two dots, as two dots close under magnification seen with eye together appear two dots can be

Microscopy and Cell Diversity Microscopy and Cell Diversity OBJECTIVES: • To learn the parts of the microscope and their function. • Gain experien...
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Microscopy and Cell Diversity

Microscopy and Cell Diversity OBJECTIVES: •

To learn the parts of the microscope and their function.



Gain experience using the compound microscope.



Learn the basic differences between prokaryotic and eukaryotic cells.



Recognize differences between plant and animal cells.



Understand why cells are so small.

INTRODUCTION To investigate the living world biologists use a variety of tools. The microscope is the instrument of choice so we begin here with a lesson in microscopy. Microscopes are a system of lenses that magnify objects that are too small to see. In this exercise, you will become familiar with compound microscope. Magnification is an expression of how much larger an image appears relative to its actual size. Magnification is determined by multiplying the power of the eyepiece lens (also called the ocular lens) by the power of the objective lens. Typically, ocular lenses have a power of 10X while objective lenses can vary from 1X to 100X. If you are viewing a slide with a 10X ocular lens and a 4X objective lens, the total magnification is 40X. In the case of dissecting microscopes, magnification is calculated by multiplying the ocular lens power (10X) by the setting on the zoom control. Another important concept in microscopy, and one that limits the usefulness of magnification, is resolving power. To understand this, think of two small dots placed close together on a piece of paper--with your eye you can still distinguish them as separate points. In contrast, if the two dots are placed closer together, they will appear as a single dot; however, if you examined this single dot under a microscope, the increased resolution would allow you to see both dots again. •• two dots, as seen with eye

• two dots close together appear as one

•• under magnification two dots can be seen again

The resolving power of your eye is only about 0.1 millimeter (mm), whereas the resolving power of a good compound microscope is 0.0002 mm or 0.2 micrometers (µm). Note that 1 millimeter = 1000 micrometers. There is a limit to increased resolution. Most laboratory microscopes are only capable of magnifying up to about 1000X. This is not a limitation of the lenses to magnify, but rather a limit of resolving power. It would be of little use to enlarge something if you could not distinguish one structure from another next to it. 1

Microscopy and Cell Diversity

Magnification and resolving power are also limited by the wavelength of light used; shorter wavelengths provide more resolution. Electron microscopes use a beam of electrons with shorter wavelengths than visible light to allow much smaller objects to be seen (e.g. cell organelles). The most sophisticated electron microscopes can view details as small as the electron clouds that surround individual atoms.

A Word about Biological Drawing: Biological drawing is different from artistic drawing in that it is primarily a means to record information. There are three things that all biological drawings should have: • • •

A clear rendering of the important details or structures Labels for all identifiable structures An estimate of the scale or size of the image

Your scientific drawings require neither artistic talent nor do they have to be elaborate; however, they should give a clear picture of what you see. Common mistakes include making drawings that are too small to be useful or redoing drawings after lab. The time to finish your drawing is in lab while you are still looking at the subject! If you are looking at a whole organism or cell, make an outline showing its general shape and add important details such as cilia, a nucleus, vacuoles, etc. If you are looking at a tissue or multicellular structure, choose a few representative cells, tissue regions, or structures and draw them. Always use a sharp pencil (#2 or #3 preferred), not a pen, so that you can erase and correct inaccuracies in your drawings. Labels should be placed to one side of the drawing (usually the right side), with a straight line connecting the label to the appropriate structure in the drawing (see example below). Always include the magnification or an estimate of the scale of your drawing.

Review: Magnification = Ocular power x Objective power. In the example above, the ocular lens had a power of 10X and the objective lens had a power of 40X; thus, the magnification is 10X x 40X or 400X (400 times larger than life size).

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Microscopy and Cell Diversity

I. THE COMPOUND MICROSCOPE The compound microscope (see Figure 1 below) uses a double lens system to magnify specimens up to 1000X. One set of lenses, found in the eyepiece, is called ocular lens; these are usually 10X in power. A second series of lenses, called the objective lenses, are mounted on a rotating disk. These “click” into position to sit directly over the specimen being magnified. The objective lenses on a typical compound microscope are 4X and 10X (these are low power lenses for scanning), and 40X (high power, or “high and dry” lens). With the compound microscope the light is transmitted through the specimen on a slide. Hence the light source is beneath the object, shines up through it and into the lens system. This means that the object has to be thin enough to see through. In other words, you cannot examine your finger through a compound microscope.

Figure 1. A Compound Light Microscope

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Microscopy and Cell Diversity

A. PARTS OF THE COMPOUND MICROSCOPE The compound microscope is a delicate and expensive instrument and it must be treated with utmost care. •

ALWAYS carry the microscope with TWO HANDS. One hand should support the base and the other should hold the arm of the microscope.



DO NOT TILT THE MICROSCOPE as the oculars may fall out.



Use only LENS PAPER to clean the optics (other types of paper will irreversibly scratch the lenses). Gently wipe off smudges.



REMOVE any stain or water from the stage IMMEDIATELY (use a Kimwipe).



Contact your TA if you are having problems focusing or have questions about what you see.

PROCEDURE 1. Obtain a compound microscope from the cabinet and set it up at your lab bench. 2. Using Figure 1 as a guide, locate the following parts of the microscope: Arm - the supporting body of the microscope. Oculars or Eyepieces - some oculars have a pointer or scale etched onto the lens. This gives you a point of reference for finding and/or measuring structures. Record the ocular magnification on your scope here: _______ X.



On binocular microscopes, you can adjust the distance between the oculars to accommodate the distance between your eyes. This is done by gently sliding or pushing the oculars closer together or farther part. Body tube - hollow tube providing the right optical distance between the ocular and objective lenses. Revolving nosepiece - the mount for the objective lenses; revolves to bring the various objective lenses into position for viewing the object. Objective lenses - lenses of various sizes and magnification powers. Note that the lowest power lens has the shortest tube but provides the largest viewing diameter. Record the magnifications of these lenses from lowest to highest power: ____ X, ____ X, ______X. Stage – the platform on which the microscope slide is placed under the objective lens. Notice the hole in the stage that allows light to pass up through the specimen on the slide.

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Microscopy and Cell Diversity Clamp for microscope slide - secures your slide for viewing. This may be either a spring-type clamp or two manual clips. Stage manipulator knobs - allow you to move the slide right or left, forward or backward, on the stage Substage lamp - the light source. The lamp is incorporated into the base of the microscope. Before turning it on, make sure the light intensity dial is set to “1”, its lowest setting. This will prolong the life of the bulb. If you suspect the bulb has burned out (no light visible from the base when the microscope power is ON and the intensity setting is at “10”) contact your TA for assistance. Substage condenser - a lens system used to focus the light beam up into the iris, and the slide. For your purposes, the condenser should be positioned high, with its uppermost lens just beneath the slide on the stage. Practice moving the condenser up and down, but leave it in the high position. Iris diaphragm – regulates light intensity (it is located just under the stage). The diaphragm can be adjusted to control the amount of light transmitted through the slide. Turn on the light source, locate the lever on the diaphragm, and look through the microscope. Open and close the diaphragm by moving the lever back and forth. If your field of view is too dark, open the diaphragm more.



A common problem is the tendency to leave the diaphragm wide open. When you first use the microscope, close the diaphragm and slowly open it to achieve a light level that is comfortable for viewing. Coarse Focus adjustment knob - adjusts the coarse focusing. With the low power objective in place, move the coarse adjustment knob while viewing from the side. The lens moves up and down very quickly. This control is only used with the low power (4X) objective lens. Fine Focus adjustment knob - adjusts the fine focus. Once you have an object in focus under the scope, use the fine adjustment to better focus the image for viewing. Carefully adjust this control while looking at the lens from the side. Note that it does not move up and down as quickly as the coarse control. Use this adjustment with the 10X, 40X, and 100X objective lenses.

B. USING THE MICROSCOPE If you have a monocular microscope you should look into the single ocular lens with the eye that is opposite to the hand you draw with (e.g. if right handed, look through the ocular with your left eye); keep both eyes open. This may seem awkward at first; if you have trouble, place a card over the free eye, but don’t develop the habit of closing the unused eye. Once you get used to this practice, you will be able to look at the specimen with one eye and draw what you see. 5

Microscopy and Cell Diversity

If you have a binocular microscope, first adjust the width of the two eyepieces (by either pulling them apart or pushing them together) until the interpupillary distances match that of your eyes. You will find this when the position of your eyes is comfortable and the images from the two eyepieces fuse into one. If you have difficulty with this, it is probably because your eyes are not used to adjusting their muscles to allow the two images to align correctly. Try looking at a wall, then shift your gaze to the microscope without changing your eye position. If you continue to have difficulty viewing through both eyepieces, use the scope as if it were monocular and try again the next time you use the microscope. Most importantly, don’t strain your eyes for a prolonged period of time. PROCEDURE 1. Check the revolving nosepiece and make sure the lowest power objective (4X) is in place over the viewing hole in the stage. 2. Use the coarse focus knob to move the stage to its uppermost position (turn the knob away from yourself). 3. Obtain a letter “e” slide1, place in on the stage, and secure it with the clamp. Center the slide over the viewing hole using the stage manipulator knobs. 4. Turn the COARSE adjustment knob toward you to bring the image roughly into focus. Then, turn the FINE adjustment knob to bring the image into sharper focus. 5. In which direction does the image move as you move the slide to the right ________________? When you move the image to the left ________________? 6. Which way does the image move if you move the slide towards you ________________? When you move the slide away from you ________________? 7. Turn the revolving nosepiece to move the 10X objective into place. These microscopes should be parfocal. This means that once the proper focus is adjusted for one objective lens, the image should remain relatively sharp when switching to another objective lens. Is your microscope parfocal? _____ Now, switch to the 40X objective lens. 8. While you still have the letter “e” slide on the stage, adjust the iris diaphragm to familiarize yourself with regulating the light. Lenses of lower magnifying power require less light. With a 4X objective lens, you should be comfortable viewing with the diaphragm almost completely closed. 9. Looking back to page 1 or 2, calculate the magnification you are using. ___________X

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Microscopy and Cell Diversity

C. PREPARING A WET MOUNT The technique for making a wet mount allows you to examine material by keeping the sample in water or an isotonic saline solution (which approximates physiological fluids). You can use this technique to examine many types of materials including pond water and microscopic organisms you want to see while they are still alive. PROCEDURE: 1. Use a dropper or transfer pipet to place 1-2 drops of pond or aquarium water (or other live sample) on the center of a clean, dry glass slide. 2. Take a clean coverslip (coverglass) and hold it at a 45-degree angle to the surface of the slide (Fig. 2a). Bring the lower edge up to the edge of the water droplet (Fig. 2b), and slowly lower the coverslip over the drop (Fig. 2c). Be careful not to trap air bubbles under the coverslip.

a

b

c

Figure 2. Preparation of a wet mount. 3. Use a small piece of paper towel or bibulous paper to blot away any excess water around the coverslip. If necessary, wipe any water off the underside of the slide. 4. Now mount the slide on the microscope stage. Note that the light source will generate heat where it is focused on the slide. Hence, your living specimens in the water will heat up and the water will evaporate. Keep the light OFF until you are ready to make your observations to avoid drying out and cooking your specimen! 5. Examine your preparation under low power (4X or 10X objective) and look for visible cell structures. If you are examining pond water, you should see a variety of organisms including: filamentous organisms made up of many cells (either green algae or cyanobacteria); single-celled diatoms with curved, boxlike outlines; protozoa - single celled organisms that move about by cilia or flagella. You may also see various types of debris; these may represent the partial remains of decayed organisms or inorganic particles. Scan your slide to see how many different organisms you can find. 6. Locate an organism you find interesting and switch to a higher power objective lens. You will see more details, but you will notice that when you focus at one part of the organism, the other parts go out of focus. This reflects the property of lenses called depth of field: The lower the magnification of a lens, the greater the depth of field. The higher the magnification of a lens, the shallower the depth of field.

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Microscopy and Cell Diversity

D. DIFFERENTIAL STAINING In some cases, a specimen may need to be stained to improve the contrast of internal structures. Cytologists use a wide variety of stains to add contrast to cells and tissues for microscopy. Simple stains – Many colored (dyes) will bind irreversibly to biological molecules, just as they will to fabrics or your hair. These stains usually kill living cells because they bind to molecules and block them from cellular activity. Methylene blue is a common positively charged dye that will bind to negatively charged molecules such as nucleic acids, many proteins, and some polysaccharides. In contrast, Sudan black is a fatsoluble dye that selectively binds to fatty materials, Vital stains are stains that do not kill cells; for example, Neutral red becomes concentrated in the vacuoles of plant cells and Janus green B is oxidized in the mitochondrion to form a blue stain.

E. SECTIONING Some materials are either too thick to fit under the compound microscope or too opaque to transmit light. Such materials are often cut into a series of very thin sections, which can then be mounted on glass slides and examined. PROCEDURE: 1. To better understand the terms used for sections, consider a familiar object like a carrot. If you were to make thin sections through the middle, these would be cross sections (c.s.). If you were to cut a thin section from the top to the pointed tip of the carrot, this would be a longitudinal section (l.s.). In the space provided, draw a simple illustration of each of these sections. Carrot (c.s.)

Carrot (l.s.)

2. There are certain problems in interpreting a three-dimensional structure from a thin section. What do you think these difficulties are? (Hint: How would you know what a carrot looked like if all you had to base it on was the cross section in the previous step?)

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Microscopy and Cell Diversity MICROSCOPY SUMMARY SHEET: Answer in your own words and in complete sentences. 1. The metric system is based on powers of ten. Complete the following: 1 meter = _________ cm = ___________ mm = ___________ µm 2. What is the magnification if you use a 5X ocular lens and a 20X objective? ________?

A 40X objective ___________?

3. Write a brief function for the following microscope parts: Substage condenser -

Iris diaphragm -

Objective lens 4. What happens to the working distance between the objective lens and the slide preparation as you increase magnification? 5. Arrange the following steps in the correct order (1-6). _____ _____ _____ _____ _____ _____

Mount slide on stage Make sure the 4x (lowest power objective) is in place over the viewing hole Adjust the coarse focus Adjust the light intensity setting to “5” and turn on the power. Adjust the fine focus Look through the oculars

6. What is the purpose of using stains in microscopy? 7. On the specimen below, indicate which plane shows a cross section (A) and which shows a longitudinal section (B).

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Microscopy and Cell Diversity

II. CELL DIVERSITY The basic unit of life is the cell. This exercise will introduce you to the three major types of cells (Domains of life) and introduce you to some of the variety within one of them, the Eukaryotes. We will start with the Prokaryotes.

PROKARYOTES Bacteria are one example of prokaryotic cells: cells that lack membrane bound organelles. Prokaryotic cells reproduce asexually by binary fission and are most notable for their ability to take and use DNA from other bacteria and their environment. The prokaryotes are generally divided into two main groups called Domains, the Bacteria and the Archaea. Although many bacteria can cause illness or disease, many species are beneficial to humans. Bacteria typically have one of three shapes (round, rod, or spiral) and can be stained in a fourstep procedure called the Gram stain, which was devised by a Danish physician, Dr. Christian Gram. Some bacteria develop a purple color (gram-positive) as a result of this staining procedure, while other bacteria develop a pink color (gram-negative). Gram staining is routinely used as a first step in the identification of bacteria, including many potential pathogens.

PROCEDURE 1. Examine a demonstration slide showing gram-stained bacterial cells. This slide may be under immersion oil (i.e. the 100X objective lens) so you can see the cells more clearly. If you need to adjust the focus, do so ONLY with the FINE focus knob. DRAW a few of these bacterial cells to note the shape. Are the cells you see gramnegative or gram-positive? _________

Mag. _______

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Microscopy and Cell Diversity

EUKARYOTES Eukaryotic cells represent the third Domain of life. Eukaryotic cells are found in plants, animals, fungi, and the protists. The most conspicuous features of Eukaryotic cells is that they contain membrane bound organelles (nucleus, endoplasmic reticulum, mitochondria, chloroplasts, etc.) and they can reproduce asexually by mitosis and sexually by the process of meiosis.

A. FUNGI---YEAST CELLS Yeasts are common single-celled members of the kingdom Fungi. Several species of yeasts are especially important to humans in the production of bread, wine, and several carbonated beverages. By-products of their metabolic activity are responsible for producing carbon dioxide gas (as for leavening bread, making homemade soda) and alcohol (for wine and beer). Other species of yeast are pathogenic causing a variety of skin infections, for example Athlete's foot is caused by the fungus Trichophyton spp. Yeast are capable of reproducing both sexually and asexually (budding), the latter is the most common. The bud emerges as a side growth from the parent cell. When the parent cell undergoes mitosis, one nucleus migrates into the growing bud cell and is walled off. The bud eventually separates from the parent cell to form a new yeast cell. PROCEDURE 1. Refer back to the section entitled 'Making a Wet Mount' and make a wet mount from the liquid suspension of Baker’s yeast. Place a small drop on a clean microscope slide and add a drop of Methylene blue stain. Apply a coverslip, blot off any excess liquid with Bibulous paper, and examine the cells under the microscope (400x mag.). Increase the contrast (close aperture diaphragm) to improve viewing. In the space below, draw a few yeast cells including one that is budding.

Mag. _______

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Microscopy and Cell Diversity

B. PLANT CELLS – Elodea or Egeria, a common aquatic plant Plant cell membranes are enclosed by a rigid cell wall composed largely of cellulose fibers. The cell wall not only protects the cell membrane but also provides structural support for the cell and allows the passage of water and materials into and out of the cell. PROCEDURE 1. Set your microscope to low power; use the 4X objective lens (40X magnification). 2. Obtain a plain glass slide and a plastic coverslip. 3. Using forceps, remove a single leaf from the aquatic plant and make a wet mount using distilled water. 4. Study the leaf under 40X and 100X magnification. Examine your leaf mount near the edge (margin) of the leaf to see the plant cell structures most clearly. 5. In the space provided, DRAW a few plant cells in the leaf and LABEL the key features (cell wall, nucleus, chloroplasts, vacuole). 6. Discard the coverslip in the wastebasket provided in lab. SAVE the glass slide wash and dry it so you can use it for the next activity (Part C). Basic Plant Cell

Egeria leaf cells

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Basic Animal Cell

Mag. _______X

Human cheek cells

Mag. ________X

Microscopy and Cell Diversity

C. ANIMAL CELLS – Human cheek (epithelial) cells) You have a ready supply of epithelial cells inside your cheek. Epithelial tissues are sheets of cells that either cover or line body surfaces, both internally and externally. These include the cells lining the oral cavity, the surface of the lungs, the interior of blood vessels, and the outer layer of skin, just to name a few. PROCEDURE: 1. Obtain a clean toothpick. 2. Gently scrape the inside of your mouth (cheek area) with the BLUNT end of the toothpick (unless you want blood cells too!). Smear the scrapings over the center of the glass slide and let it dry for about 1 minute. Discard the toothpick into the trash. 3. Add 2 drops of dilute Methylene blue stain to the cell smear and carefully lay a coverslip over the drop. 4. If the stain seeps beyond the edges of the coverslip, either blot away the excess stain with a small piece of paper towel, or lay the slide between two sheets in a book of Bibulous paper. Gently press the Bibulous paper down to blot the excess stain from the slide. 5. Mount the slide on the stage of the microscope and examine your cheek cells. Begin at the lowest power objective lens, 4X (i.e. 40x magnification). Change to the 10x objective and then to the 40X objective (What will your magnification be now? _______ X). 6. Note the cell membrane, nucleus, and granular cytoplasm. If you have trouble seeing these structures, try adjusting the light intensity and contrast (by turning down the iris diaphragm). You may notice some very tiny, rod or round shaped, darkly staining structures in you slide preparation; these are bacteria from your mouth. 7. In the space provided above, DRAW 2 - 3 cheek cells and LABEL the cell membrane, cytoplasm, and nucleus. Record the magnification. 8. When you are done, discard the slide preparation in the container of Bleach water. 9. Now that you have been able to visualize an animal cell and a plant cell, take a moment to compare your drawings of these two cell types. NAME at least three structural differences between the animal cells and plant cells. a. b. c.

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Microscopy and Cell Diversity CELL DIVERSITY SUMMARY SHEET: Complete the following table by writing a brief function for the cell structures listed and “yes” or “no” if each structure is found in prokaryotic and eukaryotic cells.

Prokaryotes Cellular Structure Plasma membrane

Function

Eukaryotes

Bacteria

Plant Cell

Animal Cell

Size: 1 – 5 µm

Size: 10 – 100 µm

Size: 10 – 100 µm

Cell wall Cytoplasm Nucleus Mitochondria Chloroplasts Large central vacuole

 IMPORTANT things to remember BEFORE putting the microscope away: • Make sure the LOWEST objective lens (4X) is in place over the viewing hole. • Remove any slide left on the stage and return it to the box or tray in lab. • Make sure the light intensity dial is at its LOWEST setting. • NEATLY wrap the cord around the base, and replace the dust cover. • Set the microscope in the cabinet with the OCULARS facing the BACK wall of the cabinet.

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Microscopy and Cell Diversity

III. LIMITS TO CELL SIZE and DIFFUSION Cells need to move substances across their membranes. Various transport processes are involved and this exercise will only focus on passive diffusion. The concept of diffusion is sometimes difficult to grasp. Diffusion is defined as the random movement of molecules from an area of high concentration to an area of low concentration. The diffusion rates of substances are affected by the medium involved (gas, liquid, solid), the size of the molecule (small, large), the charge of the molecule (+/-), and the temperature. Ultimately, net diffusion stops when the molecules are equally distributed and the system is said to be at equilibrium. You will perform an experiment using phenolphthalein2 agar and a dilute sodium hydroxide solution (0.1M NaOH). You will be given agar cubes (“cells”) of two sizes and you will investigate the effect of cell size on the diffusion rate of sodium hydroxide. MATERIALS:

phenolphthalein agar cubes plastic beaker ruler spoon

graduated cylinder bottle of 0.1M NaOH timer razor blade

PROCEDURE: The experiment will run for 10 minutes. 1. Measure and record the dimensions of each cube (length, width and depth, in cm). Cube A:

L = _____

W = _____ D = _______

Surface area (SA) = ______ Volume = ________ Cube B:

L = _____

W = _____ D = _______

Surface area (SA) = ______ Volume = ________ 2. Measure 100 ml of 0.1M NaOH and pour into the beaker provided. 3. Using the spoon, carefully place the two agar “cells” in the beaker. 4. Start the timer (Note the time ____ : _____ ) and allow the “cells” to incubate with periodic gentle stirring for 10 minutes.

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Phenolphthalein is a pH indicator that is pink when the pH is 8 or greater and colorless at pH values below 8. 15

Microscopy and Cell Diversity 5. While you are waiting you can compute the total surface area, volume, and surface area-to-volume ratios for the two cells. 6. After 10 minutes, remove the two cells and blot them dry with a paper towel. Diffusion will still be taking place so you will need to work quickly. If necessary, adjust your elapsed time. 7. Using a razor blade (a plastic ruler works well too), carefully cut a slice of agar, approximately 3 mm thick from the middle of the cube (see diagram below). Note the pink and colorless regions. razor blade

8. Measure the depth of the pink region (mm) and the dimensions of the colorless region. 9. Determine the rate of diffusion of NaOH (in mm/minute) and the volume of the cube through which the NaOH has diffused.

Start time _____:_____

Final time _____:_______

Area of one Total Cell Agar side of cell Surface Area cube (l x w) (SA = 6 x l x w) in cm2 in cm2 A B

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Volume of Cell (V = l x w x h) in cm3

Elapsed time _______ min.

Ratio of SA to Volume (SA : V) : :

Distance NaOH traveled, mm

Diffusion rate for NaOH, mm / min.



What volume of the cube remains colorless? A:_________ cm3 B: ________ cm3



What volume of the cube has turned pink?

A:_________ cm3 B: ________ cm3

Microscopy and Cell Diversity CELL SIZE SUMMARY SHEET: 1. What happens to the SA to V ratios as the cells become larger? Smaller?

2. How many 1 cm3 cells would fit into a single cell that is 3 cm x 3 cm x 3 cm?

3. Which option gives the greater surface area, a single large cell (3cm x 3cm x 3cm) or multiple small cells that occupy the same volume? Why? Show the math to prove your point.

4. Give two reasons why it is advantageous for cells to be microscopic.

5. For multicellular organisms, diffusion is not an efficient means of transport for nutrients or gases. What strategies do multicellular organisms use to transport materials?

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