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LAB MANUAL FALL 2010

Exercise 11 Genetics

Laboratory Manual for General Biology I (BSC 1010C)

Lake-Sumter Community College

Science Department

Leesburg

24 Aug 2010

Table of Contents

Note to Students3Exercise 1 - Measurements and Lab Techniques4Exercise 2 - Functional Groups, Organic Molecules, Buffers, and Dilutions13Exercise 3 - Qualitative Analysis of Biological Molecules25Exercise 4 - The Microscope36Exercise 5 - Cell Structure and Membrane Function51Exercise 6 - Enzyme Activity61Exercise 7 - Respiration68Exercise 8 - Photosynthesis72Exercise 9 - Cell Division81Exercise 10 - DNA Fingerprinting89Exercise 11 - Genetics104

A significant portion of this lab manual is used with the kind permission of the Science Department at Seminole State College, Sanford, Florida.

Note to Students

Students should read and study the exercises before coming to the laboratory.

Students should supply themselves with the necessary materials for laboratory including the text book, lecture notes, laboratory manual, calculators, pens, pencils.

This image in the left margin indicates a procedure that must be completed in the laboratory.

All other procedures can be accomplished through information gathered from web resources, including videos and animations, lecture notes and the text book. The instructor may also suggest additional materials to be examined.

Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C (24 Aug 2010)2

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.)

1000 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 1795. Since this 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)

Table 1.1. Metric System Units

Exponential multiplier

Length

Volume

Mass

103

kilometer (km)

kiloliter (kl)

kilogram (kg)

102

hectometer (hm)

hectoliter (hl)

hectogram (hg)

101

decameter (dam)

decaliter (dal)

decagram (dag)

100 = 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)

10-4

These units have no prefixes

10-5

10-6

micron ()

microliter (l)

microgram (g)

10-7

These units have no prefixes

10-8

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 (da)deci (d)centi (c)milli (m)micron ()nano (n)m, l, g)

Move up the staircase to larger units, down to smaller ones. As example, to convert 37.35 decimeters (dm) to millimeters (mm), move the decimal point 2 places to the right (3735).

Fill in the basic metric unit for each measurement in Table 1.2

Table 1.2 Basic Metric Units

Measurement

Basic Metric Unit

Length

Volume

Mass

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 l

______________________________ ml

67.5 dam

______________________________ m

500

______________________________ 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 1.754 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. Obtain a metric ruler

2. Obtain three rectangular blocks (small, medium, large)

3. Measure the three dimensions (length, height, width) of the block to the nearest tenth of a centimeter of the three blocks

4. Record these measurements in Table 1.4

Table 1.4 Block Dimensions

l (cm)

w (cm)

h (cm)

Small

Medium

Large

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 = _______________ cm2

SA = _______________ cm2

SA = _______________ cm2

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 _______________ cm3

Vol _______________ cm3

Vol _______________ cm3

Calculating Surface Area : Volume (SA : Vol)

Divide the surface area (cm2) by the volume (cm3) recording your answer in Table 1.7

Table 1.7 Surface Area, Volume, and SA: Vol

Surface area (cm2)

Volume (cm3)

SA : Vol

Small

Medium

Large

Use the data from Table 1.7 to construct a bar plot in Fig. 1.2.

Fig. 1.2 Relationship Between SA : Vol and Volume (cm3) of Blocks

(SA : Vol)

(Volume (cm3))

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 cells transportation requirements through diffusion. But, as you noticed in this procedure an objects 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.

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

A submerged object will displace an amount of water equal to its volume

1 ml = 1 cm3

Procedure

1. Obtain a 1000 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 Sample Graduated Cylinder Readings

36 ml53 ml6.6 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 cm3).

7. Repeat these procedures for the rest of the irregularly shaped objects.

8. Calculate the volume of a block of wood using the procedure described previously (Part B2) and using this method.

9. Record your data in Table 1.8

Which method is most accurate for determining the volume of the block of wood?

Table 1.8 Water Displacement Data

Irregularly shaped object

Volume (cm3)

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 1.9.

2. Calculate the volume of these objects using the methods described previously.

3. Calculate density of each object.

Density = mass (g) / volume (ml or cm3)

4. Record your answers in Table 1.9.

Table 1.9 Mass, Volume, and Density of Various Objects

Mass (g)

Volume

(cm3 or ml)

Density

(g / cm3 or ml)

irregularly shaped object _______________________

irregularly shaped object _______________________

small block

medium block

The density of water is 1 g /ml or cm3.

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.

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 121.6 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 cm2 and cm3). The mass of this block is 121.6 g. What is its density and will it sink or float in water?

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 cm3)? 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 Bergmanns 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.

Exercise 1 Measurements and Lab Techniques


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. Basic principles of pH and buffers, as well as dilutions will also be covered.

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

Bogens 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 its 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.

Table 2.1 Biologically Important Functional Groups

Hydroxyl

Carbonyl

Carboxyl

Aldehyde

Ketone

Amine

Phosphate

Sulfhydryl

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

Color

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.

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. 2.1.

Fig. 2.1 Some Biologically Important Organic Molecules

(fructose chain(hydroxyl, ketone)) (glucose chain(hydroxyl, aldehyde))

(fructose ring(hydroxyl)) (glucose ring(hydroxyl))

(glycine(amine, carboxyl))

(glycerol(hydroxyl))

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 Bogens Universal Indicator to determine pH of specific solutions.

Bogens 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 Bogens 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 2.3

8. 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? __________ Lets see.

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 2.3

11. 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

unbuffered, pH 4 solution

B

buffered, pH 4 solution

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 50%, 25%, and 10% solution from an available stock solution of 100%?

The algebraic equation C1V1 = C2V2 provides our tool to answer this question, where

C1 = concentration (%) of stock solution

V1 = volume (ml) or stock required to prepare the solution (you typically are solving for this variable)

C2 = concentration (%) of dilution you wish to prepare

V2 = 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%, 25%, and 10% dilution. Record your answers in Table 2.4.

Table 2.4 Volumes Needed to Prepare Dilutions

Concentrations C2

10%

25%

50%

Volume of stock solution (ml) - V1

Volume of water (ml)

Total volume of dilution (ml) - V2

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 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 cuvettes so that each cuvette is approximately full

7. Calibrate the spectrophotometer and place each cuvette into the machine one at a time. Follow the instructions for using the spectrophotometer at the end of this exercise

8. Read the % light transmittance for each dye solution you prepared and record your results in Table 2.5

Table 2.5 % Light Transmittance Associated with Various Concentrations of Dye

Dye Solution

% Concentration of Dye

% Light Transmittance

1

0 (DH2O only)

100

2

10

3

25

4

50

5

100 (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%, 50%, and 100% data from Table 2.5 on Fig. 2.2

2. 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

Fig. 2.2 Standard Curve Relating Dye Concentration to % Light Transmittance

(% Light Transmittance)

(Dye Concentration (%))

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:

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 2.5

3. Use the calibrated spectrophotometer to read the % transmittance of your unknown dye concentration solution. Record in Table 2.5

4. 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 2.5

5. 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

Using the Spectrophotometer

The spectrophotometer (Fig. 2.3) is a device to determine the percent transmittance (passing through) and/or absorbance (optical density) of various wavelengths of light through a sample. The machine you will be using, the Spectronic 20 is so named because it sends a band of light with a wavelength spread of 20 nanometers through a sample. That light passes through a prism generating a spectrum much the way a rainbow is formed. A fine slot in a wall of the machine allows only a thin slice of that spectrum through the sample. When you turn the wavelength know you are adjusting the prism so the proper 20 nanometers of light spectrum passes through the slot.

Fig. 2.3 The Spectronic 20.

Instructions

1. After plugging in the instrument, turn the On/Off switch - Zero Control knob clockwise until it clicks on. Allow a minimum of 10 minutes of warm up time before recording any measurements.

2. After warm up, select the desired wavelength by turning the Wavelength Control knob. The desired wavelength reads out on the Wavelength Readout. For this lab, the wavelength will be kept constant at 540 nm. Do not adjust the wavelength.

3. Adjust the percent transmittance to 0.0 (displayed in Absorbance Readout) using the Zero Control knob. Be sure there is no cuvette in the Sample Holder and that the lid is closed completely prior to performing this procedure.

4. While holding near the top, wipe the outside of each cuvette with Kimwipes or other lint free soft tissue to remove any fingerprints or other marks. Place a blank cuvette (one containing only distilled water) into the Sample Holder being sure the index line on the cuvette lines up with the mark on the holder. Never fill a cuvette more than full to prevent spills and never hold a cuvette anywhere other than at the very top. Always wipe down a cuvette to remove marks prior to reading in the machine.

5. Use the 100% Transmittance Control knob to set the percent transmittance to 100 (displayed in Absorbance Readout). You have now established the range of light for your samples.

6. Insert a wiped cuvette containing your sample in the Sample Holder in place of the blank. Once inserted and with the lid closed, read the resultant value from the Absorbance Readout. You may read several samples consecutively, though the machine calibration should be checked periodically to be sure it has not drifted from 100%. If it has, the machine should be adjusted as per the preceding directions. Be sure to wipe the cuvettes regularly to keep them clean. If solutions are very dark or differ greatly in color, you should rinse out the cuvettes with distilled water between readings.

7.

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

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 505 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?

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

Exercise 2 Functional Groups, Organic Molecules, Buffers, and Dilutions



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

Question

A

B

Are proteins present in this solution?

How many mg of protein are dissolved in this 100 ml solution?

Thinking

Would smelling, tasting, or touching the solution help determining if it has proteins or not? (not a good idea in lab)

The best thing to do is add a protein indicator. If the solution changes color, then proteins are present.

Changing the solutions color indicated proteins are present, but it does not detect exactly how much protein is present.

An analytical test giving the answer in numbers, not just by presence or absence, needs to be done.

Response

A qualitative analysis must be performed.

A qualitative analysis must be performed.

Materials

Exercise 3 Qualitative Analysis of Biological Molecules

Equipment

test tubes and racks

2 ml pipettes

5 ml pipettes

10 ml pipettes

pipette pumps


marking pencils (Sharpie)

filter paper disks

Petri dish

forceps

droppers

spot plates

water baths at 95C


1% dextrose (glucose)

1% starch (amylose)

6% starch (amylose)

concentrated HCl (in buret)

1 M NaOH (in buret)

apple juice

chicken broth

egg white

whole milk

vegetable oil

distilled water

Benedicts

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:

Benedicts 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 1% dextrose (glucose) solution to test tube #1

4. Use a different (why?) pipette to transfer 1 ml of the 1% starch solution (swirl to mix before transferring) to test #2

5. Use a 10 ml graduated cylinder to measure and transfer 1 ml of Benedicts 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 95C water bath for two minutes

8. Observe and record any color change in Table 3.2

Part A2: Detection of Starch

Procedure

1. Use a 2 ml pipette to transfer 0.5 ml of 1% dextrose solution to test tube #3

2. Use a different 2 ml pipette to transfer 0.5 ml of 1% starch solution (swirl to mix before transferring) to test #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 2 ml pipette to transfer 1 ml of 1% dextrose to both test tubes #5 and #6

2. Use a different 2 ml pipette to transfer 1 ml of 1% starch to both test tubes #5 and #6

3. In test tube #5, perform the Benedicts test first

4. Make note of any color changes

5. After the Benedicts test perform the IKI test in test tube #5

6. Make note of any color changes

7. Record your observation in Table 3.2

8. 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?

Obtain a simple sugar / starch unknown (labeled A, B, C, and D) and test it using the proper sequence of Benedicts and IKI reagent

Record the letter of your unknown and any color changes in Table 3.2

What (sugar, starch, or both) was in your unknown?

Table 3.2 Qualitative Analysis of Simple Sugars, Starch, and a Carbohydrate Unknown

Test Tube

Test Solution

Reagent

Hypothesis

Results

1

2

3

4

5

Benedicts 1st

IKI 2nd

6

IKI 1st

Benedicts 2nd

Unknown (_____)

Part B: Starch Hydrolysis

Many complex organic polymers in the presence of water can be broken down (hydrolyzed) into their simpler monomers by treatment with hydrochloric acid (HCl) and heat. Such an acid hydrolysis is a gradual decomposition (i.e., a few small monomers are broken off at a time until the polymer is completely converted into monomers).

For example, the hydrolysis of polysaccharides (long chains of monosaccharides) is a stepwise reaction in which the long chains are hydrolyzed into shorter oligosaccharids, then even smaller disaccharides and finally into individual monomers or monosaccharides (Fig. 3.1). In this way, starch, a polysaccharide, can be ultimately converted into its glucose monosaccharide (simple sugar) units.

By using the Benedicts and IKI tests in combination, the process of hydrolysis can be followed. One of these tests should become progressively less positive while the other should become more positive as the starch hydrolysis proceeds.

Which test would do what?

Procedure

1. Obtain a clean test tube

2. Swirl the flask containing starch solution and transfer 5 ml of it to the test tube using a pipette

3. Pipette 5 ml of distilled water into the same test tube

4. Shake thoroughly

5. Wearing protective gloves and eyewear, go to the fume hood and carefully dispense 3 ml of concentrated HCl from the buret into the test tube. Be very careful not to spill any HCl and make sure to properly close the buret. Notify your instructor immediately of any spills

6. Mix the solution in the test tube gently for 15 seconds

7. Use a clean dropper to place one or two drops of the solution from the test tube onto a spot plate and test it with IKI. Record the results in Table 3.3 as positive (+) or negative (-) at time 0 minutes

8. Place the test tube into a hot water bath

9. After 1 minute, remove one or two drops of the solution from the test tube, place onto the spot plate in the depression adjacent to the first sample, and test with IKI. Record the results in Table 3.3

Fig. 3.1 Starch Hydrolysis

10. Leave your test tube in the hot water bath and repeat the above procedure with successive samples taken from the test tube every minute

Try to obtain a succession of IKI tests showing gradual breakdown of starch from the blue-black color of unhydrolyzed starch, to the re-brown color of the oligosaccharide (partially hydrolyzed starch), to the amber color of IKI which indicates the absence of starch.

11. Record your results in Table 3.3

Table 3.3 Starch Acid Hydrolysis as Evidenced by an IKI Test

Time (min)

0

1

2

3

4

5

6

7

8

9

10

IKI Result (+ or -)

12. After the IKI test becomes negative, use a pipette to transfer 2 ml of the remaining test solution into a separate clean test tube

13. Wearing protective gloves and eyewear, go to the fume hood and carefully dispense 2 ml of concentrated NaOH from the buret into the test tube. This will neutralize the acid solution

14. Perform the Benedicts test on this neutralized solution

15. Are simple sugars present in this solution? __________

16. Record your answer in Table 3.4

17. Transfer 1 ml of the original (unhydrolyzed) starch solution to separate test tube

18. Perform a Benedicts test on this solution (this is your negative control)

19. Are simple sugars present in this solution? __________

20. Record your answer in Table 3.4

Table 3.4 Progress Results of Starch Acid Hydrolysis as Evidence by an IKI and a Benedicts Test

Test of Original (Unhydrolyzed) Starch

Test of Completely Hydrolyzed Starch

IKI

( + or - ); test color _______________

( + or - ); test color _______________

Benedicts

( + or - ); test color _______________

( + or - ); test color _______________

Part C: Detection of Lipids

A lipid is a non-polar (hydrophobic) organic molecule which is insoluble in water. One type of lipid is 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.

(AWOACEM)

Procedure

1. Obtain a blank filter paper disk

2. Mark the disk with a pencil following the pattern as shown in this figure

A apple juice

C chicken broth

E egg white

M whole milk

O vegetable oil

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 3.5

5. 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 tap 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)


12. Compare your results to your hypothesis

Table 3.5 Sudan IV Test for Lipids

Substance Tested

Hypothesis

Result

Apple juice

Chicken broth

Egg white

Whole milk

Vegetable

Distilled water

Part D: 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.

Biruet reagent, which is pale blue, contains copper sulfate (CuSO4). 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 3.6

5. Transfer 1 ml (approximately 20 drops) of the appropriate solution to properly marked test tube

6. Wearing protective gloves and eyewear, go to the fume hood and carefully dispense 1 ml of 1M NaOH from the buret into each test tube

7. Swirl gently to mix

8. Add 10 drops 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

Whole milk

Vegetable

Distilled water


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

Protein

Lipid

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?



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 microscopes

dissecting microscopes

microscope slides

coverslips

droppers

lens paper

forceps

toothpicks

Biological Specimens

Allium (onion)

pond water

Prepared Slides

newspaper print

colored threadsReagents

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.

Fig. 4.1 The Compound Light 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 focus adjustment knob 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 focus adjustment knob. 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

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 examining a microscope, as magnification increases, the working distance ______________________.

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:

M1 x DFV1 = M2 x DFV2

where M1 and DFV1 = magnification and diameter of the field of view, respectively, of objective 1, M2 and DFV2 = 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

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 ()

Scanning

4

5

__________

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 microscopes field of view becomes smaller, thinner, and darker (Table 4.3).

Table 4.3 Changes in a Microscopes Field of View as a Function of Magnification Power

Scanning

Low Power

High Power

Depth of Field of View (DFV)

(Gets Darker) (Gets Smaller) (Gets Thinner)

Depth of Focus

Light

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 D: Estimate of Cell Size

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:

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?

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

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-endParamecium cells arranged side-to-side

Paramecium Length __________ (in microns)Paramecium Width __________ (in microns)

Part E: Dry Mounts, Preparation of Wet Mounts, and Replacement Staining

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.

Part E1: 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 E2: 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

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. Wait a few minutes and then repeat the process but replacing the IKI under the coverslip with water

e. Once all the yellow IKI has been removed, dry the upper surface of the slide

f. 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)

Part E3: 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. Place a coverslip over your cheek cells

5. Observe and sketch the unstained cheek cells

6. Stain your cheek cells with methylene blue stain using the replacement staining technique

7. Observe and sketch the stained cheek cells. Identify the nucleus, cytoplasm, and cell membrane

8. How do these cells differ from onion cells?

9. Estimate the diameter of one of your cheek cells (in microns)

Cheek Cell Diameter __________ (in microns)

Part E: 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

Part F: The Dissecting Microscope

It is possible to have too much magnification when viewing some specimens. For example, how would you use your compound light microscope to view an entire earthworm? Larger specimens may require lower magnification. For this, biologists use dissecting microscopes (Fig. 4.4). Fill in Table 4.4 and notice the diameter of the field of view in these microscopes is substantially larger than that in compound light microscope. Your instructor will describe the use and features of this microscope.

Fig. 4.4 The Dissecting Microscope

Table 4.4 Diameter of the Field of View for the Dissecting Microscope

Objective Lens

Magnification

DFV (mm)

DFV ()

Lowest Power

2

10

10,000

Highest Power

4

5

__________

Procedure

1. Obtain a dissecting microscope using two hands to carry it

2. Identify the parts as per Fig. 4.4 and their functions

3. Observe the various objects made available in lab using the dissecting microscope

Using the information in Table 4.4, complete this sentence for the dissecting microscope

As magnification increases, DFV __________

How you seen this relationship before? __________ Where? __________________________


1. What is the total magnification of an object if the ocular lens magnification is 20x and the objective lens magnification is 45x?

2. Which objective lens is in place if the object you are viewing is magnified 1000x assuming an ocular lens magnification of 10x?

3. What is the diameter of the field of view (DFV) of a 1000x objective lens if the DFV of a 400x objective lens is 500 ? Express your answer in mm.

4. What is the DFV of a 40x objective lens if the DFV of a 10x objective lens is 3 mm? Express your answer in .

5. When viewing an organism using the 40x objective lens from question 5, you estimate 6 organisms could fit across the DFV if they were laid end-to-end and 20 could fit is stacked side-by-side. What is the length and width of this organism (in microns)?

6. What is the DFV of a 25x objective lens if the DFV of a 100x objective lens is 1.5 mm?

7. Using the 100x objective lens from question 6, you estimate 12 organisms could fit across the DFV if they were laid end-to-end and 30 could fit is stacked side-by-side. What is the length and width of this organism (in microns)?

8. What is the total magnification of an objective lens with a DFV or 0.8 mm if the DFV of a 100x objective lens is 2 mm?

Exercise 4 The Microscope

Exercise 5 - Cell Structure and Membrane Function

Introduction

The cell is the lowest level of biological organization performing all activities of life. Therefore, it is the fundamental unit of structure in living things. As such, the characteristics of cells are of monumental concern to the understanding of biology. The structure of cellular components reflects adaptation to accomplish those functions necessary for life. The collective functions of individual cells allow for the activity and behavior of the entire organism of which those cells are a part.

In this laboratory exercise, you will use a compound light microscope to examine cells and observe cellular activity. You will also conduct experiments illustrating some of the basic mechanisms of cellular transport.

Materials

Equipment

compound light microscope

microscope slides

coverslips


150 ml beakers

droppers

dialysis tubing

dental floss or string

scissors

forceps

triple beam balances

95C water bath


Elodea

Prepared Slides

Amoeba

Euglena

ParameciumReagents and Solutions

Benedicts

IKI

5.0% saline solution

concentrated glucose

concentrated starch

Part A: Cellular Transport

Cellular transport mechanisms are typically divided into two categories: Passive Transport and Active Transport. The basic differences between them is summarized in Table 5.1

Table 5.1 Basic Differences Between Passive and Active Transport

Passive Transport

Active Transport

Types and Direction of Transported Substances

Involves the movement of water or solute through a semi-permeable membrane down their concentration gradient (i.e., from regions of higher concentration of water or solutes to regions of lower concentration).

Involves the movement of solutes through a semi-permeable membrane against their concentration gradient (i.e., from regions of lower concentration of solutes to regions of higher concentration).

Cellular Energy

Does NOT require cellular energy in the form of ATP.

Requires cellular energy in the form of ATP.

Membrane Transport Proteins

Passive transports systems include two types of diffusion and osmosis:

Simple diffusion membrane transport proteins not required

Facilitated diffusion membrane transport proteins required

Osmosis specific to the passive transport of water from an area of higher water concentration to an area of lower water concentration (lower to higher concentration of solutes). Water moves through protein channels known as aquaporins.

Requires membrane transport proteins.

Solutions are often described using the terms hypotonic, hypertonic, and isotonic. Tonicity is a comparative term related to the concentration of solutes in a solution. It may be defined as the ability of a solution to cause a cell to gain or lose water. Hypotonic solutions contain less solute by % (i.e., more water) when compared to hypertonic solutions, which contain more solutes by % (i.e., less water). With a hypotonic solution that is separated from a hypertonic one by a selectively permeable membrane that allows water molecules to pass through but not solutes, the net movement of water molecules will be from a region of high water concentration (i.e., low solute hypotonic) to a region of lower water concentration (i.e., high solute hypertonic). Isotonic solutions are equal to one another in solute concentration; therefore, a concentration gradient does not exist and water moves in equal rate back and forth across the membrane.

This exercise will explore of these basic principles of cellular transport.

Part A1: Passive Transport in a Model Cell

Procedure

1. Obtain a piece of dialysis tubing

2. Working quickly so the dialysis tubing wont dry out, fold one end and tie off with floss or string

3. Open the other end of the tubing by sliding your fingers back and forth across the top

4. Place 10 ml of concentrated glucose and 10 ml of concentrated starch into the bag

5. Squeeze out the excess air from the bag before folding its other end and tying off

6. Rinse the bag gently under running water at the sink and blot dry with a paper towel. Make sure the bag is not leaking

7. Weigh the bag to the nearest 0.1 g and record as initial mass of the bag in Table 5.2

8. Fill a 150 ml beaker with distilled water

9. Add enough IKI to the beaker water to turn it yellow

10. Place the dialysis bag in the beaker. The bag should be fully submerged

11. Let your beaker sit no less than 30 minutes

This model cell system consists of four different molecules which could possibly move through the small holes in the dialysis bag. What are they?

1. _______________ 2. _______________ 3. _______________ 4. _______________

Based on the molecular size of these four molecules, develop a hypothesis to describe which molecules will move into the bag, which will move out and why. Record your hypothesis in Table 5.3

12. After your bag has soaked for the appropriate amount of time (no less than 30 minutes), remove it from the beaker and gently blot dry with a paper towel

What color is the solution in the bag? _______________

What color is the solution in the beaker? _______________

13. Weigh the bag again to the nearest 0.1 g and record in Table 5.2

14. Calculate the change in the mass of the bag by subtracting the initial mass from the final mass of the bag and record in Table 5.2

15. Calculate the % mass change of the bag using this formula and record in Table 5.2

% mass change of bag

Table 5.2 Mass and Time of Dialysis Bag Experiment

Mass

Time (hr : min)

Final

__________ g

_____ : _____

Initial

__________ g

_____ : _____

Change in Mass of Bag

__________ g

% Mass Change of Bag

__________ %

16. Pour some of the contents of the bag into a test tube

17. Test the bag contents with Benedicts reagent

18. Pour some of the contents of the beaker into a test tube

19. Test the beaker contents with Benedicts

20. Fill in Table 5.3

Table 5.3 Results of Dialysis Bag Experiment

Molecular Component of the Dialysis Bag System

Net Movement of Molecules Across the Dialysis Bag (In / Out / None)

Hypothesis

Final Results Based on Observations and Testing

Explanation

Part A2: Osmosis in Elodea

Elodea is a common aquatic plant related to Hydrilla. It has leaves of only two layers of thickness.

In this exercise, the thin leaves of Elodea will be useful in exploring some of the principles of osmosis. As seen under the compound microscope, the movement of cytoplasm with the Elodea leaf cells along the perimeter of the cell called cyclosis or cytoplasmic streaming will be observed.

Procedure

1. Using forceps, remove one leaf from an Elodea plant

2. Prepare a wet mount of the leaf

3. Observe the leaf at high power under the microscope

4. Identify the parts of an Elodea leaf (Fig. 5.1)

Where are the chloroplasts located? _______________

Do you see cyclosis (cytoplasmic streaming)? _______________

5. Draw your Elodea cell and label the visible parts

6. Using the replacement staining technique, replace the water under your coverslip with the 5% saline solution

7. After 5-10 minutes, observe the cells again and make note of any changes that have occurred

8. Draw the cell again

Where are the chloroplasts located now?

What cellular structure (not visible previously) has receded from the cell wall?

What happened to the volume of the central vacuole to cause this change?

In what type (hypotonic, hypertonic, isotonic) of environment is the Elodea cell?

Fig. 5.1 Elodea Cell

Part A3: Osmoregulation in Protists

Some single-celled organisms live in a fresh water environment that is hypotonic to their cellular fluid which means they are continually taking on water through osmosis. They stay alive because they possess abilities to regulate internal water pressure using contractile vacuoles. These contractile vacuoles remove excess water from the cell. Contractile vacuoles typically appear a fluid-filled bubbles in the cytoplasm that slowly get large and then suddenly disappear.

Procedure

1. Using the web, find and view pictures and video clips of contractile vacuole function in Paramecium. Your instructor may also make some clips available online or have you view them in class

2. Draw and label the structures in the Paramecium below. Note the contractile vacuole

What is its function?

Part B: Structure and Motility in Protists

Most groups of protists are capable of movement. This motility is made possible by one of three types of structures. Organisms like Amoeba move by means of pseudopodia (false foot) which are extensions of the cytoplasm. Paramecium and similar organisms move using cilia, fine hair-like structures covering the cell membrane. Organisms typically have many, many cilia. Other protists, such as Euglena, move using flagella, which are whipped back and forth. Organisms usually have one or just a few flagella. Finally, some protists lack the ability to move at all.

Procedure

1. Using the web, find and view pictures and video clips of protist structure and movement. Your instructor may also make some clips available online or have you view them in class

2. Using online resources and the text book, draw and label the following parts for Amoeba, Paramecium, and Euglena

Exercise 5 Cell Structure and Membrane Function

Lake-Sumter Community College, Leesburg Laboratory Manual for BSC 1010C


cell membrane

cytoplasm

pseudopod (Amoeba)

cilia (Parmecium)

flagella (Euglena)

contractile vacuole

nucleus

chloroplast (Euglena)

food vacuole

Amoeba

Paramecium

Euglena


1. If the initial mass of a dialysis bag was 8.2 g and final mass was 10.9 g, what is the % mass change of the bag?



4. A pre-weighed dialysis bas which contained a solution of 10% glucose was placed in a beaker containing a solution of 20% glucose. After one hour, the bag was weighed again. Calculate the % mass change of this dialysis bag from the following information:

Mass of bag before experiment: 15.3 g

Mass of bag after experiment: 12.7 g

5. Was the beaker solution in question 4 hypertonic, hypotonic, or isotonic to the dialysis bag contents?

6. What are the major differences between the following pairs of cells?

prokaryotic and eukaryotic

plant and animal

protists and generalized animal cells

7. How was the dialysis bag in your experiment an example of a semi-permeable membrane?

8. Define these terms:

hypertonic

hypotonic

isotonic

9. Complete the following sentence: When two aqueous solutions are separated by a semi-permeable membrane, the net water movement is always from a ________tonic to a ________tonic solution.


Exercise 6 - Enzyme Activity

Introduction

Enzymes are biological catalysts that regulate the rate of chemical reactions. Their 3-dimensional conformation and therefore their function can be affected by several variables.

In this laboratory exercise, you will manipulate various factors that affect an enzymes activity. The enzyme is catalase, which is found in most all living organisms. Catalase decomposes hydrogen peroxide (H2O2), a toxic compound into water and oxygen:

2H2O2 + catalase 2H2O + O2

The amount of oxygen created is directly proportional to the rate of the enzymatic reaction. Therefore, measuring the amount of oxygen produced provides a measure of the speed at which the reaction is proceeding.

The effect of external factors such as substrate concentration, temperature, and pH will be examined.

Materials

Equipment


metric rulers


droppers

marking pens (Sharpies)

thermometers

ice water bath

warm (40C) water bath

hot (95C) water bath


catalase

hydrogen peroxide (H2O2)

pH 2 buffered H2O2 solution




Exercise 6 Enzyme Activity




Part A: Enzyme Activity as a Function of Substrate Concentration

An enzyme requires a substrate which it converts into product.

Drawing from what you have learned about enzymes so far, develop a hypothesis regarding the effect of substrate (H2O2) concentration on enzymatic reaction rate.

Hypothesis: As substrate concentration increases, reaction rate will _______________.

Procedure

1. Obtain six test tubes and a test tube rack per group

2. Using a graduated cylinder, measure 1 ml of catalase and add to each of the six tubes

3. Using a dropper, add 1 drop of H2O2 to test tube #1

4. After timing for 30 seconds, mark the maximum height of the bubble column with a Sharpie

5. Doing each tube one at a time, repeat steps 3-5 above to the remaining five test tubes increasing by one drop the amount of H2O2 in each test tube (i.e., test tube #2 receives 2 drops of H2O2, test tube #3 receives 3 drops of H2O2, etc.)

6. Upon completion, return to each test tube and measure in mm the distance from the bottom of the test tube to the height of the mark you made

7. Fill in Table 6.1 and plot the results as a bar chart in Fig. 6.1

Table 6.1 Reaction Rates for Catalase at Various Substrate Concentrations

Test Tube

Catalase (ml)

H2O2 (# drops)

Height of Bubble Column (mm)

1

1

1

2

1

2

3

1

3

4

1

4

5

1

5

6

1

6

Fig. 6.1 Reaction Rate of Catalase as a Function of Substrate Concentration

(Height of Bubble Column (mm))

(# of Drops of Substrate (H2O2))

Make a general statement regarding the effect of substrate concentration on enzymatic reaction rate:

Part B: Enzyme Activity as a Function of Temperature

Temperature is a measure of the speed at which molecules are moving. As temperature increases, the molecular movement speed does so as well. Increasing temperature increases the probability and rate at which enzyme and substrate come together, thereby increasing the reaction rate. However, enzymes are subject to denaturation at excess temperatures. A denatured enzymes active site conformation is changed not allowing the substrate to bind. The result is that at these temperatures, the reaction will decrease. An enzymes optimum temperature is that point which has the greatest reaction rate but does not denature the enzyme.

Drawing from what you have learned about enzymes so far, develop a hypothesis regarding the effect of temperature on enzymatic reaction rate.

Hypothesis: As temperature increases, reaction rate will _______________ and then _______________.

Procedure

1. Obtain eight test tubes and a test tube rack per group

2. Using a graduated cylinder, measure 1 ml of H2O2 into four of the eight test tubes

3. Using a dropper, add 1 drop of catalase to each of the other four test tubes

4. Place one test tube each of H2O2 and catalase into each of the three water baths (make note of which tubes are yours for later retrieval). Leave the remaining pair of test tubes in the rack

5. Allow all tubes to acclimate for 15 minutes

6. After 15 minutes, proceeding one pair of tubes at a time, pour the H2O2 into the catalase




Table 6.2 Reaction Rates for Catalase at Various Temperatures

Temperature (C)

Catalase (# drops)

H2O2 (ml)


Cold

Room

Warm

Hot

Fig. 6.2 Reaction Rate of Catalase as a Function of Temperature


(Temperature (C))

Make a general statement regarding the effect of temperature on enzymatic reaction rate:

What is the optimum temperature for catalase? __________Part C: Enzyme Activity as a Function of pH

Another variable that can affect enzyme conformation and therefore activity levels is pH. Like with temperature, enzymes also have an optimum pH.

Drawing from what you have learned about enzymes so far, develop a hypothesis regarding the effect of pH on enzymatic reaction rate.

Hypothesis: As pH moves away from optimum, reaction rate will _______________.

Procedure

1. Obtain five test tubes and a test tube rack per group

2. Using a graduated cylinder, measure 1 ml of buffered (2, 4, 7, 9, 11) H2O2 and add to each of the six tubes

3. Using a dropper, add 1 drop of catalase to test tube #1


5. Doing each tube one at a time, repeat steps 3-5 above to the remaining four test tubes



Table 6.3 Reaction Rates for Catalase at Various Temperatures

Test Tube

pH

Catalase (# drops)

H2O2 (ml)


1

2

1

1

2

4

1

1

3

7

1

1

4

9

1

1

5

11

1

1

Fig. 6.3 Reaction Rate of Catalase as a Function of pH


(pH)

Make a general statement regarding the effect of pH on enzymatic reaction rate:

What is the optimum pH for catalase? __________


1. What is meant by an organic catalyst?

2. List and describe the affect of the three major factors that cause changes in rate of enzymatic activity.

3. Why did certain temperatures and pH exhibit little or no activity at all?

4. During the reaction, you may have noticed a slight bit of heat given off. Explain the source of this heat.

5. What is the general equation for all enzymatic reactions?

6. Fill in these blanks

The rate of enzymatic reaction is _______________ (directly / inversely) proportional to substrate concentration.

At optimum, enzymatic reaction rate is __________ (greatest / least).

Exercise 6 Enzyme Activity


Exercise 7 - Respiration

Introduction

Regardless of species, all living organisms carry on the process of respiration. The equation for respiration is

C6H12O6 + 6O2 6CO2 + 6H2O + energy

In this process, the energy released from food molecules is coupled into the synthesis of ATP. The energy in ATP is then used to power metabolic reactions. Respiration can occur with oxygen (aerobic) or without oxygen (anaerobic) respiration. In anaerobic respiration, only glycolysis occurs while in aerobic respiration glycolysis is followed by the Krebs cycle and a process known as oxidative phosphorylation. During glycolysis, 2 ATPs for each glucose molecules are produced. This is the total ATP production for anaerobic respiration. Aerobic respiration increases that output approximately 19 fold (38 ATPs).

In this laboratory exercise, you will examine aspects of respiration by qualitative examination of carbon dioxide production.

Materials

Equipment

large test tubes and racks

bean seeds

ungerminated

germinated

germinated, boiled

150 ml flasks

rubber stoppers

thistle funnels

glass tubing


Exercise 7 Respiration

phenol red



Part A: Carbon Dioxide Production

Seeds contain stored food material in the form of carbohydrates. When a seed germinates, the carbohydrate is broken down liberating energy (ATP) needed for growth of the enclosed embryo.

For this procedure, dry bean seeds have been soaking for some time in water to begin the germination process. Another set of beans was not soaked and is, therefore, not germinating. A third set was allowed to germinate then was boiled.

Procedure

1. Obtain three respiration flask setups (Fig. 7.1)

2. Place about 30 ml of ungerminated seed into one of the flasks. Repeat this for the other two flask placing germinated seeds in one and the germinated and boiled seeds in another

3. Fit rubber stoppers securely into the flasks and the thistle funnels

4. Add enough water to each test tube to cover the ends of the glass tubing coming out of the flask

5. Set the flasks aside for approximately 1.5 hours

6. After this time, replace the water in each test tube with phenol red solution. Phenol red is a pH indicator that changes color in response to changes in pH. Red indicates a neutral pH. A yellow color signifies an acid

7. Pour water through the thistle funnels into each flask forcing the gas in the flask out through the glass tubing and into the test tube. Carbon dioxide (CO2) when bubbled through water forms a mild acid called carbonic acid (H2CO3). Any CO2 given off by the seeds in the flask will interact with the water in the test tube creating carbonic acid changing the phenol red solution to yellow


Fig. 7.1 Respiration Procedure Setup

Table 7.1 Carbon Dioxide (CO2) Production in Bean Seeds

Ungerminated

Germinated

Germinated, boiled

color

CO2 present?

Which set of seeds was respiring?

What happened during the boiling of the germinated seeds that caused the results you found?


1. What is the chemical equation for respiration?

2. Define or describe the following:

aerobic

anaerobic

NADH

FADH2

ATP

3. How many ATPS are produced during

glycolysis?

Krebs cycle?

oxidative phosphorylation?

aerobic respiration?

anaerobic respiration?

4. Some wines are marketed as sparkling wines. Sparkling wines are considered so due to a dissolved gas that makes them fizzy. Apply what you know about the process of respiration to explain the type and origin of this gas.

5. Animals are either homeothermic or poikilothermic (homeo same, poikilo varied, thermic warm), more commonly known as warm-blooded or cold-blooded, respectively. Homeothermic animals include mammals and birds. Examples of poikilothermic species would include the invertebrates, reptiles, fish, amphibians, etc. Using what you have learned about respiration and enzymes, explain why poikilotherms move more slowly in the winter than the summer.

6. Very small mammals such as shrews and many rodent species have very high metabolic rates compared to larger mammals like ourselves or elephants. Based on what youve learned about temperatures effect on the rate of enzymatic reactions, as well as the relationship between surface area and volume, provide an explanation.

Exercise 7 Respiration

Exercise 8 - Photosynthesis

Introduction

The equation for photosynthesis is essentially the reverse of respiration.

6CO2 + 6H2O C6H12O6 + 6O2

In this process, energy from the sun is used to reduce carbon dioxide (CO2) into glucose (C6H12O6). Photosynthesis is best understood as a set of two linked sets of chemical reactions. The light reactions require sunlight, carbon dioxide and water as inputs and output energy in the form of ATP, NADPH as a reducing agent. Oxygen is also formed during the light reactions. The outputs of the light reactions then become inputs for the dark reactions. Dark reactions, as the name suggests, do not require sunlight. Instead, the dark reactions reduce carbon dioxide into glucose using the ATP and the electrons carried by NADPH from light reactions.

In this laboratory exercise, you will examine aspects of photosynthesis by spectral examination of the pigment chlorophyll. A qualitative analysis of carbon dioxide production as evidence of the process of photosynthesis will also be conducted.

Materials

Equipment

large test tubes and racks

ring stands

test tube clamps

rubber test tube stoppers

chromatography paper

scissors

capillary tubes

metric rulers

spectrophotometer

kimwipes

cuvettes

light source

razor blades or scalpel

1000 ml beakers

drinking straws


Elodea


chromatography solvent (acetone)

chlorophyll

distilled water

Exercise 8 Photosynthesis

phenol red



Part A: Photosynthetic Pigments

Part A1: Paper Chromatography of Photosynthetic Pigments

The chloroplasts of higher plants contain photosynthetic pigments that capture light energy from the sun. Chlorophyll a is the main photosynthetic pigments. Chlorophyll b, carotenes, and xanthophylls are all considered secondary pigments. A mixture of all four pigments is found together in one leaf or quantity of chlorophyll extract. If one wishes to examine these mixed pigments separately, the process of paper chromatography provides a solution. This process separates the individual pigments. For this procedure, a concentrated amount of chlorophyll will placed onto a spot near one edge of a strip of chromatography paper. This will then be placed in a chromatography apparatus (Fig. 8.1). The solvent at the bottom of the tube will be drawn up the chromatography paper by capillary action. As the solvent passes through the concentrated chlorophyll the pigments will travel along with the solvent at a rate and to a distance based upon their solubility and molecular size. Therefore, the

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