PART V: MEASURING LIVING CELLS
6.10 Exercise 10 – Preparing a wet mount of a naturally pigmented (red onion) cell.
To view living cells under a microscope, you first have to “wet” mount them on a glass slide. Prepare wet mounts of onion cells as follows: 1. Place a clean microscope slide on a paper towel. Put a drop of water (less than 1 cm diameter) in the center of the slide. 2. Obtain a wedge of red onion from the Central Study Area. With your fingers or forceps, remove a portion of the thin (cellophane-like) tissue that lines the outer surface of each scale-like leaf. With scissors or a razor blade, cut off a small (0.5 cm square) piece of epidermis and place it in the drop of water on the slide. 3. Place a cover glass over the specimen by holding the cover glass at a 45-degree angle against one edge of the water drop. Let the water spread along the width of the cover glass before slowly lowering the glass down over the material. Try to avoid trapping air bubbles under the cover glass. With your compound microscope, view your wet mount of onion epidermis. Use the circles below to represent the field of view (circle of light) as seen through your microscope. In the left-hand circle, sketch a few of the onion epidermal cells under high power. Record the magnification you are using (100x, 430x). For the onion cells, draw the highest magnification that allows you see at least one whole onion cell in the field of view. Use the circle on the left for this drawing.
Onion epidermal cells ( x) Human epidermal cells (430 x)
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6.11 Exercise 11 – Staining a wet mount of an unpigmented (human cheek) cell.
1. Place a second clean microscope slide on a paper towel. Put a drop of water (less than 1 cm
diameter) in the center of the slide.
2. Obtain human epidermal cells from the inside of your own mouth. Shake a clean toothpick out of the dispenser. Gently scrape the inside surface of your cheek with the toothpick. Put the cheek cell scrapings into the water droplet. Spread out the scrapings by gently tapping the toothpick in the water droplet.
3. Place a cover glass over the specimen by holding the cover glass at a 45-degree angle against one
edge of the water drop. Let the water spread along the width of the cover glass before slowly lowering the glass down over the material. Try to avoid trapping air bubbles under the cover glass.
4. Unlike red onion cells, human cheek cells are not naturally pigmented. Cheek cells are almost
completely transparent and need to be stained. Place a small drop of methylene blue dye at one edge of the cover slip and touch a paper towel to the opposite side of the cover slip. The dye will be drawn under the cover slip and across the cheek cells, staining them.
5. After the dye has been in place for one minute, put a drop of clean water near the edge of the cover
slip and again touch a paper towel to the opposite side. This will remove excess dye, but leave the cells stained.
6. View the slide under the microscope. In the right-hand circle above, sketch a few of the cheek cells
under high power (over 400 x)
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Table 3: Relative sizes of cells: List the lengths of several kinds of cells, including your three protozoans and any other specimens measured by other students in the lab.
Specimen Cell length (µm) Key features
Onion epidermis
Human epidermis (cheek cells)
A protozoan with cilia (Paramecium)
A protozoan with a flagellum Name:
A protozoan with pseudopodia Name:
Virus (influenza) Limit for light microscopes Bacteria (anthrax) Red blood cell (human) Airborne pollen Limit for naked eye Human ovum Grain of sand
0.02 µm 0.5 µm 1.0 µm 7.0 µm 25 µm 100 µm (= 0.1 mm) 100 µm (= 0.1 mm) 500 µm (= 0.5 mm)
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6.12 Exercise 12 – Estimating the size of a unicellular organism
1. Water from ponds contains many interesting unicellular organism. One of the most common is the single-celled Paramecium – a fast moving organism covered with beating, hair-like cilia. Place a drop of Paramecium culture onto a clean microscope slide. For best results, take a sample of the “sludge” from the bottom of the container. 2. To see the fast-moving Paramecium better, add a drop of “Proto-slo” to the sample on you slide and stir using a dissecting needle or toothpick. Proto-slo slows down swimming organisms by “thickening” (increasing the viscosity of) the water. 3. Add a cover slip by holding a cover glass at a 45-degree angle against one edge of the water drop. Permit the culture solution to spread along the width of the cover glass. Then slowly lower the cover glass. Try to avoid trapping air bubbles under the cover glass. 4. Use the low power objective to find a Paramecium. Center the Paramecium in the field of view before switching to higher power. 5. Sketch the Paramecium under medium and high power in the circles below. Estimate its length, using the diameter of the field of view. Enter your estimate in Table 3. Paramecium (medium x) Paramecium (high x)
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Sketch two additional protozoans (other than Paramecium). Use your drawings to estimate the size of these protozoans, and enter results in Table 3. Name _______________________ ( x) Name ____________________ ( x) Length ________ µm Length ________ µm Locomotion type ___________________ Locomotion type __________________ Based on the cells you have measured, are all protozoans about the same size? Are any of the species significantly larger — say, 10-times larger — than the others? CHECK OUT PASS: 1. Return your carrel to its original condition. An instructor will check your carrel and give you a “check out pass” to turn in at the front desk as you leave. 2. When you have finished using the compound microscope: a. Turn off the light source. b. Rotate the nose piece to the low power objective. c. Unplug the microscope and coil the wire loosely over the body tube. d. Cover the microscope. 3. Rinse off and dry the slides and cover slips you used for wet mounts. Any broken cover slips should be placed in the “broken glass” container near the fish tanks.
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Lab 2 – Enzymes and Cell Function 7
YOU NEED TO WORK WITH A PARTNER FOR THIS LAB AND ALL REMAINING LABS THIS SEMESTER. Please plan ahead. Partners share the data they collect, but the reports you write for labs #2 and #3 must be written individually.
Growing, reproducing, digesting, and the many other processes of “life” involve thousands of different biochemical reactions. Without enzymes, almost none of these biochemical reactions would proceed quickly enough to sustain life. Enzymes are catalysts that can help break larger molecules into smaller molecules, or help join two molecules together, all while remaining unchanged themselves. Enzymes are involved in everything from photosynthesis to the fertilization of eggs by sperm, from the digestion of food to the clotting of blood. Enzymes are proteins that catalyze (speed up) vital biochemical reactions by reducing the “activation energy” needed to get the reaction going.
Without enzymes, the temperature inside living cells is too low, and the concentration of reacting molecules is too dilute, to sustain the biochemistry of life. Enzymes are extremely efficient. Minute quantities of an enzyme can accomplish at low temperatures what otherwise would require much higher temperatures and/or harsh chemical reagents. For example, one ounce of the stomach enzyme pepsin can digest two tons of egg white in a few hours. Without pepsin, digesting this much egg white would require 10-20 tons of strong acid working for 24-48 hours at high temperature. Enzymes are so extraordinarily efficient for four reasons: (1) Enzymes can be used over and over again because they are not themselves changed by the reactions they catalyze. In the process of converting one molecule (the substrate) to another (the product), the enzyme binds temporarily with the substrate to form an enzyme-substrate complex. The enzyme returns to its original form as soon as the transformation of the substrate into the product is complete.
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Figure of “Lock-and-key model” is from Wikipedia.com
(2) Enzymes are extremely specific. They are very choosy about what substrates they will bind with and what reactions they will catalyze. Most enzymes bind with only one particular kind of molecule (like a “lock and key”) and cause only one particular kind of change in that molecule. Some enzymes specialize in synthesis (joining two substrates) while others specialize in splitting the substrate into products. (3) Enzymes are extremely reactive, much more reactive than ordinary chemical catalysts. For example, hydrogen peroxide (H2O2) by itself slowly decomposes into water and oxygen. A small
amount of powdered iron will act as a catalyst and speed up this decomposition several fold. But a single molecule of the enzyme catalase (found in human blood) can, in one minute, split more than five million peroxide molecules! Catalase is one of the fastest acting enzymes known. Other enzymes operate on their substrates at rates ranging from 1000 to 500,000 molecules per minute. (4) Most enzymes function best within a narrow range of temperature and pH (acidity). For example, as temperature rises, the rate of an enzyme-catalyzed reaction will at first increase because the enzyme and substrate molecules move around more quickly and so encounter each other more often. But above a certain temperature most enzymes become denatured (lose their shape) and so lose their catalytic activity. In this lab, you will learn about some of the qualitative characteristics of enzyme catalyzed reactions (Part I) and then quantify the effects of environmental factors (temperature, pH) on the rate of enzyme- catalyzed reactions (Part II).
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Lab learning objectives
You will have mastered the content of this minicourse when you are able to: 1. Explain why life on earth could not exist without enzymes. Using specific examples, explain how the
metabolic efficiency of living organisms is improved by the extreme reactivity and specificity of enzyme catalysts (See Introduction.)
2. Use a spectrophotometer and other qualitative and quantitative techniques to measure the activity
of an enzyme under different environmental conditions. 3. Graph and analyze the effects of temperature and acidity (pH) on the rate of an enzyme catalyzed
reaction. 4. Explain, at the molecular level, why enzymes work better at certain temperatures and pH’s. Include
the effects of pH and temperature on molecular motion and shape.