ap biology lab-12

7/16/2019 AP Biology Lab-12

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Big Idea 2: Cellular Processes: Energyand Communication

What factors affect the rate of photosynthesis in living leaves? What factors affectthe rate of cellular respiration in multicellular organisms?

Please be sure you have read thestudent intro packet before you do this lab.

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations Summary Pre-lab and Questions

Part A: Photosynthesis

What is a product of photosynthesis in a water plant and what factorsincrease or decrease the rate of production of this product?

Part B: Cellular RespirationHow does exercise affect disposal of waste products from cellularrespiration?

Lab Investigation 6.1Part 1 - Floating Leaf Disk

Te Floating Leaf Disk echnique for Quantifying Net OxygenProduction during Photosynthesis

Part 2 - Cellular RespirationCellular Respiration – Leaf Disk Method Extended to CellularRespiration

Part 3 - Student Guided Inquiry Student Guided Inquiry - esting a variable that affects the Rate ofPhotosynthesis.

LAB 6Photosynthesis and

Cellular Respiration



LAB 6 - Photosynthesis andCellular Respiration

Big Idea 2: Cellular Processes: Energy and Communication

What factors affect the rate of photosynthesis in living leaves? What factorsaffect the rate of cellular respiration in multicellular organisms?

BACKGROUND

Living systems require free energy and matter to maintain order, to grow, andto reproduce. Organisms use different strategies to capture, use, and store freeenergy. Autotrophic organisms capture free energy from the environment through

photosynthesis and chemosynthesis, whereas heterotrophic organisms harvest freeenergy from carbon compounds produced by other organisms. In multicellularplants, photosynthesis occurs in the chloroplasts within cells.

Photosynthesis – the Light Reaction. White light is composed of all the colors present in the visible spectrum. Some

light wavelengths can be used by the photosynthesis pigments present in greenplants to produce sugars. Because there are different pigments present in greenleaves and these pigments absorb different wavelengths of visible light, a largeportion of white light can be used during the light reaction of photosynthesis.

Chloroplast Structure



Photosynthesis Rate (as measured by oxygen production)

L i g h t A b s o r p t i o n

Wavelength of Light (nm)

Chlorophyll a

Chlorophyll b

Beta-Carotene

During the light reaction of photosynthesis, chlorophyll molecules in thethylakoids of chloroplasts transfer electrons through a series of electron acceptorsto NADP+ (nicotinamide adenine dinucleotide phosphate) and ADP (adenosine

diphosphate). During this process NADP+ is reduced to NADPH (the reducedform of NADP+) and ADP to AP (adenosine triphosphate), which then transfersenergy-rich electrons to the Calvin cycle intermediates to eventually produce sugar(glucose).

O 2

CO 2

G3P

Calvin Cycle

L i g h t

R e a

c t i o

n s

r u b i s c o

H 2O H

2O

E n e r g y f r o m S u n l i g h t

A T P

A D P

N A D P H

N AD P +



Te process of photosynthesis occurs in a series of enzymatic reactions thatcapture light energy to build energy-rich sugars and store energy in the molecules AP and NADPH. Te overall process is summarized in the following reaction:

water + carbon dioxide + light → sugars + oxygen6 H

2O + 6 CO

2 + light → C

6H

12O

6+ 6 O

2

How can you quantify the rate of photosynthesis? (How many moles of O2 are

produced for every mole of glucose?) o determine the net rate of photosynthesis,one could measure one of the following:

• Te production of oxygen - Te difficulty in measuring the production ofoxygen from photosynthesis is the simultaneous consumption of O

2 in aerobic

respiration.

• Te consumption of carbon dioxide - Measuring the consumption of CO2

is more generally practiced but equipment and procedures are not usuallyavailable in most labs.

In the following photosynthesis labs, you will practice a technique called “floatingleaf disk” to indirectly measure the rate of photosynthesis by the accumulation ofoxygen gas. Normally the leaf disks float due to the gases in the spongy mesophyll. A vacuum is used to remove trapped air in the spongy mesophyll layer of the leafand replaced by a solution of sodium bicarbonate ions that serve as a carbon dioxidesource for photosynthesis. Te disks will initially sink (since the gases have beenremoved by the vacuum). As photosynthesis proceeds, O

2 again accumulates in

the air spaces of the spongy mesophyll, and the leaf disks will once again becomebuoyant and float. In this way, the rate of photosynthesis can be indirectly measured

by the rate of rise of the leaf disks.However, cellular respiration is also taking place simultaneously in themitochondria and O

2 is being consumed. Terefore, the rise of the floating disks

indirectly measures the NE rate of photosynthesis occurring in the leaf tissue.



PREPARAION

Materials and Equipment are listed with each individual lab.

iming and Length of LabsPre-lab A and B are paper scenarios and do not require lab bench time.

Lab Investigation 6.1Part 1 - Floating Leaf echnique Practice wo lab periods are needed for all students to practice the techniques involved

in getting the vacuum and leaf disks to sink; as well as collect data, graph, anddiscuss data analysis.

Part 2 - Extension of Photosynthesis Leaf Floating echnique to measure

net rate of cellular respiration Tis lab could be piggybacked with Part 1 with results in the dark obtained in

less than 30 minutes. Otherwise, if started from scratch with new floating disksand then put into the dark, it could take 60-90 minutes to complete.

Part 3 - Student Guided Inquiry – Quantitative Net Rate of Photosynthesis Tis activity will take a minimum of two lab periods – one for design and

discussion and one for conducting the experiment and collecting data. Data analysiscould be done out of class.



Learning objectives aligned standards and sciencepractices (SP):

• Design and conduct an experiment to explore the effect of certain factors,including different environmental variables, on the rate of cellularphotosynthesis and cellular respiration (4A2 and SP 6.2).

• Connect and apply concepts, including the relationship between cell structureand function (chloroplasts and mitochondria); strategies for capture, storage,and use of free energy; diffusion of gases across cell membranes; and thephysical laws pertaining to the properties and behaviors of gases (2A2 and SP1.4, SP 3.1).

• Learn how a respirometer system can be used to measure respiration rates inplant seeds or small invertebrates, such as insects or earthworms (2A1 and SP6.1).

General Safety Precautions:• Safety goggles must be worn when working in close proximity to exposed

light bulbs that can easily shatter.

• Keep solutions away from the electrical cords of light sources;

• If temperature is the chosen variable to test, it is not necessary to heat anysolution beyond 60 °C; and

• Most but not all syringes are capable of withstanding the vacuum created in thisprocedure without failure, however, you should test the syringes beforehand.



Pre-lab and Questions: Part A Photosynthesis

What factors increase or decrease the rate of production of oxygen gas productionin the photosynthesis reaction in a water plant?

You are going to make predictions of the rate of photosynthesis based on theamount of oxygen produced at an increased or decreased rate. Tis can be observedby counting the number of bubbles released from the plant. Placing a water plantlike Elodea or Anacharis in a cup half full of water and a 0.2% solution of bakingsoda (sodium bicarbonate, NaHCO

3) gives the plant an instant source of dissolved

carbon dioxide in the water.

Chart descriptions 1-10: 1. Original set up with bright light, 0.2% sodium bicarbonate

(NaHCO3).

2. Predict how results would differ if the light source was very closeto the plant and super intense;

3. What if light was further away from the plant;4. What if the light was very dim (no artificial light, just natural

lighting in the room); 5. What if plant was put in the dark. 6. Predict what would happen if the sodium bicarbonate was much

more concentrated (10%);7. What if no NaHCO

3 was used.

8. Predict what would happen if the 0.2% sodium bicarbonatesolution was near freezing temperature (5 °C)? or

9 What if the solution was a hotter temperature (no greater than60 °C)?

10. Predict what would happen in an acid solution pH by addingdrops of HCl to the 0.2% sodium bicarbonate water until thepH was around 4.

Pre-lab Data able 1: Photosynthesis increase? Slow? None?

Increase Slow or decrease No gas released

1. Original set-up

2. Super intense light3. Artificial light 10 feet away

4. Natural room light

5. No light (dark)

6. 10% NaHCO 3

7. No NaHCO 3

8. 5 °C

9. 60 °C

10. pH 4.0



Questions

1. What are the gases involved before and after photosynthesis?

2. What is the gas product of photosynthesis that accumulates onthe leaves of the original set up?

3. What plant organelle carries out photosynthesis?

4. What variable change out of the 9 different scenarios in yourprediction chart increased photosynthesis?

5. Did any of the variables appear to inhibit photosynthesis?



6. Was this a quantitative or qualitative experiment? Does countingbubbles actually measure the amount of gas produced?

In Lab Investigation 6.1 using the floating disk technique, you will practice atechnique that measures the accumulation of oxygen. However, it will be the NEaccumulation since respiration is going on at the same time in the mitochondriaand oxygen is being used up for cellular respiration to convert AP to ADP andproduce energy.


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Pre-lab Part B: Cellular Respiration

How does exercise affect disposal of waste products from cellular respiration?

Carbon dioxide is one of the products of cellular respiration. Because CO2is

slightly acidic when dissolved in water, pH indicators such as bromothymol blue(BB) can detect its presence.

“Cellular Sue” obtained a small water bottle of distilled water with 10 drops ofbromothymol blue (BB). BB is an indicator that is very sensitive to changes inpH and turns blue if slightly basic (pH greater than 7.0) and starts turning green to yellow as pH changes from base to acid (pH value below 7.0).

After blowing into the straw for five minutes, the BB liquid gradually turnedfrom blue to green and finally to yellow; demonstrating the production of CO

2 from

respiration

Before After



Questions:

1. After running in place for 1 minute and jumping rope for another

minute, “Cellular Sue” once again blew her breath through thestraw into a blue-colored solution of BB. Do you think it tookfive minutes for the solution to change color this time? Wouldthere be more or less CO

2 in her breath compared to the first

trial before exercising?

2. How does exercise affect the amount of CO2 produced from

cellular respiration? Where in your cells is AP being convertedfor quick energy and what are the products of this reaction?



Lab Investigation 6.1:Part 1 - Floating Leaf Disk

Te Floating Leaf Disk echnique for Quantifying Net Oxygen Productionduring Photosynthesis.

In this part of the lab, you will practice the floating leaf disk technique to measurethe net rate of photosynthesis by testing a variable that you know affects photosynthesis.For this technique, the most challenging skill is to get the disk to sink initially.

When immersed in water, oxygen bubbles are usually trapped in the air spaces ofthe spongy mesophyll in the plant leaf.

By creating a vacuum in this experimental procedure, the air bubbles can be drawnout of the spongy mesophyll, and the space is refilled by the surrounding solution. Tis allows the leaf disks to sink in the experimental solution.

If the solution has bicarbonate ions and enough light, the leaf disks will begin toproduce sugars and oxygen through the process of photosynthesis. Oxygen collectsin the leaf as photosynthesis progresses, causing the leaf disks to float again. Telength of time it takes for the leaf disks to float again is a measure of the net rate ofphotosynthesis.



Floating Disk Technique set-up

Materials:

• Baking soda* (sodium bicarbonate - NaHCO 3) (1 g)

• Liquid soap - Dawn®* (approx. 5 mL of dish washing soap in 250 mL of water)

• Plastic syringes, 60 cc (2)

• Pipets (2)

• Living leaves* (spinach, ivy, etc.)

• Hole punch*

• Clear plastic cups (2)*

• Stopwatch

• Light source**item not included

Procedures:

1. Prepare 300 mL of 0.2% bicarbonate solution for each experiment(0.2 g/100 mL). What is the purpose of the bicarbonate solution

for the leaf disks while they are in solution (see Pre-lab)? 2. Pour the bicarbonate solution into a clear plastic cup to a depth

of about 3 cm. Label this cup “With CO 2.” Fill a second cup

with only water to be used as a control group. Label this cup“Without CO

2.” Troughout the rest of the procedure, you will

be preparing material for both cups, so do everything for bothcups simultaneously.

3. Using a pipet, add one drop of a dilute liquid soap solution tothe solution in each cup. It is critical to avoid suds. If eithersolution generates suds, then dilute it with more bicarbonate or

CO2 in Solution

Bubbles of O2 Forming

Solution



water solution. Why use soap? Te soap acts as surfactant or“wetting agent” — it wets the hydrophobic waxy surface of theleaf, allowing the solution to be drawn into the leaf and enablingthe leaf disks to sink in the fluid.

4. Using a hole punch, cut 10 or more uniform leaf disks for eachcup. Avoid major leaf veins. Te choice of plant material to cut

is perhaps the most critical aspect of this procedure. Te leafsurface should be smooth and not too thick.

5. Draw the gases out of the spongy mesophyll tissue and infiltrate

the leaves with the sodium bicarbonate solution by performingthe following steps:

• Remove the piston or plunger from both syringes. Placethe 10 leaf disks into each syringe barrel.

• Replace the plunger, but be careful NO to crush theleaf disks. Push in the plunger until only a small volumeof air and leaf disk remain in the barrel (less than 10%).

• Pull a small volume (5 cc) of sodium bicarbonate plussoap solution (previously prepared) up into one syringeand a small volume of water plus soap into the other

syringe. ap each syringe to suspend the leaf disks inthe solution. Make sure that, with the plunger inverted,the disks are suspended in the solution. Make sure noair remains. Move the plunger to get rid of air from theplunger before you attempt the next step.

• You now want to create a vacuum in the plunger to drawthe air out of the leaf tissue. Tis is the difficult step tomaster. Once you learn to do this, you will be able tocomplete the entire exercise successfully.



Create the vacuum by holding a finger over the narrowsyringe opening while drawing back on the plunger (seeabove figure). Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf disks to suspendthem in the solution. Now release the vacuum by lettingthe plunger springs back. Te solution will infiltrate theair spaces in the leaf disk, causing the leaf disk to sink inthe syringe (see above figure). You may have to repeatthis procedure two to three times in order to get the disksto sink.

If the plunger does not spring back, you did not havea good vacuum, and you may need a different syringe.If you have difficulty getting your disks tosink after three tries, it is usually becausethere is not enough soap in the solution. ry adding a few more drops of soap to thecup and replacing the liquid in the syringe.

Placing the disks under vacuum more thanthree times can damage the disks and youmay need to cut new ones.

6. Pour the disks and the solution from thesyringe into the appropriate clear plasticcup. Disks infiltrated with the bicarbonatesolution go in the “With CO

2” cup, and

disks infiltrated with the water go into the“Without CO

2”cup (control).

7. Place both cups under the light source and

start the timer. At the end of each minute,record the number of floating disks. Tenswirl the disks to dislodge any that stuckagainst the side of the cups. Continue untilall of the disks are floating in the cup withthe bicarbonate solution.

8. o make comparisons between experiments, a standard pointof reference is needed. Repeated testing of this procedure hasshown that the point at which 50% of the leaf disks are floating(the median or E

50, the Estimated ime it takes 50% of the

disks to float) is a reliable and repeatable point of reference forthis procedure.

Data able 6.1a: Number of Floating Leaf Disks

Minutes 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 E50

Cup With

CO2 0Cup Without

CO2

0



Analyzing Results

Graph your results in the following format. Te point at which 50% of theleaf disks are floating (the median) is the point of reference for this procedure.By extrapolating from the graph, the 50% floating point is about 11.5 minutes.Using the 50% point provides a greater degree of reliability and repeatability for this

procedure. As Steucek, et. al. (1985) described, this terms is referred to as the E50.How do your results and your graph compare?

One of the problems with the E50

graph is that it is an inverse relationship tophotosynthesis. In other words, as the E

50 number of minutes goes down, the rate

of photosynthesis goes up. o correct for this representation of data, Steucek, et.al. 1985 showed a positive slope for photosynthesis with the E

50 by calculating the

inverse or 1/E50

for his graphs.Keep this in mind as you analyze your results in the next Lab Investigations

2.2, 2.3.

5 10 1500

2

4

6

8

10

12

Time in Minutes

N u m b e

r o f D i s k s

Rate of Photosythensis

E T

1 / E T

Rate of Photosythensis



Part 2 - Cellular Respiration

BACKGROUND

Living systems require free energy and matter to maintain order, to grow, andto reproduce. Organisms use different strategies to capture, use, and store freeenergy. Autotrophic organisms capture free energy from the environment throughphotosynthesis and chemosynthesis, whereas heterotrophic organisms harvest freeenergy from carbon compounds produced by other organisms. Te process ofcellular respiration harvests the energy in carbon compounds to produce AP thatpowers most of the vital cellular processes. In eukaryotes, respiration occurs in themitochondria within cells.



Mitochondria are located in both plant and animal eukaryotes. Respiration isnot just a process for animals. Plants consume oxygen and release carbon dioxidethe same way as humans and other animals. Teir growth, maintenance andreproduction rely on respiration as a way of breaking down the molecules producedduring photosynthesis and using them for energy.

Plant cells not only convert energy from the sun into sugars in the chloroplasts,but they also go through the breakdown of the sugar molecules to produce energyfor growth in the mitochondria. Cellular respiration occurs at all times if the plantcell has sufficient water and sugars. In many plants, it most often occurs at night.

Cellular respiration is the process that releases energy by breaking down glucoseand other food molecules in the present of oxygen (oxidation). Cellular respirationrequires oxygen, a sugar or other complex food molecules (carbohydrates, lipids)and gives off CO

2, water, and energy. Te equation for cellular respiration is:

glucose (sugar) + oxygen gas→ carbon dioxide gas + water + energy C

6H

12O

6+ 6 O

2(g) → 6 CO

2(g) + 6 H

2O + 686K cal/mole of glucose

Te three main stages of cellular respiration are shown in the following diagram:Glycolysis, the Krebs Cycle, and Electron ransport Chain. Te energy received istrapped in small packages of AP. Tis is sometime referred to as chemiosmosis,in which the energy available in electrons pump protons across a membrane andprovides the energy for AP synthesis. You can see from the diagram that about38 AP for each glucose molecule oxidized is formed during the cycle.

Illustration by RegisFrey



During glycolysis, one molecule of 6-carbon glucose sugar is broken in half,producing two molecules of pyruvic acid, a 3-carbon compound. During theKrebs cycle, pyruvic acid is broken down further into CO

2 in a series of energy-

extracting reactions. Te Krebs cycle generates high-energy electrons that arepassed to NADH and FADH

2. Te electrons are then passed from those carriers

to the electron transport chain. Te electron transport chain uses the high-energyelectrons from the Krebs cycle to convert ADP to AP, for a grand total of about36-38 AP per glucose molecule. Tese 36-38 AP molecules represent about38% of the total energy of glucose. What happens to the remaining 62%? It isreleased as heat. Although 38% does not seem like much, the cells are actuallymore efficient at using food than the engine of a typical automobile is at burninggasoline.



Procedure

Leaf Disk Method Extended to Cellular Respiration

• Why do the floating disks, now filled with oxygen — a product ofphotosynthesis, represent only the net rate of photosynthesis?

• What other process is going on in the plant cells in the mitochondria?

• What would happen if the floating disks from your photosynthesis lab wereimmediately placed in a dark cabinet after all 10 disks started to float? Would you be able to estimate the rate of cellular respiration using the floating leafdisk technique?

ry it in this lab. Either continue from Lab Investigation 6.1 Part 1, or start anew “floating leaf technique.” Once the 10 leaf disks have all floated, immediatelyput them in the dark and continue assessing every minute until all floating diskshave sunk. Make sure to follow the same procedures for recording the number offloaters each minute and stirring the disks so they do not stick to the sides of thecup.

1. Graph your results.

2. What is your E50

for cellular respiration (sinking instead offloating)?

3. How does it compare to net photosynthesis in the same plant?4. Describe what is happening in the plant cell when placed in the

dark to cause the floating leaf disks to sink.

Part 3 - Student Guided Inquiry

esting a variable that affects the Rate of Photosynthesis

During the Pre-lab, you investigated several variables that might influence therate of photosynthesis. Based on your findings and questions raised in that lab, you will now design your own experiment using the floating leaf technique to assessthe net rate of photosynthesis. Here is a list of ideas to consider in the followingcategories: environmental variables, plant or leaf variables, and methods variables inusing the floating disk technique:

Environmental Variables: light intensity (brightness), direction of light or angleof insolation, light colors and different wavelengths, temperature, concentration ofCO

2 (sodium bicarbonate), pH (acid or basic);



Plant or Leaf Variables: leaf colors (amount of chlorophyll), leaf size, stomatadensity, stomata distribution, light-starved leaves vs. leaves kept in bright light, typeof plant (house, desert, full-sun, succulent, etc.), leaf age, leaf variegation, role ofrespiration and measuring the gross photosynthesis;

Methodology Variables in Floating Disk echnique: size of leaf disk, depthof CO

2 solution, methods of cutting disks, leaf disk overlap, soap amount, repetition

of experiment using the same disks, how long can disks stay sunk (overnight?),methods of collecting data.

Materials: One advantage of the leaf floating disk technique is that equipment and supplies

required are inexpensive.

If the experimental design is looking at quantifying light intensity differences,it is highly recommended that a PAR meter (photosynthetically active radiation) beused instead of foot-candles (which is more of an outdated, subjective measure of

luminance). A PAR meter counts photons in the PAR spectrum and will greatlyfacilitate the experimental design. Tese can be purchased for around $35.

Procedures:Design and conduct an experiment to test a variable that affects the Rate of

Photosynthesis.Step 1:

Chose your driving question to investigate.

Step 2: Fill in the ExD (Experimental Design) form on the next page

to plan your experiment. Make sure you are only testing one variable at a time and include a control, identify your independentand dependent variables.

Step 3:

Design data tables and collect data.

Step 4:

Analyze the data.

Step 5: Summarize your conclusions.



Experimental Design (ExD) FormComplete this ExD pre-lab planning form before beginning your lab

1. Independent variable: (What is the cause agent? What are you changing? )

2. Dependent variable: (What is being measured? )

3. Lab set-up:

ExperimentalGroups

Number of Trials

4. Control: (What is the experimental group being compared to? )

5. Hypothesis: (Use an “if ” [Independent Variable], “then” [Dependent Variable] format.State the cause and effect relationship between the I.V. and the D.V. It must be testable.)

6. Lab title: (e effect of ____[I.V.] ____on ____[D.V.]____.)

7. Experimental constants: ( Name at least six variables NOT altered during the experiment.)

8. Sketch of experimental set up with labels:

9. Detailed procedure:



Analysis and Conclusions:

1. Graph your E50

data.

2. Make a second graph with E50

(1/E50

) inverted to show a direct

relationship between the E50 and the rate of photosynthesis.


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Post-lab Connections and QuestionsComparing Photosynthesis and Cellular Respiration

1. How is the energy flow in photosynthesis and cellular respirationrelated?

2. Exactly how is the equation for photosynthesis different fromthe equation for cellular respiration? Compare their reactants,products and energy flow.

3. Compare photosynthesis to cellular respiration in terms oflocation within the cells, and what types of organisms canphotosynthesize and what types of organisms can oxidize sugarsand other food molecules to produce AP during cellularrespiration?

4. Compare the function AP in the light dependent and lightindependent(Calvin Cycle) reaction of photosynthesis to thethree main activities of respiration (glycolysis, Krebs Cycle, andelectron transport).

5. What is the difference on a global scale in the atmosphericcontributions of photosynthesis and cellular respiration?

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