Laboratory Manual for General Biology I (BSC 1010C) Lake-Sumter Community College Science Department Leesburg

Transcription

1 Laboratory Manual for General Biology I (BSC 1010C) Lake-Sumter Community College Science Department Leesburg

2 Table of Contents Note to Students... 3 Exercise 1 - Measurements and Lab Techniques... 4 Exercise 2 - Functional Groups, Organic Molecules, Buffers, and Dilutions Exercise 3 - Qualitative Analysis of Biological Molecules Exercise 4 - The Microscope Exercise 5 - Cell Structure and Membrane Function Exercise 6 - Enzyme Activity Exercise 7 - Respiration Exercise 8 - Photosynthesis Exercise 9 - Cell Division Exercise 10 - DNA Fingerprinting Exercise 11 - Genetics A significant portion of this lab manual is used with the kind permission of the Science Department at Seminole State College, Sanford, Florida. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 2

3 Note to Students Students should read and study the exercises before coming to the laboratory and should supply themselves with the necessary materials including the text book, lecture notes, laboratory manual, calculators, pens, and pencils. All students are required to wear appropriate clothing to lab as outlined by the lab instructor as well as follow all safety precautions during laboratory exercises. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 3

4 Exercise 1 - Measurements and Lab Techniques Introduction In scientific experiments, observation and accurate measurements are essential. The investigations in this exercise will familiarize you with some of the methodologies and equipment in use in biology laboratories. Your objective is to learn to correctly select and use equipment to obtain accurate results, while avoiding damage to the equipment or yourself. Materials Equipment meter sticks metric rulers blocks of various sizes irregularly shaped objects (fossils, rocks, bones, etc.) 500 ml graduated cylinders triple beam balances Part A: The Metric System Scientific measurements are expressed in the units of the metric system or its modern day successor, the International System of Units (SI). We will use this system exclusively throughout this course. The metric system was invented by the French vicar Gabriel Moutin in 1670 and officially adopted as the standard for weights and measures in France in Since then it has spread throughout much of the rest of the world. Although the United States traditionally uses the English system, its use has become more common in recent years. You may have even noticed canned goods and drinks in grocery stores are given in metric as well as English units. Just like in the English system, the metric system has three categories of units. For distance, it is meter, for volume, liter, and for mass, gram. The metric system makes use of prefixes to change the value of the unit in multiples of 10 (Table 1.1) Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 4

5 Exercise 1 Measurements and Lab Techniques Table 1.1. Metric System Units Exponential multiplier Length Volume Mass 10 3 kilometer (km) kiloliter (kl) kilogram (kg) 10 2 hectometer (hm) hectoliter (hl) hectogram (hg) 10 1 decameter (dam) decaliter (dal) decagram (dag) 10 0 = 1 meter (m) liter (l) gram (g) 10-1 decimeter (dm) deciliter (dl) decigram (dg) 10-2 centimeter (cm) centiliter (cl) centigram (cg) 10-3 millimeter (mm) milliliter (ml) milligram (mg) These units have no prefixes 10-6 micron (µ) microliter (µl) microgram (µg) These units have no prefixes 10-9 nanometer (nm) nanoliter (nl) nanogram (ng) Use this mnemonic device to remember the order of the prefixes: kids have dropped over dead converting many blank blank metric blank blank numbers Conversion between related units is accomplished by moving the decimal point the appropriate number of places left or right (Fig. 1.1). Fig. 1.1 Metric Unit Conversion Staircase kilo (k) hecto (h) deca (dam) m, l, g deci (d) centi (c) milli (m) micron (µ) nano (n) Move up the staircase to larger units, down to smaller ones. As example, to convert decimeters (dm) to millimeters (mm), move the decimal point 2 places to the right (3735). Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 5

6 Exercise 1 Measurements and Lab Techniques Fill in the basic metric unit for each measurement in Table 1.2 Table 1.2 Basic Metric Units Measurement Length Volume Mass Basic Metric Unit Carry out the metric conversions in Table 1.3. Table 1.3 Practice Metric Conversions 550 ml l 3.7 g mg 20 km m 78.4 cm mm 212 µl ml 67.5 dam µm 500 µm mm Part B: Length Measurements Length measurements are made with a metric ruler. When using a linear device, you should extend your answer at least to the finest divisions on the device. For example, if you have a meter stick with markings to the millimeter, you could measure your height to the nearest millimeter (e.g., 1754 millimeters or meters). The size of objects falling between marked divisions may be interpolated. Interpolation is an estimation how the distance an object extends between the smallest marks on the device. Part B1: Metric Height Procedure 1. Obtain a meter stick 2. Find a partner and stand them with their back against a wall or door frame 3. Make a small mark at the level of the top of their head 4. Measure this height in centimeters making the most accurate measurement you can with the meter stick 5. Repeat the procedure with yourself and record your height here cm Part B2: Calculating Surface Area to Volume Ratios (SA : Vol) w h l Procedure 1. Use the dimensions given in table 1.4 for various block sizes, calculate total surface area and volume and enter in Tables 1.5 and 1.6 Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 6

7 Exercise 1 Measurements and Lab Techniques Table 1.4 Block Dimensions Small Medium Large l (cm) w (cm) h (cm) Calculating Surface Area: Surface area of a rectangular block = 2 (l x w) + 2 (l x h) + 2 (w x h). Use the data in Table 1.4 to fill in Table 1.5. Table 1.5 Surface Area Calculations Small Medium Large calculations calculations calculations SA cm 2 SA = cm 2 SA = cm 2 Calculating Volume: Volume of a rectangular block = l x w x h Fill in Table 1.6 Table 1.6 Volume Calculations Small Medium Large calculations calculations calculations Vol cm 3 Vol cm 3 Vol cm 3 Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 7

8 Exercise 1 Measurements and Lab Techniques Calculating Surface Area : Volume (SA : Vol) Divide the surface area (cm 2 ) by the volume (cm 3 ) recording your answer in Table 1.7 Table 1.7 Surface Area, Volume, and SA: Vol Surface area (cm 2 ) Volume (cm 3 ) SA : Vol Small Medium Large Use the data from Table 1.7 to construct a bar plot in Fig Fig. 1.2 Relationship Between SA : Vol and Block Size The plot just constructed provides a visual illustration of the changes in SA : Vol with blocks of different volumes. Describe the kind of relationship you see: This SA : Vol ratio is very important in biology and helps to explain why cells have typically not grown larger than microscope size. The SA : Vol affects the movement of materials in and out of cells. Very small cells have high ratios and can usually supply most all the cell s transportation requirements through diffusion. But, as you noticed in this procedure an object s ratio decreases relatively quickly as it grows in size. This larger size means less surface area is available per unit of volume. The result is as cells grow larger, diffusion is not longer sufficient to meet all the cells needs. Cells must either divide to maintain that larger ratio or develop elaborate internal transport mechanisms. These topics will be discussed further in later sections of this course. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 8

9 Exercise 1 Measurements and Lab Techniques Part C: Measuring Volume of Irregular Shaped Solids Calculation of the volume of regularly shaped objects like rectangular blocks or spheres is straightforward. However, how can we obtain the volume of something like a piece of bone, or rock, or a fossil? Their irregular shapes preclude the use of any formula. However, two important facts are useful to remember o A submerged object will displace an amount of water equal to its volume o 1 ml = 1 cm 3 Procedure 1. Obtain a 500 ml graduated cylinder 2. Fill cylinder to about the midway mark with tap water 3. Note the level of water in the cylinder in ml Reading a graduated cylinder Graduated cylinders are marked off in volume units Larger units are indicated (e.g., 10 ml, 20 ml, 50 ml, etc.) Smaller units are not marked but are indicated You must pay attention to these smaller, unmarked units to get an accurate reading for volume Due to capillary attraction, a liquid in a graduated cylinder will not form a flat surface. Instead, it curves up the sides forming a dip or meniscus. By convention, we always read the volume of the liquid from the bottom of the meniscus (Fig. 1.3) Fig. 1.3 Graduated cylinder readings (record you readings in the blanks) _ ml _ ml _ ml 4. Being careful not to splash out any of the water in the cylinder, submerge the irregularly shaped object. Make sure it is completely underwater. Objects that float should be held underwater 5. Make note of the level of water in the graduated cylinder again 6. Subtract the initial volume of water from this final reading (express your answer in cm 3 ) 7. Record your data in Table 1.8 Table 1.8 Water Displacement Data Irregularly shaped object Volume (cm 3 ) Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 9

10 Exercise 1 Measurements and Lab Techniques Part D: Measuring Mass and Density Procedure 1. Use a triple-beam balance to determine the mass (in grams) of the objects listed in Table Calculate the volume of these objects using the methods described previously 3. Calculate density of each object Density = mass (g) / volume (ml or cm 3 ) 4. Record your answers in Table 1.9 Table 1.9 Mass, Volume, and Density of Various Objects irregularly shaped object _ small block medium block Mass (g) Volume (cm 3 or ml) Density (g / cm 3 or ml) The density of water is 1 g /ml or cm 3. In comparing the densities of the objects in Table 1.9 to the density of water, Which objects float? The densities of these objects are than that of water. Which objects sink? The densities of these objects are than that of water. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 10

11 Exercise 1 Measurements and Lab Techniques Practice Problems 1. Calculate the surface area and volume of a rectangular solid measuring 8.6 cm in length, 2.4 cm in width, and 3.8 cm in height (use appropriate units). The mass of this block is g. What is its density and will it sink or float in water? 2. Calculate the surface area and volume of a rectangular solid measuring 43 mm in length, 12 mm in width, and 19 mm in height (report your answer in cm 2 and cm 3 ). The mass of this block is 8.5 g. What is its density and will it sink or float in water? Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 11

12 Exercise 1 Measurements and Lab Techniques 3. Initial volume of water in a graduated cylinder is 0.26 l. Completely submersing an irregularly shaped object into the water raises the water level to 512 ml. What is the volume of the object (express your answer in cm 3 )? The mass of this object is 60 g. What is its density and will it sink or float in water? 4. A principle of ecology known as Bergmann s rule states an organism of a given species will be larger in colder latitudes than those in warmer ones. For example, grey squirrels (Sciurus carolinensis) in Florida are significantly smaller than their counterparts in New York. Using what you have learned about changes in surface area with volume and its implications for membrane transfer, provide a scientifically reasonable explanation for this observation. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 12

13 Exercise 2 - Functional Groups, Organic Molecules, Buffers, and Dilutions Introduction An overwhelming majority of the elements listed on the periodic table are naturally occurring. A much smaller proportion of those are found in living systems in anything other than trace amounts. Six of those elements are most abundant (CHNOPS): Carbon (C) Hydrogen (H) Nitrogen (N) Oxygen (O) Phosphorus (P) Sulfur (S) Other elements of biological significance include sodium, potassium, calcium, magnesium, iron, and chlorine. Atoms of these elements combine through bonding in a variety of ways to form molecules. This exercise will examine some of the basic combinations of atoms that form molecules. principles of ph and buffers, as well as dilutions will also be covered. Basic Materials Equipment spectrophotometers molecular model kits cuvettes cuvette racks Kimwipes Test tubes and racks 10 ml pipettes pipette pumps 50 ml beakers marking pencils Reagents and Solutions Bogen s Universal Indicator 1M NaOH 1M HCl ph 4 buffered solution ph 4 unbuffered solution colored dye stock solution, 100% distilled water unknown dye solutions Part A: Functional Groups and Biologically Important Molecules Most biological molecules are held together by covalent bonds. Covalent bonds result in relatively stable molecules that do not dissociate in aqueous (water) environments. These stable molecules can serve as monomers (building blocks or subunits) for the synthesis of larger dimers (2 monomers) or polymers (chains of many monomers). Biological molecules are classified according to their functional groups. Functional groups are clusters of atoms bonded to carbon backbones and are most commonly involved in chemical reactions. They impart particular characteristics to larger molecules to which they are attached. For example, any molecule with a carboxyl group behaves as an organic acid like fatty acids or amino acids. Those with a hydroxyl group are considered alcohols (e.g. glycerol). Carbohydrates contain a carbonyl group (either an aldehyde if it s at the end of the molecule or a ketone if not) along with a number of hydroxyl groups. Table 2.1 illustrates some of the more biologically important functional groups. In this table, each line represents one covalent bond. Single and double bonds can exist. Each functional group bonds to a carbon backbone, often symbolized by the letter R (e.g. R-OH would be a molecule containing a hydroxyl functional group). Each functional group must have at least one covalent bond available for attachment to this carbon backbone. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 13

14 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions Table 2.1 Biologically Important Functional Groups Carbonyl Hydroxyl Aldehyde OH C O H Ketone C O C O Carboxyl OH Amine Phosphate Sulfhydryl N H H H O O P OH O S H Procedure 1. Fill in Table 2.2 using the periodic chart in your text. Table 2.2 Elements Represented in Molecular Model Kits Element Atomic Symbol Atomic Number # of Valence Electrons # of e - s needed to fill valence shell Carbon Hydrogen Nitrogen Oxygen Phosphorus 2. Obtain a molecular model kit 3. Examine the colored balls to determine the number of holes in each. Each ball represents an atom of a particular element. The holes represent the valence (bonding capacity) of the atom. Using the information in Table 2.2, you should be able to determine which elemental atom is represented by each ball Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 14

15 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions 4. Use the molecular kit to construct models of each of the functional groups in Table 2.1. Use the appropriate colored ball to represent each atom. The grey sticks are bonds. Use the longer sticks to bend to create double bonds. When building functional groups, you will always have one free end of a stick that represents the attachment point of the functional group to the carbon backbone ( R ). Pay attention to the content and shape of each functional group Circle and label the functional groups within these biologically important molecules in Fig Fig. 2.1 Some Biologically Important Organic Molecules H HO H H H H C C C C C C H O OH H OH OH OH glucose chain (hydroxyl, aldehyde) H H OH H C H OH H OH O H OH glucose ring (hydroxyl) H OH H HO H H H H C C O C C C C H OH H OH OH OH fructose chain (hydroxyl, ketone) H H C H OH OH O H OH OH H fructose ring (hydroxyl) H C OH H H H N H C H O C glycine (amine, carboxyl) OH H H H C C C H glycerol (hydroxyl) OH OH OH Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 15

16 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions Part B: Buffers The ph of blood and other body fluids is relatively insensitive to the addition of acids or bases. This is due to the presence of buffers in living systems which help to maintain homeostasis by maintaining normal ph levels. The ph of a solution can be determined in a variety of ways, including the use of ph meters, litmus paper, and chemical reagents. In this exercise, we will use the chemical reagent Bogen s Universal Indicator to determine ph of specific solutions. Bogen s Universal Indicator changes color at specific ph end points: Pink = ph 4 Yellow = ph 6 Green = ph 7 Blue = ph 9 Violet > ph 9 In order to determine the effect of buffers on ph, we will attempt to raise the ph of an unbuffered acid solution by adding small amounts of a base. For comparison, we will repeat this procedure with a buffered acid solution. Once both solutions are basic, we will attempt to return them to the original ph by adding small amounts of acid. Procedure 1. Obtain two 50 ml beakers and label them A and B 2. Pipette 10 ml of an unbuffered ph 4 solution into beaker A 3. Pipette 10 ml of a buffered ph 4 solution into beaker B 4. Add 3 drops of Bogen s Universal Indicator to each beaker 5. Note the color. Is this color expected? 6. Slowly add 1M sodium hydroxide (NaOH) one drop at a time to beaker A, swirling the beaker between each drop. Do until you detect a permanent color change to violet 7. Record the number of drop required to change the color to violet in Table Repeat the last two steps with beaker B The test you just performed illustrated the effect of a buffer when you attempted to increase the ph (make it more basic). Did the buffered solution require more or less (circle one) drops to change the ph? Do you suppose buffers would resist ph changes in either direction? Continue the procedure from above 9. Slowly add 1M hydrochloric acid (HCl) one drop at a time to beaker A, swirling the beaker between each drop. Do until you detect a permanent color change to pink 10. Record the number of drops required to change the color to pink in Table Repeat the last two steps with beaker B Table 2.3 The Effect of Buffer on ph Change Beaker Contents # drops to violet # drops back to pink A B unbuffered, ph 4 solution buffered, ph 4 solution Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 16

17 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions Part C: Dilutions Part C1: Basic Dilutions During scientific experiments, it is often necessary to dilute the solution provided (the stock solution). For example, such a dilution might be made to reduce chemical concentrations so the rate and intensity of reactions can be controlled. A stock (100%) dye solution and distilled water will be used in this lab. How would you go about preparing 10 ml each of 75%, 25%, and 10% solution from an available stock solution of 100%? The algebraic equation C 1 V 1 = C 2 V 2 provides our tool to answer this question, where C 1 = concentration (%) of stock solution V 1 = volume (ml) or stock required to prepare the solution (you typically are solving for this variable) C 2 = concentration (%) of dilution you wish to prepare V 2 = volume (ml) of dilution you wish to prepare Procedure 1. Use the algebraic equation to determine volumes of 100% stock (ml) and distilled water (ml) required to create 10 ml each of 0%, 10%, 25% and 75% dilution. Record your answers in Table 2.4. Table 2.4 Volumes Needed to Prepare Dilutions Concentrations C 2 10% 25% 75% Volume of stock solution (ml) - V 1 Volume of water (ml) Total volume of dilution (ml) - V 2 2. Obtain 3 test tubes and a test tube rack 3. Prepare the three dilutions from Table 2.4 by pipetting the correct amount of stock in the test tube first and then diluting the stock with the correct amount of distilled water. There should be the same amount of liquid in each test tube when you are finished 4. Obtain 5 cuvettes on a cuvette rack 5. Transfer distilled water (0% dye solution) to the first cuvette up to about ¾ full. Distilled water is used as a blank solution to calibrate the spectrophotometer 6. One at a time and in order of increasing concentration, transfer enough of the other 4 solutions so that each cuvette is approximately ¾ full 7. Set the spectrophotometer to a wavelength of 450nm 8. Read the % light transmittance for each dye solution you prepared and record your results in Table 2.5 Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 17

18 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions Table 2.5 % Light Transmittance Associated with Various Concentrations of Dye Dye Solution % Concentration of Dye % Light Transmittance 1 0 (DH 2 O only) (stock) Unknown A, B, C, D (circle yours) What relationship exists between concentration of dye and % light transmittance? Part C2: The Standard Curve Procedure 1. Plot the 0%, 10%, 25%, 75%, and 100% data from Table 2.5 on Fig Attempt to draw a best fit line through the scatter of data points. Do not simply connect the dots. Make your line pass through the average spread of the dots. This line represents a standard curve and illustrates the relationship between percent concentration of a dye solution and percentage of light transmitted. Use this standard curve to complete Part C3 Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 18

19 % Light Transmittance Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions Fig. 2.2 Standard Curve Relating Dye Concentration to % Light Transmittance Dye Concentration (%) Describe the kind of relationship you see: Part C3: Determination of Unknown Dye Concentration Procedure 1. Select a cuvette of unknown dye concentration (letters A-D) from the samples available 2. Record the letter of your unknown in Table Use the calibrated spectrophotometer to read the % transmittance of your unknown dye concentration solution. Record in Table Determine the concentration of your unknown by finding the value of % transmittance on the Y- axis of Fig. 2.2 and drawing a perpendicular line down from that point to where it crosses the X- axis. That intersection point is the percent dye concentration of your unknown. Record that in Table Return your unknown cuvette to your instructor and tell them your result 6. Rinse out the rest of the cuvettes and place them on the cuvette rack. Do not scrub them with a test tube brush as it will scratch and render them useless Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 19

20 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions Practice Problems and Review Questions 1. Given a stock solution of 2.0% dextrose, how would you prepare 10 ml of each of the following solutions? a. 0.1% dextrose solution b. 1.0% dextrose solution c. 0.5% dextrose solution 2. Given a stock solution of 5.0% sodium chloride (NaCl), how would you prepare 20 ml of each of the following solutions? a. 2.0% sodium chloride solution b. 0.5% sodium chloride solution c. 3.0% sodium chloride solution Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 20

21 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions 3. Given a stock solution of 10% dextrose, how would you prepare 5 ml of a 0.9% dextrose solution? 4. Given a stock solution of 0.9% dextrose, how would you prepare 5 ml of a 0.5% dextrose solution? 5. Given a stock solution of 0.5% dextrose, how would you prepare 5 ml of a 0.004% dextrose solution? 6. How would you prepare 25 ml of a 15% dye solution beginning with a 20% stock dye solution? 7. How would you prepare 9 liters of a 50% dye solution beginning with a 60% stock dye solution? Express your answer in ml. 8. How would you prepare 600 ml of a 20% starch solution beginning with a 50% stock starch solution? Express your answer in liters. 9. You have 10 ml of a 60% stock dye solution. What is the maximum amount of a 12% dye solution you could prepare? Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 21

22 Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions 10. How would you go about preparing the 12% dye solution in question 9? 11. What are buffers and why are they biologically important? 12. List the functional groups present in each of these molecules glucose fructose glycine glycerol 13. List some possible polymers that can be formed from each of these monomers glucose fructose glycine glycerol Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 22

23 Exercise 3 - Qualitative Analysis of Biological Molecules Introduction Macromolecules are large molecules formed from aggregates of smaller ones. Biological macromolecules are typically classified as carbohydrates, lipids, proteins, and nucleic acids. It is possible to identify macromolecules and monomers by using chemical indicators. Reagents used as chemical indicators express their results either qualitatively or quantitatively by determining the presence or relative amount of a substance in a solution. The example in Table 3.1 should help you understand the basic difference between qualitative and quantitative analyses. The reagents used in this exercise provide qualitative results. Each reagent exhibits a visible color change in the presence of a specific substance; however, it does not provide an amount (quantitative) result. A qualitative test will also be used to track the step-by-step hydrolysis of the polymer starch, a polysaccharide, into its glucose (monosaccharide) monomers. Table 3.1 A Case Study Illustrating the Difference Between Qualitative and Quantitative Analyses Case Study You are given a beaker containing 100 ml of an aqueous solution A B Question Are proteins present in this solution? How many mg of protein are dissolved in this 100 ml solution? Would smelling, tasting, or Changing the solution s color touching the solution help indicated proteins are present, determining if it has proteins or but it does not detect exactly not? (not a good idea in lab) how much protein is present. Thinking Response The best thing to do is add a protein indicator. If the solution changes color, then proteins are present. A qualitative analysis must be performed. An analytical test giving the answer in numbers, not just by presence or absence, needs to be done. A quantitative analysis must be performed. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 23

24 Exercise 3 Qualitative Analysis of Biological Molecules Materials Equipment test tubes and racks 10 ml pipettes pipette pumps 10 ml graduated cylinders marking pencils (Sharpie) filter paper disks Petri dish water baths at 95 C Reagents and Solutions 1% dextrose (glucose) 6% starch (amylose) 1 M NaOH apple juice chicken broth egg white whole milk vegetable oil distilled water Benedict s IKI Biuret Sudan IV Part A: Detection of Carbohydrates Carbohydrates are molecules consisting of one (monosaccharide), two (disaccharide), or many (polysaccharide) simple sugars. Examples of carbohydrates include glucose, sucrose, glycogen, maltose, and starch (amylose). In this exercise, you will experiment with two carbohydrate reagents: Benedict s reagent usually light blue in color, forms a yellow-green, orange, or red precipitate when boiled in the presence of reducing sugars such as simple sugars (e.g. glucose) Iodine-Potassium Iodide (IKI) amber colored, forms a dark purple or black precipitate in the presence of starch. Read the information on the following pages (Parts A1, A2, and A3) and fill in the first three columns of Table 3.2 before performing the experiments. Part A1: Detection of Simple Sugars Procedure 1. Obtain a test tube rack and six test tubes per group 2. Label the test tubes 1 through 6. #1 and #2 will be used in this part 3. Use a 10 ml pipette to transfer 1 ml of the dextrose (glucose) solution to test tube #1 4. Use a different (why?) pipette to transfer 1 ml of the starch solution (swirl to mix before transferring) to test #2 5. Use a 10 ml graduated cylinder to measure and transfer 1 ml of Benedict s reagent to each test tube. Swirl to mix 6. Note the color of each solution 7. Gently heat the contents of each test tube in a 95 C water bath for two minutes 8. Observe and record any color change in Table 3.2 Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 24

25 Exercise 3 Qualitative Analysis of Biological Molecules Part A2: Detection of Starch Procedure 1. Use a pipette to transfer 1 ml of dextrose solution to test tube #3 2. Use a different pipette to transfer 1 ml of starch solution (swirl to mix before transferring) to test tube #4 3. Add one drop of IKI reagent to each test tube and swirl gently 4. Observe and record any color change in Table 3.2 Part A3: Identification of a Carbohydrate Unknown If you were given an unknown solution and had to perform both the simple sugar (Part A1) and the starch (Part A2) tests in the same test tube, which test would you perform first? The following experiment will help to answer this question. Procedure 1. Use a pipette to transfer 1 ml of dextrose to both test tubes #5 and #6 2. Use a different pipette to transfer 1 ml of starch to both test tubes #5 and #6 3. In test tube #5, perform the Benedict s test first 4. Make note of any color changes 5. After the Benedict s test perform the IKI test in test tube #5 6. In test tube #6, perform the IKI test first 7. Make note of any color changes 8. After the IKI test perform the Benedict s test in test tube #6 9. Make note of any color changes 10. Record your observation in Table From the results of test tubes #5 and #6, determine which test you should run first if you were limited to using just one test tube and had to test for both simple sugars and starch. Only one of these two test tubes will allow you to see the results of both tests correctly Which test would you perform first and why? 12. Obtain a simple sugar / starch unknown (labeled A, B, C, and D) and test it using the proper sequence of Benedict s and IKI reagent 13. Record the letter of your unknown and any color changes in Table 3.2 What (water, glucose, starch, or both) was in your unknown? Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 25

26 Exercise 3 Qualitative Analysis of Biological Molecules Table 3.2 Qualitative Analysis of Simple Sugars, Starch, and a Carbohydrate Unknown Test Tube Test Solution Reagent Hypothesis Results Benedict s 1 st IKI 2 nd 6 IKI 1 st Benedict s 2nd Unknown (_) Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 26

27 Exercise 3 Qualitative Analysis of Biological Molecules Part B: Detection of Lipids A lipid is a non-polar (hydrophobic) organic molecule which is insoluble in water. One type of lipid are fats, also called triglycerides or triacylglycerols. A fat molecule is composed on one glycerol and three fatty (palmitic) acid molecules. Sudan IV-lipid complex will produce an orange spot on filter paper to which lipid has been added. Procedure 1. Obtain a blank filter paper disk A C 2. Mark the disk with a pencil following the pattern as shown in this figure A apple juice C chicken broth W E egg white O E M whole milk O vegetable oil M W distilled water (control) 3. Make a hypothesis as to which of the above substances you would expect to contain lipids 4. Record this hypothesis in Table Transfer a small drop of each substance to the appropriate circle on the filter paper 6. Allow the filter paper to dry 7. Once dry, soak the filter paper for 3 minutes in a petri dish containing Sudan IV reagent. Leave the dish on the counter where it was originally to avoid spillage 8. Remove the filter paper disk with forceps and gently rinse with distilled water over the sink for one minute 9. Hold the filter paper over something white for contrast and observe the results 10. Examine the color for the six spots and indicate whether the substances contained lipid using the by indicating - for negative (no color change; no lipid) and + for positive (color change; lipid) 11. Record your results in Table Compare your results to your hypothesis Table 3.5 Sudan IV Test for Lipids Substance Tested Hypothesis Result Apple juice Chicken broth Egg white (albumin) Whole milk Vegetable oil Distilled water Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 27

28 Exercise 3 Qualitative Analysis of Biological Molecules Part C: Detection of Proteins Proteins are polymers of amino acids in which the carboxyl functional group of one amino acid forms a peptide bond with the amine functional group of another amino acid. H H N HR C H O C OH + H H N HR C H O C OH H 2 O H H N HR C H O C HOH H N peptide bond HR C H O C OH Biuret reagent, which is pale blue, contains copper sulfate (CuSO 4 ). The Biuret reaction is based on the complex formation of cupric ions with proteins. In this reaction, copper sulfate is added to a protein solution in strong alkaline solution. A purplish-violet color is produced, resulting from the complex formation between the cupric ions and the peptide bond. Procedure 1. Obtain a test tube and rack and six clean test tubes per group 2. Mark the test tubes with the same symbols used in the lipid experiment (Part C) 3. Make a hypothesis as to which of the above substances you would expect to contain proteins 4. Record this hypothesis in Table Transfer 1 ml (approximately 20 drops) of the appropriate solution to properly marked test tube 6. Dispense 1 ml of 1M NaOH into each test tube 7. Swirl gently to mix 8. Add 0.5 ml of 1% Biuret reagent to each test tube 9. Swirl gently to mix 10. Look for any instant change in color from blue to violet. This is the positive test for proteins 11. Record your results in Table 3.6 using the same symbols (- and +) as described in Part C 12. Compare your results to your hypothesis Table 3.6 Biuret Test for Proteins Substance Tested Hypothesis Result Apple juice Chicken broth Egg white (albumin) Whole milk Vegetable oil Distilled water Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 28

29 Exercise 3 Qualitative Analysis of Biological Molecules Practice Problems and Review Questions 1. Explain the difference between a qualitative and quantitative analysis test. 2. What substance is used as a control in the a. Sudan IV test? b. Biuret test? 3. Complete the following table concerning the reagents used in detecting these test substances. Test Substance Reagent Test Procedure Color of Positive Result Color of Negative Result Starch Sugar Lipid Protein Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 29

30 Exercise 3 Qualitative Analysis of Biological Molecules 4. In which order must the sugar and starch test be run? Why? 5. What are the differences among polysaccharides, oligosaccharides, disaccharides, and monosaccharides? 6. What are the two primary components of a triglyceride? 7. What are the monomers that make up proteins? 8. List and briefly describe the four levels of protein structure. 9. How do proteins of foods differ from those of the organism consuming them? 10. Name a molecule of living systems other than protein which contains nitrogen. 11. What is hydrolysis? Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 30

31 Exercise 4 - The Microscope Introduction The microscope is an essential tool in modern biology. It allows us to view structural details of organs, tissue, and cells not visible to the naked eye. This laboratory exercise is designed to demonstrate some of the potential uses of various types of light microscopes and to help you become familiar with proper microscopic techniques. Materials Equipment compound microscope dissecting microscope microscope slides coverslips droppers lens paper forceps toothpicks Biological Specimens Allium (onion) pond water Prepared Slides newspaper print colored threads Paramecium Reagents IKI methylene blue Detain (or Protoslo) Part A: Care and Use of the Compound Microscope ALWAYS CARRY THE MICROSCOPE UPRIGHT WITH TWO HANDS, ONE ON THE BASE, THE OTHER ON THE ARM MAKE SURE YOUR WORKBENCH IS FREE OF CLUTTER BEFORE YOU PLACE THE MICROSCOPE ON THE BENCH DO NOT DRAG OR SHOVE THE MICROSCOPE ACROSS THE LAB BENCH ALWAYS LIFT TO MOVE OR TURN IT The steps on the next few pages represent the correct procedure for viewing a specimen under a compound microscope. Your instructor will demonstrate the proper use of the microscope as well as describe its features. Refer to Fig. 4.1 to familiarize yourself with the parts of the microscope as you study each step in the procedure. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 31

32 Exercise 4 The Microscope Fig. 4.1 The Compound Light Microscope Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 32

33 Exercise 4 The Microscope Viewing a Specimen with a Compound Light Microscope Procedure 1. Clean the slide and coverslip by rubbing them gently with lens paper 2. Use the coarse focus adjustment knob to maximize the working distance (the distance between the stage and the objective lens) 3. Rotate the revolving nosepiece into position with the scanning power (4x) objective lens in the viewing position 4. Center the slide holder of the mechanical stage on the microscope stage 5. Place the slide between the stage clip and push it all the way back to the bar 6. Plug in the microscope and turn on the light switch 7. Using the mechanical stage drive knobs, center the coverslip and specimen over the stage aperture 8. While carefully watching the slide on the stage, use the coarse focus adjustment knob to move the specimen towards the scanning objective lens until it stops. The stage will come close to the lens but will not touch it 9. Adjust the interpupillary distance until you see a single circle while looking through the microscope with both eyes open. This circle of light is called the field of view 10. While looking through the ocular lenses, turn the coarse and fine focus adjustment knobs of the microscope until you see something you believe is the specimen. Stop. Move the slide back forth using the mechanical stage drive knobs. The item you thought was specimen should likewise be moving back and forth 11. Cover of close the eye that is not looking through the ocular containing the diopter ring. Viewing with only that eye focus using the coarse and fine focus adjustment knobs. Adjust the light using the iris diaphragm adjustment lever and/or the light adjustment. Then close your other eye adjusting the diopter ring on that ocular lens to bring the object into focus 12. Adjust the condenser to the highest position 13. Using the mechanical stage drive knobs, center the specimen of choice in the viewing area 14. These microscopes are parfocal (if one lens is in focus, all other lenses are, at least, close to focus). In order to change to the next highest magnification, simply rotate the nosepiece to the low power (10x) objective lens 15. These microscopes are also parcentral (if an object is in the center of the field of view for one lens, it will be, at least, close to the center of the field of view at other lenses) 16. Using the mechanical stage drive knobs, re-center the specimen in the viewing area 17. With the low power (10x) objective, use the coarse and fine focus adjustment knobs to focus the view of the specimen and the iris diaphragm adjustment lever to increase the light intensity on the specimen 18. Re-center the specimen in the field of view. Rotate the nosepiece to the high power (40x) objective lens. Use the FINE FOCUS ADJUSTMENT KNOB ONLY to focus and the iris diaphragm adjustment lever to increase the light intensity on the specimen. If needed, use the light adjustment to provide additional light 19. When removing the slide, rotate the nosepiece so the scanning power (4x) objective is in the viewing position, then use the coarse focus adjustment knob to maximize the working distance 20. After you have completed the laboratory activity, turn the light switch off. Clean all microscope lenses (objective and ocular) with lens cleaner and lens paper Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 33

34 Exercise 4 The Microscope 21. Prepare the microscope for storage using the checklist below. Be sure a. The scanning power (4x) objective is in the viewing position b. The mechanical stage has been positioned so the stage arm is flush with the right side of the stage c. The cord is wrapped securely around the microscope arm d. The stage has been adjusted all the way down e. The condenser has been adjusted all the way up f. The light adjustment is turned all the way down and the light is turned off Part B: Magnification There is a set of three objective lenses on your microscope. The magnification (or power) of each objective lenses is engraved on the side of the objective. The ocular lens is also normally engraved with its magnification (typically 10x). To determine the total magnification of a specimen, use the following formula: Total Magnification = Ocular Magnification x Objective Magnification Procedure 1. Use Table 4.1 to record the magnification values for each objective lens and the ocular lens on the microscope 2. Calculate total magnification (using the formula above) for each objective lens and record n Table 4.1 Table 4.1 Total Magnification of Microscope Objective Lens Name Magnification Objective Lens Ocular Lens Total Scanning Low Power High Power Part C: Working Distance and Diameter of the Field of View Part C1: Working Distance Working distance is the distance between the stage and objective lens (Fig 4.2). Because objective lenses vary in lengths, the working distance will change as you switch from one objective lens to the next. In a microscope, as magnification increases, working distance. Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 34

35 Exercise 4 The Microscope Fig. 4.2 Working Distances with Various Objective Lenses Part C2: Diameter of Field of View The approximate size of a specimen can be estimated if the diameter of the field of view (DFV) is known. In parfocal microscopes, if we know the magnification and DFV for one objective lens, we can calculate the DFV for a second objective on the same parfocal microscope using the following formula: M 1 x DFV 1 = M 2 x DFV 2 where M 1 and DFV 1 = magnification and diameter of the field of view, respectively, of objective 1, M 2 and DFV 2 = magnification and diameter of field of view, respectively, of objective 2. As magnification increases, the diameter of the field of view (Fig. 4.3). Fig. 4.3 Diameter of the Field of View (DFV) with Various Objective Lenses 4x 10x 40x Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 35

36 Exercise 4 The Microscope Fill in Table 4.2 for your microscope using the values given for the scanning objective and the above formula. Table 4.2 Diameter of Field of View (DFV) for the Compound Microscope Objective Lens Magnification DFV (mm) DFV (µ) Scanning 4 4 Low Power 10 High Power 40 Part C3: Depth of Focus The depth of focus for a particular objective refers to the power of the objective to produce an in-focus image from objects that are slightly different distances away from the objective lens. As magnification power increases, the depth of focus decreases. When viewing specimens under a microscope, it is beneficial to keep in mind that as magnification power increases the microscope s field of view becomes smaller, thinner, and darker (Table 4.3). Table 4.3 Changes in a Microscope s Field of View as a Function of Magnification Power Scanning Low Power High Power Diameter of Field of View (DFV) Depth of Focus Gets Smaller Gets Thinner Light Gets Darker Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 36

37 Exercise 4 The Microscope Part D: Newsprint (dry mount) Procedure 1. Obtain a prepared slide of newspaper print 2. View the newsprint under the microscope using the scanning power (4x) objective Move the slide slowly to the right as you view the image in the field of view. In which direction do the letters appear to move? Move the slide slowly away from you as you view the image in the field of view. In which direction do the letters appear to move? Part E: Depth of Focus Procedure 1. Obtain a prepared slide of colored threads. The threads have been arranged to intersect at a single point 2. Focus on the intersection of the three threads first with the scanning power (4x) objective lens and then the low power (10x) objective lens 3. Very slowly rotate the fine focus adjustment knob while looking at the intersection of the threads Which thread is on bottom? In the middle? On top? Part F: Viewing specimens Specimens are often mounted in water (or other liquids) on a glass slide and then covered with a small thin glass or plastic coverslip to prepare for microscopic viewing. These wet mounts are unstained and sometimes difficult to see. Replacement staining can add color and contrast enhancing the detail of the specimen. It is important to be able to estimate the sizes of different specimens under the microscope. Already knowing the diameter of the field of view for a particular objective (Table 4.2), we can utilize the following formula to estimate size: FV si e of cell of cells across FV At which magnification do you think you are able to get the most accurate estimate of cell number and thus the most accurate estimation of cell size?. Why? Part F1: Paramecium Procedure 1. Obtain a prepared slide of the single-celled protozoan, Paramecium 2. Use the correct focusing technique to find the Paramecium at high power 3. Estimate the # of Paramecium cells required to fill across the DFV end-to-end 4. Use the formula to calculate Paramecium length in microns Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 37

38 Exercise 4 The Microscope 5. Estimate the # of Paramecium cells required to fill across the DFV side-by-side 6. Use the formula to calculate Paramecium width in microns Paramecium cells arranged end-to-end Paramecium cells arranged side-to-side Paramecium Length (in microns) Paramecium Width (in microns) Part F2: Allium (onion) epidermis (wet mount) Procedure 1. Prepare a wet mount of Allium (onion) epidermis 2. Place one or two drops of water on a clean slide 3. Peel the epidermis (thin skin) off the inside of a piece of sliced onion using forceps 4. Place the epidermis carefully in the water on the slide 5. Place a coverslip over the epidermis 6. Observe the cells under the microscope and sketch what you see Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 38

39 Exercise 4 The Microscope 7. Stain the onion cells with IKI using the replacement staining technique a. Place a few drops of IKI on the slide against one edge of the coverslip b. Place the smooth edge of a single layer of paper towel up against the opposite edge of the coverslip. The paper towel will pull the water out from underneath the coverslip. In turn, the water as it exits will drag the IKI stain underneath the coverslip c. Continue this process, adding more IKI if necessary, until the stain covers the area under the coverslip d. Examine under the microscope 8. Observed the cells under the microscope again and sketch what you see 9. Can you see more or less detail after staining compared to the unstained cells? _ 10. Estimate the length and width of an onion cell (in microns) Onion Cell Length (in microns) Onion Cell Width (in microns) Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 39

40 Exercise 4 The Microscope Part F3: Cheek Cells (wet mount) Procedure 1. Place one or two drops of water on a clean slide 2. Obtain a clean toothpick and collect cheek cells by gently scraping the inside of your cheek 3. Swirl the tip of the toothpick in the water on the slide (immediately discard your toothpick) 4. Stain your cheek cells with methylene blue stain 5. Place a coverslip over your cheek cells 6. Observe and sketch the stained cheek cells. Identify the nucleus, cytoplasm, and cell membrane 7. How do these cells differ from onion cells? 8. Estimate the diameter of one of your cheek cells (in microns) Cheek Cell Diameter (in microns) Part G: Pond Water Although staining cells makes it easier to see their detail, most staining techniques also kill any live specimens. Thus, looking at microorganisms can be a challenge. Living microorganisms are also difficult to see clearly because many of them are motile and must be chased around the slide while you are focusing. Procedure 1. Place a drop of pond water on a clean microscope slide. Try to obtain a sample that is near any floating debris and organisms tend to congregate there. Be careful not to shake the jar 2. Add a coverslip 3. Examine under the microscope 4. Try to keep motile microorganism in focus by following them around as they move on the slide. If they move too quickly, carefully lift up the coverslip and add a drop of Detain (or Protoslo) 5. Draw a few of the critters you see in space provided Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C 40