of stain. Place the used toothpick directly in a garbage can. Do NOT place it on the lab bench. 3. Place a cover slip over the preparation and examine under low and then high power. 4. On your slide you should find both single cells and groups of cells that are clustered together. Locate the nucleus, plasmalemma (plasma membrane) and cytoplasm. A. Before applying the coverslip Add a drop of methylene blue to the specimen and then position coverslip as described for a wetmount B. After applying the coverslip

Figure 4.4. Two methods of staining wet mounts.

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- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 9 Measure the diameter of a typical epithelial cell. How does it compare in size and shape to the plant cells? What structures are lacking or are different than those you observed in the plant cells? What structures are present in both the animal and the plants cells? Answer question 11 on the lab assignment pages. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 10 Using high power, make a detailed drawing of a clump of two or three epithelial cells from your cheek. Label plasma membrane, cytoplasm, nucleus. Don’t forget a proper title, scale, etc. Refer to page A-8 and 9 in the appendix of this lab manual for instructions for making proper drawings. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Permanent Slides Permanent slides are ones in which the specimen has been fixed or preserved and dehydrated before being mounted on a slide using a drop of transparent permanent mounting medium. Many tiny organisms can be mounted whole, that is without being cut into sections. In such a case, the slide is termed a whole mount (w.m.) because the whole uncut specimen is on the slide. Often, entire organisms and pieces of tissue are too thick or opaque to be observed as whole mounts. They must be "sectioned" or cut into thin slices. After fixation and dehydration the specimen is infiltrated with a sectionable medium (paraffin or epoxy resins). The specimen and the hardened medium are then cut into a block from which thin slices (l0 microns thick) are shaved. These sections are then mounted on a glass microscope slide in a drop of transparent medium as before. Both whole mounts and sectioned specimens may be found as permanent slides in your slide box. The label on the slide should tell you whether the material to be studied is a whole mount (w.m.) or a cross section (c.s.; t.s.; x.s.) or longitudinal section (l.s.). - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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* * * * * TASK 8 * * * * ** * * * * TASK 8 * * * * *

PUTTING YOUR MICROSCOPE AWAY WHEN YOU ARE FINISHED with the microscope, prepare it to be put away. In putting a microscope away, BE SURE that: 1. The shortest objective lens is in line with the body tube. 2. No slide is left on the stage. 3. The microscope is clean - no water or dust on the stage or lenses. Make a routine habit of cleaning both the lenses and the stage of your scope with lens tissue after each use. Boxes of lens tissue and lens cleaner are stored in the cupboard of each lab bench. Remember to always use a liquid with the tissue when you clean a lens. 4. The stage is in a lowered position. 5. The cord is coiled around the base to avoid entanglement in the cabinet. Make sure the cord is not pinched under the condenser gears or under the bottom of the stage. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LABORATORY ASSIGNMENT 11 When you are finished with your microscope, clean it and arrange it for storage. Have a demonstrator check it and sign your assignment page. - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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LABORATORY 4 ASSIGNMENT 1 1. This student's microscope was properly set up. Demonstrator's signature_______________________ ASSIGNMENT 2 2. A represents the letter e as it appears on your slide. In B print the letter e as it appears in your microscope. A B 3. On the e you put in Figure 2B, draw a dotted circle to mark the area you observed under the 10x objective lens. Next draw a solid circle to mark the area you observed under the 40x objective lens. 4. Make a summary statement about the relationship between magnification and field of view. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ASSIGNMENT 3 5. Objective lens Diameter of Field 4x __________ mm 10x __________ mm 40x __________ mm

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6. If you were observing a live specimen under the 40x objective lens and it moved out of your field of view, explain how could you find it again quickly and easily? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ASSIGNMENT 4 7. On the crossed thread slide, which thread is in the middle? _________________________ ASSIGNMENT 5 8. Diameter of thread: Ocular Units Diameter 4x objective _________ _______ 10x _________ _______ ASSIGNMENT 6 9. Elodea whole leaf: length ______________ width ______________ number of layers ______________ ASSIGNMENT 7: Cell Measures: 10. Elodea cells: Typical midrib cell - length __________ - width ____________ Typical blade cell -length __________ - width ____________ 11. Why did you observe chloroplasts only around the edges of the plant cells? ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________

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ASSIGNMENT 9 12. Cheek epithelial cell: diameter ____________ 13. Compare and contrast the plant cells and the animal cells you observed. ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 11. This student's microscope was cleaned and put away properly. Demonstrator's signature __________________________________