Microscopy and the Metric System

Transcription

1 Experiment Microscopy and the Metric System Margaret E. Vorndam, M.S. Version Review the safety materials and wear goggles when working with chemicals. Read the entire exercise before you begin. Take time to organize the materials you will need and set aside a safe work space in which to complete the exercise. Experiment Summary: Students will have the opportunity to use the metric system to measure length, volume, mass, and temperature. Students will learn how to convert between Metric and English units and identify the types of equipment and glassware used in measurement. In addition, students will learn about the different types of microscopes and their functions, the parts of the microscope, and how to calculate total magnification, depth of field, and field of view. They will practice using a microscope by preparing a wet mount slide, a slide of cheek cells, and a slide of onion cells Hands-On Labs, Inc.

2 Objectives To perform English to metric and Fahrenheit to Celsius temperature scale conversions, To suggest the correct microscope to use in a particular situation, To operate a microscope, To prepare a simple specimen wet mount and view it under a compound light microscope, and To outline the use of common experiment instruments. Time Allocation: Four to eight hours total 40 Hands-On Labs, Inc.

3 Materials Materials From: Qty Student Provides 1 Cutting board 1 Glass to hold water 1 Object to measure 1 Onion Item Description: 1 Disposable magazine or newspaper 1 Isopropyl alcohol 1 Paper towels 1 Sugar 1 Toothpicks 1 Liquid detergent for cleaning 1 Tap water of varied temperatures 1 Thread, 1 long pieces (three colors) 1 Microscope 1 Yardstick with metric scale (optional) 1 Apron From LabPaq 2 Beaker, 50 ml, plastic 1 Cylinder, 50 ml - Graduated, Plastic Dissection-kit with 7-tools - including the following: Bent Probe, 1 Dropping Pipette, Probe, Ruler in pocket, Scalpel with 2 Blades - Note blades are in the pocket, Scissors, Tweezers 1 IKI Indicator, 2.1% - 13 ml in Glass Vial in Bubble Bag 10 Pipette, Long Thin Stem 1 Scale-Spring-10-g in Box Labeled Slim Pen Scale 1 Slide - Cover Glass - Cover Slip Cube 1 Prepared Slide, any 1 Gloves packages 6 pairs 1 Thermometer-in-cardboard-tube Misc. Papers Bag 1 Lens-paper-pack-50-sheets Note: The packaging and/or materials in this LabPaq may differ slightly from that which is listed above. For an exact listing of materials, refer to the Contents List form included in the LabPaq. Safety Issues: Specifically review Cutting, Chemical, and Glassware safety issues Lab Hint: When using IKI iodine indicator solution wear apron, safety glasses and rubber gloves plus cover work surfaces with white plastic garbage bags to avoid stains 41 Hands-On Labs, Inc.

4 Exercise 1: Measuring Length, Weight, Volume, and Temperature Search Key Words: metric, metric measurement, experiment equipment, metric length, metric weight, metric volume, temperature, Celsius, metric conversion Metric Terminology and Unit Conversions: Scientific measurement is based on the International System of Units commonly referred to as the metric system. A French vicar, Gabriel Mouton, conceived of the system around In 1790, Louis XVI authorized the use of the system for scientific pursuits, and France was the first country to adopt the metric system as its formal method of measurement in Today, the metric system is used worldwide, and many countries have adopted it as their measurement method. Students in the United States generally have not had the opportunity to use the metric system in their daily lives. Although the U.S. adopted the system in 1972, it has never actually used it as the unit of measurement. However, it is important for students of the sciences to understand the metric system and its relative relationship to the English system that is commonly used in the U.S. Students may be familiar with computer storage terminology which uses metric, as in byte, megabyte, gigabyte, etc. The versatility of the metric system is attributable to its decimal system base. For example, the centimeter is used for measuring length; it is abbreviated as cm, and 2.54 cm = 1 inch. Ten centimeters are equal to one decimeter (dm). Ten dm (or 100 cm) are equal to one meter (m). There are similar relations for weight and volume measurements. We will examine each of these in turn. But first, let us look at the relationships between the metric and English systems and relationships within the metric system. Not all types of measurements use every increment of the metric system. For instance, in the length category, cm and m are commonly used, but dm is not. Convention dictates that liter (L) is always capitalized. Periods are not used after units, except at the end of a sentence, of course. The metric system is based on the following prefix terms that are associated with changes in the powers of ten. Metric Prefixes Pico Nano Micro Milli Centi Deci Deka Hecto Kilo Mega Giga Symbol p n m(mu) m c d da h k M G P o w e r of 10 Mathematical Equivalent ,000 1,000, Hands-On Labs, Inc.

5 Common Units of Metric Measurement Length nm mm mm cm Meter, m km Weight pg ng mg mg Gram, g kg Volume ml ml Liter, L Note: The grey areas above are not commonly used as a unit for that particular measurement. The student must also be familiar with conversions between different powers of ten. When doing conversions, determine the conversion factor first and then do the calculation. Conversions can be determined using proportional equations. For instance, how do we determine the number of ml in 5 liters of fluid? First, determine the conversion factor. The direction of the conversion determines which unit is in the numerator and which is in the denominator. The numerator will be the unit that we are converting to, and the denominator will be the 1 unit equivalent for the unit we are converting from. It is easy to remember that the units that we are converting from must cancel out, which is accomplished by dividing by that unit. Thus, the unit we are converting from must appear in the denominator. We know that there are 1,000 ml in 1 L, so, in this example, the needed conversion factor is 1,000 ml/1 L. The proportional equation will be: 1,000 ml = X ml 1 L 5 L Thus: X ml = 1,000 ml/1 L x 5.0 L and 5.0 L x (1000 ml/l) = 5000 ml, with the liter units canceling out each other. This equation can also be written as: 5.0 L x 10 3 ml/l = 5 x 10 3 ml. How many ml (microliters) are there in 2.4 liters? There are 1,000,000 ml, also written as 1x10 6 ml, in 1 L. The conversion factor will be 1x10 6 ml/1 L, so _1x10 6 ml_ = X ml 1 L 2.4 L 2.4 L x (1x10 6 ml/l) = 2,400,000 ml = 2.4 x 10 6 ml If the student is familiar with decimal and power of ten conversions, this is straightforward. Shift the decimal point the appropriate number of places right or left, depending on the direction of the conversion Hands-On Labs, Inc.

6 Try the following conversions for practice. 240,000 ng = mg = g 50 cm = mm = m The table below provides comparative metric units to English units. Metric and English Unit Equivalents 1 English Unit = Metric Unit Metric Unit = English Unit 1 inch 2.54 cm 1 cm inch 1 yard 0.91 m 1 m 39 inches = 1.09 yd 1 mile 1.61 km 1 km 0.62 mile 1 ounce g 1 g 0.04 oz 1 pound (16 oz) 0.45 kg 1 kg 2.20 pounds 1 cubic inch cm 3 1 cm 3 (= 1 ml) 0.06 in 3 1 fluid ounce ml 1 ml 0.03 fl oz 1 pint (liquid) 0.47 L 1 L 2.13 pt (liquid) 1 gallon (liquid) 3.79 L 1 L 0.26 gal. = 1.1 qt (liquid) To convert units from English to metric, and vice versa, use the comparative table values, setting up the conversion factor as above for metric conversions. For instance, to determine the number of meters in 2 yards, m = X m and 2 yards x ( m/yd) = m. 1 yard 2 yards And to determine the number of ml in 3.5 oz: ml = _X ml and 3.5 fluid oz x ( ml/oz) = ml 1 fluid oz 3.5 fluid oz 1 Note that there are unit conversion differences between dry and liquid measurements in the U.S. system of weights and measures. Also, the United Kingdom (Imperial) and the US measurements differ in some cases. We use liquid volumes and U.S. scales for the units in this laboratory Hands-On Labs, Inc.

7 PROCEDURE 1. Length: A metric ruler is useful for measuring items of length. The ruler below measures in mm, indicated by the small mm near 0. a. How many mm are there in 1 cm? In a meter (m)? (Ruler is not to scale. See ruler in dissection kit.) b. Locate a measurable object to use for this exercise. If the object is long, obtain a yardstick that includes a cm scale; they can be found in local hardware stores. c. Record the length of the object below and do the conversions: Name of object cm = mm = m 2. Volume: The most commonly used metric measures of volume are the liter (L) and the milliliter (ml). Note the capitalized L, a scientific style for reference to liter. Volume can also be expressed as length x width x height, resulting in a cubic cm (cm 3 ), cubic m (m 3 ), etc., designation. One ml of liquid is equivalent to 1 cubic centimeter (cm 3 ) and is commonly written as cc for cubic centimeter. The cc designation is extensively used in the medical field. Liquid volumetric measuring devices include calibrated graduated cylinders, volumetric flasks, burets or graduated pipettes. There are many specialized variations of these basic tools in existence in today s laboratory, including automated versions Hands-On Labs, Inc.

8 Graduated Cylinder Graduated Pipette Buret Volumetric Flask Not to Scale Techniques for Liquid Measurement It is important to be as accurate as possible when measuring liquid using calibrated volumetric equipment. Since liquids tend to be slightly drawn up around the edges of the volumetric cylinder or flask due to capillary action, a bowl-shaped surface is created. This curved surface at the top of the liquid is called the meniscus. Always ensure that the bottom of the liquid s meniscus is at the desired mark on the flask or cylinder. The meniscus should be even with the volumetric mark when looking at it from a parallel, eye-level position. Practice adding tap water to the graduated cylinder. Select a ml level, and hold the cylinder mark for that ml at the level of the eye. Add water until the bottom of the meniscus is at the desired ml mark. A dropper or long-stemmed pipette can help to control the amount of liquid added, but with practice, students should become proficient at accurately pouring from a beaker. Always pour an approximate volume of liquid into a clean beaker and then from the beaker into the volumetric flask or graduated cylinder. This will minimize contamination of the parent liquid source. Dispose properly of any left over liquid. Do NOT pour it back into the original container. Why? When using a pipette or dropper to measure liquid, pour an aliquot into a clean beaker and then draw up the liquid from the beaker into the pipette. NEVER try to draw up chemicals by mouth. Why? 3. Weight: The most commonly used metric measures of weight are the gram (g), the kilogram (kg), and the milligram (mg). Metric weight is measured using a scale. Scales can be either digital or balance types and exist in all shapes and sizes. Calibrated weight sets of known 46 Hands-On Labs, Inc.

9 weights can be purchased to ensure that experiment scales weigh correctly and do not drift from true measurements. Specialized equipment such as spatulas and weighing papers are used to keep the weighing platform clean and to efficiently transfer the weighed solid material to a final container. As with liquids, it is always a good idea, but not as often done, to place an approximate aliquot of the solid material into a beaker and to use a clean knife to transfer the solid to the tared 2 weighing paper on the scale. Liquids are weighed also. In this case, the container is tared and then the liquid is added to the container until the final weight is achieved. Calibrated Weight Set Balance Scale Digital Scale Use the pen scale from the LabPaq to measure out exactly three grams of sugar. Make sure to tare the bag before adding the sugar. Why must the bag be tared before adding the sugar? How is the weight of the bag accounted for when the sugar is weighed? 2 Taring is the weighing of a measuring paper or container before it is filled with the material to be weighed so the final weight can be adjusted for the weight of the paper or container. On some scales, especially digital ones, the readout can be adjusted to zero before adding the material to be weighed. Thus, the scale is first calibrated with an empty platform at exactly g. Next, the empty weighing container is placed on the scale and the readout is reset (tared) to zero. Then the scale will show only the exact weight of the item being weighed. With other scales and the hanging scale in the LabPaq, the weight of the empty weighing container is first measured and recorded. Then the material to be weighed is placed in the container and the total gross weight is measured and recorded. The net weight of just the material equals the gross weight less the weight of the container. (gross wt. container wt. = sample wt.) 47 Hands-On Labs, Inc.

10 4. Temperature: Temperature is measured in degrees Celsius which is abbreviated as C, and also known as centigrade which means 100 graduations. The Celsius system is based on the properties of water. Pure water freezes at 0 C and boils at 100 C under conditions of standard temperature and pressure (STP). STP equals 0 C, equivalent to 273 Kelvin 3 or 32 F, and 760 mm Hg, 4 or 1 atmosphere of pressure, which is understood to be equivalent to sea level or 0 feet of altitude. STP must be specified for temperature when appropriate, because boiling point depression occurs with increasing altitude due to the drop in atmospheric partial pressure. When converting from F to C, the adjustment must include a ratio shift of the Fahrenheit scale into the Celsius scale (i.e., 32 F = 0 C and 212 F = 100 C). There are 9 F for every 5 C. We must also take into account that the Fahrenheit scale is 32 F at 0 C. The conversion from F to C is thus: C = (5 C/9 F) x (X F 32 F). For example, to convert 47 F to the equivalent C, C = (5 C/9 F) x (47 F 32 F) = (5 C/9 F) x 15 F = (5 C x 15 F) 9 F = 75 C 9 = 8.3 C Practice converting the following with this conversion formula: 45 F = C 62 F = C 98.6 F = C Use a Celsius thermometer to measure the C temperature of several different aliquots of cold and warm tap water. Make sure to allow the thermometer to remain until the temperature is stable and no longer changes. Record the temperatures: C C C 3 Kelvin temperature scale is also used by scientists. 0 K is equal to C, which physicists believe to be the lowest temperature possible in the universe. 4 Hg, is the chemical symbol for the element, mercury. Climatologists measure air pressure in mm of mercury. One atmosphere of air pressure at 0 feet elevation (sea level) is equivalent to a column of Hg inches tall = 760 mm Hg Hands-On Labs, Inc.

11 Exercise 2: Microscopy Search Key Terms: Microscope, stereomicroscope, dissecting microscope, scanning electron microscope, transmission electron microscope, compound light microscope, cheek cells Introduction: Antonie van Leeuwenhoek will forever be famous for perfecting the first working microscope. Until he began developing microscopes in 1670, previous microscopes in use since the 1590 s were only capable of magnification up to 20x (20 times or 20 power). Leeuwenhoek was able to produce a simple microscope that magnified to over 200x (200 times or 200 power). He reported the existence of bacteria in 1683 when he viewed white material scraped from his teeth with his microscope. Viewing the micro-universe has proceeded from those first attempts. The dissecting microscope, or stereoscope, efficiently magnifies objects in the range from 10x to 40x. It provides a view of organisms for purposes of closer, more detailed examination or dissection. Typical subjects might be chicken eggs, organ parts, or flower parts. Dissecting microscopes are used to magnify specimens of sizes 10 µm to 0.1 m; the equivalent in inches is to 3.9 inches. If a dissecting microscope has two ocular lenses on separate body tubes, it is referred to as a binocular dissecting microscope, or a stereoscope. The subject is lighted from the side, not from the bottom. The compound light microscope effectively magnifies in the range of 40x to 2000x. If an object under view is 10 nm in length without any magnification, what will be its viewing size at 40x? at 2000x?. What is the equivalent size at these magnifications in inches?. Show your calculations. Typical specimens used with compound light microscopes range in size from 200 nm to 5 mm; this can include large cell organelles, bacteria, and developing frog eggs. Light microscopes employ a light beam to view the specimen. In compound light microscopes, the light comes from below the specimen; thus, specimens must be small or thinly sliced to obtain a good image. We will look closer at this microscope below. The scanning electron microscope (SEM) employs electron bombardment to image very small specimens. The electrons passing through a three-dimensional specimen are read using a detection device, and a computer reconstructs the specimen image from the information gathered by the scanning process. Scanning power can reach 200,000x, and provide great detail. The transmission electron microscope (TEM) allows internal investigations of prepared specimens and has a higher magnification range than the SEM. Typical subjects may be genetic material, large molecules, viruses, and cellular organelle detail. Electron microscopes are used to image specimens that range from 1 nm to 100 µm in size. What is the equivalent in inches?. Show your calculations Hands-On Labs, Inc.

12 Compound light microscopes are so named because two sets of lenses are used during the viewing process. The ocular lens is nearest the eye, and the objective lens is nearest the specimen. Light, either from a focused mirror or electric source, is sent from below the specimen through the lenses to the eye. Light transmits through thin sections of the specimen, but is differentially transmitted through thicker portions of the specimen. Stains, dyes and contrast adjustment can be used to optimize specimen viewing. A microscope is well designed to serve its function, and should be treated with the care necessary to maintain it in working order. When moving a microscope, always support it with one hand under the base, and use the other hand to hold the microscope arm. Use lens paper to clean the lenses and light sources. The stage, arm, and base can be wiped with a soft, damp cloth. When not in use, the microscope should be stored in its case or covered to protect it from dust. Store it in a dry area away from temperature extremes and vibration. PROCEDURE 1. Parts of the Compound Light Microscope: Refer to a microscope as this section is read. Label the microscope diagram that follows as the examination of the microscope proceeds. a. Eyepiece (Ocular Lens): The magnification power is stamped on the outside of the lens. What is the power of the ocular lens?. Microscopes may have interchangeable ocular lenses of different magnification. b. Body Tube: Holds the ocular and objective lenses at the correct focal distance. c. Arm: Used to transport microscope and hold the body tube. d. Nosepiece: The revolving device that holds the objective lenses. May also be referred to as the turret. e. Objective Lenses: Consists of one or more lenses: i. The scanning power objective lens is the shortest of the lenses. What is its power? ii. The low-power objective is slightly longer than the scanning objective. What is its power? iii. The high-power objective is longer than the low-power objective. What is its power? iv. The oil immersion objective is the longest lens. Its power is approximately 95x to 100x. Not all microscopes have an oil immersion objective. f. Focus Adjustment Knobs: One or two knobs are used to focus the lens system. i. The coarse-focus knob is used only with the scanning power objective to bring specimen into view. All microscopes have a coarse focus Hands-On Labs, Inc.

13 ii. The fine-focus knob is used to adjust lenses to optimal viewing range. Used with all other objectives. (Optional) g. Condenser: A lens system below the stage used to focus the light entering from beneath the stage. (Optional) h. Diaphragm: Controls the amount of light entering from beneath the stage. (Optional) i. Light source: A lamp or mirror used to direct light up toward the stage. j. Stage & Stage Clips: The area below the objective lens that securely holds a slide in place for viewing. k. Base: Supports the working parts of the microscope. Used for transporting microscope. Label this microscope diagram with the appropriate part names and their functions: a b c d e f g h i Parts not included in microscope are: 2. Focusing the Microscope: a. Place the microscope on a stable surface. If the microscope has a mirror to focus the light source, use it near bright lighting for best viewing. But DO NOT USE direct sunlight as the light source or severe eye damage may result. b. Rotate the lowest power (scanning) objective lens into line with the ocular lens. c. Turn the coarse adjustment knob to raise the body tube to its highest position so that the objective lens is well above the stage Hands-On Labs, Inc.

14 d. Mirror light source: While looking through the ocular lens, adjust the mirror for maximum light entry through the stage area. A bright light should be observed through the ocular lens when the mirror is in the correct position. e. Electric light source: Plug the cord into an electrical outlet. f. Attempt to keep both eyes open, even with a single ocular lens, to minimize the eyestrain caused by squinting. g. Place a prepared slide on the stage. Try to locate the slide subject in the center of the viewing area. Secure the slide in place with the stage clips. h. While watching from the side of the microscope and using the coarse focus knob, lower the scanning objective lens to its lowest point above the slide. Take care not to put pressure on the slide. i. While looking through the ocular lens, slowly raise the scanning lens using the coarse focus knob until the subject material on the slide becomes clearly visible. j. If the microscope has a fine adjustment knob, use it to fine-tune the view of the subject material. Since the field of vision decreases at higher viewing magnifications, ensure that the subject material is centered below the lens. Move the slide around on the stage until the material is within the field of view and centered on the stage. k. If the microscope has either or both a diaphragm and/or condenser, adjust the settings of these items to optimize the view of the subject material on the slide. Low viewing light may be preferable, in some instances, to increase the contrast of the slide subject. l. Compound light microscopes are parfocal, meaning that if the subject is in focus at low power, it should remain nearly in focus when the objective is switched to higher power. m. Using the settings established for the scanning view above, move the low power (second shortest lens tube) objective into place. The slide subject should still be viewable. If the subject is not in focus, use the adjustment knob to slightly refocus the view, taking care not to crush the cover slip on the slide. Then, view the subject with the higher power objective lens (longest lens tube) using the same method. If the microscope has a coarse and fine adjustment knob, never use the coarse adjustment knob with higher power lenses. n. If the microscope includes an oil immersion lens, place a drop of immersion oil on the slide cover slip before rotating the lens into place. The function of the oil is to minimize light diffraction through the slide and subject so that greater detail can be seen. After using the oil immersion lens, clean excess oil off of the lens and the slide with a lens cloth. Never tilt a microscope when using oil or if viewing a wet slide. Why? o. When the observation is complete, the scanning lens should be rotated into place, and the body tube should be raised with the coarse focus knob to minimize the possibility of damaging the slide. The slide can then be removed safely Hands-On Labs, Inc.

15 3. Operating the Microscope: a. Obtain a clean slide and cover slip from the slide box. Place the slide and cover slip separately on a paper towel or other soft surface to reduce the possibility of scratching them. b. With scissors, cut a letter e from an old magazine or newspaper. c. Place the letter in the center of the slide. d. Follow the instructions in Section 6 below to make a wet mount of the letter. e. Following the directions outlined above under Handling and Focusing the Microscope, place the prepared slide on the microscope stage. Leave the scanning lens in place and focus so that the letter is clearly viewable. Make drawings of the letter in the boxes below as instructed. Side of the slide furthest away from student Look from the side of the microscope, view and then draw the letter, as it appears on the slide on the stage. Draw the letter as it appears when viewing it through the microscope. Side of the slide closest to student f. What is observed? Microscopes invert the image on the slide. This means that the subject will appear to be 180 rotated and reversed from the actual image viewed on the slide. g. While viewing the letter through the lenses, move the slide slightly. What do you observe about the movement of the letter and slide when viewed through the lenses? h. Use the directions above to view the letter at the higher objective powers. On the drawing made above, circle the portion of the letter that is viewable as successively higher power observations are made. What is your conclusion about what happens when higher power objectives are used? 4. Total Magnification Calculation: Typically, the ocular lens of a microscope will be 10x, but it may be higher or lower. The power is recorded on the side of the lens. a. Set up a data table similar to Data Table 1: Calculating Magnification, in the Lab Report Assistant section. b. What is the ocular lens power of the microscope that you are using? It may be 10x or 15x. Record it in Table 1. c. The objective lenses also have the magnification power recorded on their sides. What powers do the objective lenses on the microscope have? Record them in Table Hands-On Labs, Inc.

16 d. Now, calculate the total magnification of the viewing area by multiplying the power of the ocular lens with that of the objective lens in use. For instance, if a microscope has a 10x magnification ocular lens and a 4x objective lens in place for viewing, the total magnification will be 40x (10x multiplied by 4x). What other view magnifications are possible with the microscope? Calculate the total magnification for each set of lenses in Table Diameter of Field: a. Set up a data table similar to Data Table 2: Diameter of a Viewing Field b. With the low-power objective in viewing position, place a short transparent metric ruler on the stage. c. While viewing the ruler through the lenses, measure the low-power diameter of field of view in mm. Convert this measurement to μm and record in Table 2. d. Switch to the other higher power objectives, noting the diameter, in mm, for each in Table 2. Convert measurements to μm. How might this information be useful when viewing microscopic subjects? 6. Preparing a Wet Mount Slide: A wet mount technique is often used when mounting a specimen on a slide for viewing. This technique holds the specimen in place with a glass or plastic cover slip and minimizes light scattering through the slide. Read through this section and then practice wet mounting with water until an air bubble-free slide is achieved. a. Place the specimen in the center of the slide with tweezers or, in the case of aquatic specimens, a dropper, and add a drop of water using a dropper. b. Place an edge of the cover slip along the outer edge of the water drop on the surface of the slide. c. Slowly allow the cover slip to drop into place. This should remove the air and minimize the number and size of air bubbles trapped under the cover slip. Use a dissecting needle to control the fall of the cover slip. d. If a stain is required to enhance the image on the slide, use the same technique, but use the stain instead of a water drop as the mounting liquid. e. Wipe off the bottom of the slide before placing it on the stage. 7. Depth of Field: Prepare a wet mount slide of three differently colored crossed threads using the wet mount technique described above. Place the slide on the microscope stage with the thread crossing area in the center of the viewing area. Focus carefully, moving the scanning objective lens up and down, taking care not to break the cover slip. Depth Top Middle Bottom Thread Color 54 Hands-On Labs, Inc.

17 Record the order of the threads in this table. Note that, when one thread is in focus, the others appear blurred. Why? When you focus on another thread, what happens to the thread that you were viewing? Switch to high power and focus on one thread, then focus on another thread. What do you notice about the depth of field? Can you see as much of the thread in focus at the high power as you could at the low power magnification? 8. View an animal cell: a. Sanitize a clean toothpick with alcohol and use it to lightly scrape a small amount of epithelial cells from the inside of cheek. b. Place the scraped material in the center of a clean slide. c. Add one drop of IKI iodine indicator solution stain to the material on the slide. Use a toothpick to slightly swirl the drop, without spreading it, to separate the cells. d. Cover with a cover slip. e. Observe the prepared slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the nucleus in several cells. Locate the cytoplasm and the plasma membrane. On a sheet of paper, make a drawing of a few cells, and label the observed parts. 9. View a plant cell: a. Using the dissection kit s scalpel, remove a small piece of live skin membrane from the surface of an onion. The membrane is located just underneath the outer dead protective layers of onion skin and will appear to be nearly transparent, slightly moist, and very pliable. The membrane represents the epithelial cells common to plants. b. Place the thin membrane layer flat on a clean slide. It may help to place a drop of the IKI iodine indicator solution stain on the slide first and then place the membrane piece in it. This will enable the membrane to lie out flat instead of clumping. c. Place a small amount of the stain on the top surface and add a cover slip. d. Observe the slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the cell wall and the nucleus in several cells. Make a drawing of a few cells and label the observed parts. e. Count a column of cells, stacked end-to-end, across the field of vision under high-power magnification. f. Based on the field of vision measurement you calculated above, compute the average length of one cell in the column of cells with this formula: μm average length of cell = μm diameter of field of view total number of cells in the column. g. What differences were noted between the animal cells and the plant cells? How do the differences dictate the form of the organism? 55 Hands-On Labs, Inc.

18 Now that you have completed this lab make sure you read the lab for next week. This will help you plan your time better. Take some time and highlight anything you will need to prepare in advance. As you read the lab write out a hypothesis for each exercise Hands-On Labs, Inc.

19 Microscopy and the Metric System Margaret E. Vorndam, M.S. Version Lab Report Assistant This document is not meant to be a substitute for a formal laboratory report. The Lab Report Assistant is simply a summary of the experiment s questions, diagrams if needed, and data tables that should be addressed in a formal lab report. The intent is to facilitate students writing of lab reports by providing this information in an editable file which can be sent to an instructor. Observations Ocular Lens Magnification x Data Table 1: Calculating Magnification Objective Lenses Magnification = Total Magnification 57 Hands-On Labs, Inc.

20 Scanning Lens Low Power Lens High Power Lens Data Table 2: Diameter of a Viewing Field Magnification (ocular x objective lens powers) mm diameter of field of view μm diameter * of field of view 58 Hands-On Labs, Inc.

21 Exercise 1: Measuring Length, Weight, Volume, and Temperature Try the following conversions for practice. 240,000 ng = mg = g 50 cm = mm = m PROCEDURE 1. Length: A metric ruler is useful for measuring items of length. The ruler below measures in mm, indicated by the small mm near 0. a. How many mm are there in 1 cm?, in a meter (m)? (Ruler is not to scale. See ruler in dissection kit.) b. Locate a measurable object to use for this exercise. If the object is long, obtain a yardstick that includes a cm scale; they can be found in local hardware stores. c. Record the length of the object below and do the conversions: Name of object Volume: Always pour an approximate volume of liquid into a clean beaker and then from the beaker into the volumetric flask or graduated cylinder. This will minimize contamination of the parent liquid source. Dispose properly of any leftover liquid. Do NOT pour it back into the original container. Why? When using a pipet or dropper to measure liquid, pour an aliquot into a clean beaker and then draw up the liquid from the beaker into the pipet. NEVER try to draw up chemicals by mouth. Why? 59 Hands-On Labs, Inc.

22 Weight: Use the pen scale from the lab kit to measure out exactly three grams of sugar. Make sure to tare the bag before adding the sugar. Why must the bag be tared before adding the sugar? How is the weight of the bag accounted for when the sugar is weighed? Temperature: Practice converting the following with this conversion formula: 45 F = C 62 F = C 98.6 F = C 45 F = 62 F = 98.6 F = Use a Celsius thermometer to measure the C temperature of several different aliquots of cold and warm tap water. Make sure to allow the thermometer to remain until the temperature is stable and no longer changes. Record the temperatures: C C C Questions A. What laboratory equipment would be used to measure the following items? 5 g flour 36 ml water The length of a frog s leg 36 g water 38ºC Volume of a turtle* 125ºF Volume of blood Weight of a plant Weight of blood Temperature of a fish s body Temperature of blood *This answer may require some creativity. How could it be done? 60 Hands-On Labs, Inc.

23 B. Provide the calculation steps, including the conversion factor that would be needed to convert the following measurements, and the final answers. Use U.S. and liquid units where appropriate. 248 g = mg 145,000 μl = ml 536 ml = cc kg = g 0.75 L = μl cm = m 61 Hands-On Labs, Inc.

24 C. Provide the calculation steps, including the conversion factor that would be needed to convert the following measurements, and the final answers. Use U.S. and liquid units where appropriate. 3 cups = L 7,893 mg = lb 2.25 oz = cc 36ºC = ºF 145,000 ul = tsp 96ºF = ºC 62 Hands-On Labs, Inc.

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27 D. What advantages does the metric system have over the English method of measurement? What are the disadvantages? 65 Hands-On Labs, Inc.

28 E. Outline the steps necessary to accurately weigh 3.5 g of starch. F. Outline the steps necessary to accurately pipet 5 ml of distilled water. Pour an aliquot of distilled water into a clean beaker. Exercise 2: Microscopy The compound light microscope effectively magnifies in the range of 40x to 2000x. If an object under view is 10 nm in length without any magnification, what will be its viewing size at 40x? at 2000x? What is the equivalent size at these magnifications, in inches? Show your calculations. The scanning electron microscope (SEM) employs electron bombardment to image very small specimens. Electron microscopes are used to image specimens that range from 1 nm to 100 µm in size. What is the equivalent in inches?. Show your calculations Hands-On Labs, Inc.

29 PROCEDURE 1. Parts of the Compound Light Microscope: Refer to a microscope as this section is read. Label the microscope diagram that follows as the examination of the microscope proceeds. a. Eyepiece (Ocular Lens): The magnification power is stamped on the outside of the lens. What is the power of the ocular lens? Microscopes may have interchangeable ocular lenses of different magnification. b. Body Tube: Holds the ocular and objective lenses at the correct focal distance. c. Arm: Used to transport microscope and hold the body tube. d. Nosepiece: The revolving device that holds the objective lenses. May also be referred to as the turret. e. Objective Lenses: Consists of one or more lenses: i. The scanning power objective lens is the shortest of the lenses. What is its power? ii. The low-power objective is slightly longer than the scanning objective. What is its power? iii. The high-power objective is longer than the low-power objective. What is its power? Label this microscope diagram with the appropriate part names and their functions: 67 Hands-On Labs, Inc.

30 a b c d e f g h i Parts not included in microscope are: 68 Hands-On Labs, Inc.

31 2. Focusing the Microscope: If the microscope includes an oil immersion lens, place a drop of immersion oil on the slide cover slip before rotating the lens into place. The function of the oil is to minimize light diffraction through the slide and subject so that greater detail can be seen. After using the oil immersion lens, clean excess oil off of the lens and the slide with a lens cloth. Never tilt a microscope when using oil or if viewing a wet slide. Why? 3. Operating the Microscope: a. Obtain a clean slide and cover slip from the slide box. Place the slide and cover slip separately on a paper towel or other soft surface to reduce the possibility of scratching them. b. With scissors, cut a letter e from an old magazine or newspaper. c. Place the letter in the center of the slide. d. Follow the instructions in Section 6 below to make a wet mount of the letter. e. Following the directions outlined above under Handling and Focusing the Microscope, place the prepared slide on the microscope stage. Leave the scanning lens in place and focus so that the letter is clearly viewable. Make drawings of the letter in the boxes below as instructed. Side of the slide furthest away from student Look from the side of the microscope, view and then draw the letter here, as it appears on the slide on the stage. Draw the letter here as it appears when viewing it through the microscope. Side of the slide closest to student f. What is observed? Microscopes invert the image on the slide. This means that the subject will appear to be 180 rotated and reversed from the actual image viewed on the slide. g. While viewing the letter through the lenses, move the slide slightly. What do you observe about the movement of the letter and slide when viewed through the lenses? h. Use the directions above to view the letter at the higher objective powers. On the drawing made above, circle the portion of the letter that is viewable as successively higher power observations are made. What is your conclusion about what happens when higher power objectives are used? 69 Hands-On Labs, Inc.

32 4. Total Magnification Calculation: Typically, the ocular lens of a microscope will be 10x, but it may be higher or lower. The power is recorded on the side of the lens. a. What is the ocular lens power of the microscope that you are using? It may be 10x or 15x. Record it in Table 1. b. The objective lenses also have the magnification power recorded on their sides. What powers do the objective lenses on the microscope have? Record them in Table 1. c. Now, calculate the total magnification of the viewing area by multiplying the power of the ocular lens with that of the objective lens in use. For instance, if a microscope has a 10x magnification ocular lens and a 4x objective lens in place for viewing, the total magnification will be 40x (10x multiplied by 4x). What other view magnifications are possible with the microscope? Calculate the total magnification for each set of lenses in Table 1. Table 1: Calculating Magnification Ocular Lens Magnification x Objective Lenses Magnification = Total Magnification 5. Diameter of Field: a. With the low-power objective in viewing position, place a short transparent metric ruler on the stage. b. While viewing the ruler through the lenses, measure the low-power diameter of field of view in mm. Convert this measurement to μm and record in Table 2. c. Switch to the other higher power objectives, noting the diameter, in mm, for each in Table 2. Convert measurements to μm. How might this information be useful when viewing microscopic subjects? Table 2: Diameter of a Viewing Field Scanning Lens Low Power Lens High Power Lens Magnification (ocular x objective lens powers) mm diameter of field of view μm diameter * of field of view 70 Hands-On Labs, Inc.

33 6. Depth of Field: Prepare a wet mount slide of three differently colored crossed threads using the wet mount technique described above. Place the slide on the microscope stage with the thread crossing area in the center of the viewing area. Focus carefully, moving the scanning objective lens up and down, taking care not to break the cover slip. Record the order of the threads in this table. Note that, when one thread is in focus, the others appear blurred. Why? When you focus on another thread, what happens to the thread that you were viewing? Depth Thread Color Top Middle Bottom Switch to high power and focus on one thread, then focus on another thread. What do you notice about the depth of field? Can you see as much of the thread in focus at the high power as you could at the low power magnification? 7. View an animal cell: a. Observe the prepared slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the nucleus in several cells. Locate the cytoplasm and the plasma membrane. On a sheet of paper, make a drawing of a few cells, and label the observed parts Hands-On Labs, Inc.

34 8. View a plant cell: a. Observe the slide under the microscope, beginning with the scanning lens and then proceeding to higher magnification levels. Locate the cell wall and the nucleus in several cells. Make a drawing of a few cells and label the observed parts. b. Count a column of cells, stacked end-to-end, across the field of vision under high-power magnification. c. F. Based on the field of vision measurement you calculated above, compute the average length of one cell in the column of cells with this formula: μm average length of cell = μm diameter of field of view total number of cells in the column Hands-On Labs, Inc.

35 d. What differences were noted between the animal cells and the plant cells? e. How do the differences dictate the form of the organism? Discussion A. What is the purpose of staining cells before viewing them under a microscope? B. What type of microscope would you use to view the following organisms? There may be more than one correct response for each. Strep Throat culture Mitochondria in an animal cell Structure of a bird feather Chloroplasts in a leaf cell Earthworm digestive system DNA structure in the nucleus Cells from plant leaf Enterococcus bacteria Spores from a mushroom Herpes simplex 73 Hands-On Labs, Inc.

36 C. Summarize the capabilities of each of the microscopes listed in Table 2-3 below. Table 3: Summary of Microscope Capabilities Type of Microscope Dissecting Type of Detection Beam Magnification Range, metric Examples of Viewable Specimens Compound Light Scanning electron Transmission electron 74 Hands-On Labs, Inc.

37 Laboratory Summary 1. What have you learned from doing this laboratory? 2. Why is the information presented in this laboratory intrinsic to all future studies in biology? 75 Hands-On Labs, Inc.