biology Lab Assignment Report

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BIOL& 100L Survey of Biology- Lab #5: Photosynthesis The lab assignment for this week has three parts: (1) Evaluating leaf sources and developing a successful

system for studying photosynthesis/respiration; (2) Investigating the carbon source as a limiting factor for

photosynthesis; and (3) Demonstrating the evidence of respiration.

Review these parts of the OLI curriculum in order to prepare for this week’s lab activities: Modules 24, 25,

and 26 from Unit 5.

Part 1: Developing a photosynthesis measurement system

Overview In Part one, you will evaluate a variety of leaf sources and light arrangements to come up with an effective

demonstration of photosynthesis. This may take some time and several attempts before you achieve success;

so, be patient and persistent.

The core idea here is that fresh leaves that are viable (though detached from the plant) are still capable of

photosynthesis. Further, the oxygen that is produced as a result of photosynthesis is kept within the leaf tissue

long enough to affect the density of the leaf. Therefore, changes in leaf density can be used as an indirect

measure of photosynthetic activity and leaf density can be easily observed by the leaf buoyancy in a liquid

medium. Photosynthesis requires carbon dioxide and water which are provided in the liquid. Light can be

provided by an artificial light source. Respiration occurs simultaneously to the photosynthesis in plant cells;

however, the use of oxygen is not enough to offset the oxygen production when adequate light (i.e., adequate

intensity and wave length) and carbon dioxide are present. Carbon dioxide is adequately abundant in the air to

meet the needs of plants; however, not much of the carbon dioxide dissolves into water from the air. We will

be adding sodium bicarbonate (i.e., baking soda) to the water which dissociates as ions and reacts with the

water to provide additional dissolved carbon dioxide.

Safety The chemical used in this lab is safe; however, you will be using an electrical light source near a container of

water; so, precaution is warranted to prevent an accident. Do not use an incandescent light bulb which can

produce a lot of heat and invalidate the data collected in the experiment. Be careful with the use of the

fluorescent bulb to prevent breakage as there is a safety issue with compact fluorescent bulbs if they break.

Materials and equipment From the Lab Kit:

Plastic Cup 9 oz. (1) Don’t use a beaker

Graduated cylinder 100 mL

Syringe (10 mL)

Sodium bicarbonate (you will use ¼ tsp in this part of the lab and rest in the Part 2)

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From Home:

Desk lamp (preferably gooseneck style) with 20W fluorescent bulb (LED bulbs may work but you need to have

the equivalent amount of light as a 20W compact fluorescent bulb and you need to check to insure that it

doesn’t heat the photosynthesis chamber).

Fresh spinach and possibly other plant leaf materials

Important note about getting the spinach: Spinach that is purchased as a bunch (i.e.., leaves still attached at

the stalk) are more viable than the prewashed, separated leaves purchased in plastic containers. You might

only need to use one 5-6 inch long leaf; however, several may be required if you encounter problems. Try to get

spinach that is crisp (not wilted) and dark green. Keep the spinach cool and use them as soon as you can after

purchase. Contact the Professor if you have any questions about the right leaf source.

Hole punch (single, ¼ inch hole)

Important note about the hole punch: This will be used to cut out consistently sized leaf disks from the spinach;

so, this punch needs to cut cleanly. New ones are inexpensive ($1-2); so, if the one you have is dull and won’t

cut a clean disk from a leaf, consider buying a new one for this lab.

Stop watch (online stop watches can be used)

Spoon (just a normal cutlery spoon - metal or plastic)

Distilled water

1/4 Teaspoon measure

Blank piece of white paper

Directions Step 1: Take the fresh spinach and remove several large leaves from the bunch; place them in a bowl of cool

tap water to keep cold and hydrated. Keep the rest of the bunch refrigerated.

Step 2: Prepare the bicarbonate solution by measuring exactly ¼ teaspoon of baking soda into the plastic cup.

Use the graduated cylinder to add 100 mL of distilled water. Use a regular spoon to mix thoroughly.

Step 3: Turn on your lamp with the proper bulb installed. Bend the goose neck of the lamp down so that it

shines down onto a sheet of white paper (with the bulb around 10 cm from the paper).

Step 3: Use the hole punch to cut out 10 leaf disks from a spinach leaf (or other plant source) onto a wet paper

towel; keep leaf and disks covered with another wet paper towel.

Step 4: Extraction of gases from the leaf disks- First, pull the plunger out of the barrel of the syringe. Then,

with the forceps (i.e., tweezers,) transfer the leaf disks to the barrel part of the 10 mL syringe (See Figure 1).

Push the plunger back into the barrel to the “2 mL” mark on the syringe. Draw up some of the sodium

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bicarbonate solution from the cup into the syringe to the “9 mL” mark. Hold the syringe with the opening up

and gently push the plunger in to push out the remaining air.

Figure 1. Placement of leaf disks into the barrel of the syringe.

To remove the gas from the leaf disks so that they will sink, hold your thumb tightly over the opening of the

syringe (i.e., where the needle would be attached) and draw the plunger out to create a vacuum within the

syringe (See Figure 2A). Shake or twist the syringe as you hold the vacuum steady for several seconds so that

the leaf disks are freely suspended in the solution. Release your thumb and push any air out of the syringe

again. Tap the syringe to see of any disks are sinking. Repeat this process for at least four times. Continue the

process until all the disks clearly sink to the bottom of the syringe when the vacuum is released (See Figure

2B). Note: you might need to tap on the side of the syringe to cause the leak disks to sink.

2A

2B

Figure 2. A- Creating a vacuum in the syringe to remove gases. B- Leaf disks sink to the bottom when ready.

Step 5: Transfer the prepared leaf disks to the cup by holding the syringe over the cup and pulling the plunger

completely out allowing the contents to pour into the cup. Insure that all of the disks sink in the cup (you may

need to dab any floating disks gently with the forceps). Make sure that all the disks are evenly spread on the

bottom of the cup and none overlap (See Figure 3).

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Figure 3. Properly prepared leaf disks in cup of sodium bicarbonate solution: sunk to the bottom and evenly spread.

Step 6: Start the trial run: Take the temperature of the solution and record it in Table 1 of the Lab Report

Template (it should be the same as room temperature). Put the cup with the disks under the lamp so that the

surface of the bulb is around 2-3 cm from the lip of the cup. Immediately, start the stopwatch and make note

of when the first leaf disk rises to the surface. Continue to observe the leaf disks to confirm that their

buoyance is changing (i.e., staying at the surface). Measure the temperature at the end of the trial as well; it

shouldn’t rise more than a degree.

If the leaf viability is high and the light system adequate, you should see the leaf disks beginning to rise to the

surface in around 3-9 minutes. If they don’t all rise by 10 minutes, you need to evaluate what the problem is.

The most common problem is inadequate light and/or low viability of leaves (i.e., not fresh enough). If your

20W bulb is relatively new and appears bright, then the problem is most likely the leaves. You should find

other sources of spinach (see notes above for selection guidance) or try other plants (sometimes house plant

leaves work). Continue to try different materials and methods (recording temperature data and results in

Table 1 for each) until you have success (ideally leaf disks float within 5 minutes of light exposure).

If after several attempts of trying different things, you still do not have rising leaf disks in 3-9 minutes of

exposure to light; contact the professor.

Once you have a method and system that is working well, write a summary in Table 2 of your successful

photosynthesis system; note specific leaf source, light system features, things that you will be carefully

monitoring during the actual experimental trials of Part 2, etc.

Clean up Discard the sodium bicarbonate solution used in Part 1 and clean the cup thoroughly. You will create new

solutions for Part 2 by using the rest of the sodium bicarbonate that was included in the kit and will need all six

cups that are in your kit.

Part 2: Evaluating carbon as a limiting factor in photosynthesis

Overview Once you find a leaf source and lighting system that has reliably demonstrated evidence of photosynthesis

(ideally leaf disks float within 5 minutes of light exposure), you are now ready to experiment with the system.

In this case, you will look at the effects of varying the concentration of the sodium bicarbonate on

photosynthesis.

When we look at biological processes whether they be at the molecular, cellular, organismal, or at the

population level, we observe that the process has finite limits in how long it can occur or to what extent it can

occur under a specific set of circumstances. Plant growth is a good example and relevant to our lab here.

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When a plant is growing in a pot with set amount of nutrients under a given light in a certain environment

(moisture, temperature and atmosphere), it will grow to a certain size and then slow down. According to the

limiting factor principle, there will be only one factor (the one shortest in supply) that will be responsible for

this limitation of growth. So, if the plant is being limited by nitrogen, adding more water, light or any other

factor will not increase growth; only by adding nitrogen, will it continue to grow (at least until another or the

same factor becomes the shortest in supply). Carbon dioxide is essential for photosynthesis so it can be a

limiting factor. This is particularly true for aquatic plants and algae. Even though spinach is not an aquatic

plant, we will be studying its photosynthesis in a water medium with varying levels of carbon dioxide

(produced when sodium bicarbonate reacts with water) and see if we can detect at what concentration it

becomes limiting.

It’s essential to carefully consider the design of your experiment in terms of independent, dependent and

controlled variables. Variables are any factors that can or will be different between samples or trials of your

experiment. Ideally, and most simply, you want to only have one variable that is different between trials. In

this case, it will be the concentration of the sodium bicarbonate because we want to know the effects of

varying this sole variable. This is called the independent or explanatory variable. The effects of varying the

independent variable could include changes in many other variables; however, in our case we will be

measuring only change in timing for leaf disk floatation (called the dependent or response variable). This will

be a proxy for the rate of photosynthesis; i.e., quicker rise of leaf disks will be interpreted as a faster rate of

photosynthesis. However, there might be many other factors or variables that could also contribute to a

quicker rise of the leaf disks such as differences between trials in leaf viability, light intensity, temperature,

solution density, etc. In order to have confidence that the differences in leaf disk rise timing is due to

differences in sodium bicarbonate concentration and not these other variables, we need to make them

controlled variables. In other words, we need to keep them at the same level for all the trials.

Safety See the Part 1 Safety section for a reminder of the hazards associated with these experiments.

Materials and equipment From the Lab Kit:

Plastic Cups 9 oz. (6) Clean the one you used for Part 1 and get out the other five in the kit

Sodium bicarbonate (The remaining amount of 3 grams will be used)

Graduated cylinder 100 mL

Syringe (10 mL)

Sharpie® marker

From Home:

Lamp and bulb set up that tested successfully in Part 1

Fresh spinach or other leaf source that tested successfully in Part 1

Hole punch (single, ¼ inch hole)

Stop watch (online stop watches can be used)

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Spoon (just a normal cutlery spoon - metal or plastic)

Distilled water

Directions Follow these steps:

Step 1: Develop a hypothesis- Review the process of photosynthesis again and reflect on what you would

expect plant cells to do when carbon dioxide is low in concentration. Then answer these questions in the Lab

Report Template:

Question A: If carbon dioxide concentration is low, would expect the plant cells to not photosynthesize at all

or would you expect the rate of photosynthesis to simply slow down? Explain your reasoning.

Question B: In light of your answer to Question A, write an hypothesis that specifically states what you expect

to see in plant response (i.e., floating leaf disk rate) to varying levels of added carbon dioxide. Remember that

hypotheses are not questions, but rather are declarative statements that are testable and falsifiable.

Step 2: Prepare the sodium bicarbonate solutions by conducting a serial dilution-

a) First label the 6 plastic cups as follows: “A”,“B”,“C”, “D”, “E” and “F” with the marker.

b) Put the contents of the sodium bicarbonate bag from the lab kit into the cup labeled “A.” Use the

graduated cylinder to measure and transfer 200 mL of distilled water into this cup; use a clean spoon

to mix until fully dissolved. This will result in the addition of 0.08% equivalent mass of carbon dioxide.

c) Use the graduated cylinder to measure and transfer 100 mL of distilled water into each of the rest of

the cups (B, C, D, E and F)

d) Use the graduated cylinder to measure and transfer 100 mL of the solution in the “A” cup to the “B”

cup; mix with a clean spoon. This dilutes the concentration by half; so, cup “B” has an added 0.04%

equivalent mass of carbon dioxide.

e) Use the graduated cylinder to measure and transfer 100 mL of the solution in the “B” cup to the “C”

cup; mix with a clean spoon.

f) Use the graduated cylinder to measure and transfer 100 mL of the solution in the “C” cup to the “D”

cup; mix with a clean spoon.

g) Use the graduated cylinder to measure and transfer 100 mL of the solution in the “D” cup to the “E”

cup; mix with a clean spoon.

h) Use the graduated cylinder to measure 100 mL of the solution in the “E” and discard down the sink.

i) The cup labeled “F” will just have 100 mL of distilled water and the carbon dioxide concentration will

just be what naturally diffuses into it from the air; however, we’ll consider it as 0% as in no additional

carbon dioxide was added.

Now all of the cups have an equal amount of liquid (a controlled variable) but different concentrations of

added carbon dioxide (the independent variable) as follows:

“A” 0.08% “B” 0.04% “C” 0.02% “D” 0.01% “E” 0.005% “F” 0%

Step 3: Prepare the leaf disks- You will want to use the same leaf material that worked well in Part 1. Ideally, it

will be a single large leaf that will provide adequate number of leaf disks for all the trials (total number of leaf

disks = 60). However, if the leaves are smaller, you’ll need to use multiple leaves. The problem is that we want

leaf viability to be a controlled variable (i.e., the same among all the trials) and different leaves are likely to

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have different viabilities; so, you will have to think about a means of insuring that each cup will receive 10 leaf

disks with equal viability on average. One way to do this is to cut out the leaf disks from the leaves of the same

bunch that worked before and then mix them on the wet paper towel. Then randomly select ten for each trial.

It’s important to use uniform leaf disks; avoid using partial disks and disks that are cut from major leaf veins.

They should all be of equal green color intensity. Keep leaves and leaf disks cool and wet while you’re cutting

them out and conducting the trials.

Step 4: Conduct the photosynthesis measurement trials- Starting with cup “A,” follow the same procedures as

you did in Part 1 to remove the gases from 10 randomly selected leaf disks. Here’s the procedure again:

Pull the plunger out of the barrel of the syringe. With the forceps (tweezers,) randomly pick up ten of the leaf

disks and transfer them to the barrel part of the 10 mL syringe. Push the plunger back into the barrel to the “2

mL” mark on the syringe. Draw up some of the 0.08% solution from the cup into the syringe to the “9 mL”

mark of the syringe. Hold the syringe with the opening up and gently push the plunger in to push out the

remaining air.

To remove the gas from the leaf disks so that they will sink, hold your thumb over the opening of the syringe

and draw the plunger out to create a vacuum within the syringe. Shake or twist the syringe as you hold the

vacuum steady for several seconds. Release your thumb and push any air out of the syringe again. Tap the

syringe to see of any disks are sinking. Repeat this process for at least four times or until all the disks clearly

sink to the bottom of the syringe when the vacuum is released. You might need to tap on the side of the

syringe to cause the leak disks to sink.

Transfer the prepared leaf disks to the cup by holding the syringe over the cup and pulling the plunger

completely out allowing the contents to pour into the cup. Insure that all of the disks sink in the cup (you may

need to dab any floating disks gently with the forceps). Make sure that all the disks are evenly spread on the

bottom of the cup and none overlap. Take a photograph of Cup “A” at this point.

Take the temperature of the solution and record it in Table 3 of the Lab Report Template along with notes

about the experimental trials.

Make sure that the lamp has been on long enough (5 minutes) to come up to full light intensity. Put the cup

under the lamp so that the surface of the bulb is around 2-3 cm from the lip of the cup and start the

stopwatch. Make sure that all of the disks are fully sunk to the bottom and evenly spread still.

Collect these measurements (these are the dependent variables) and record the values in Tables 3 and 4 of

the Lab Report Template:

 Time in minutes and seconds when the first leaf disk rises to the surface. (Table 3)

 Number of leaf disks that have risen to the top at specified time intervals. This includes at time zero

when the number should be zero (i.e., all the leaf disks are sunk) and when the stop watch reaches 5

minutes, 7 minutes, 9 minutes, 11 minutes, 13 minutes, 15 minutes, and 30 minutes. (Table 4)

At the point when all of the leaf disks have reached the surface or you have reached the 30 minute time

interval, you will stop the trial. For those trials ending before 30 minutes, fill out the rest of the data sheet

with 10’s for the subsequent time intervals; e.g., if all ten disks reach the surface by 9 minutes, you would

record “10” for the 9, 11, 13, 15 and 30 minutes intervals.

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Once done with the trial for cup “A,” take a photograph of it.

Follow the same procedure with all of the cups in order from “B” to “F” (except you do not need to

photograph every cup; just “A”). Remember to measure temperature at the beginning and end of every trial.

Reflect on any factors that might be different between trials and record them in Table 3 along with any other

comments that you feel might be relevant to the study.

Step 5: Transfer data to the Excel spreadsheet and examine the data graphically- After collecting the data from

all the trials and having completed filling in Tables 3 and 4, transfer the leaf count data to the table in the Excel

file called “Lab 5 Excel Spreadsheet” and follow the directions given in that file. Copy and paste the graph into

the Lab Report Template (Table 6).

Step 6: Draw conclusions from the data- Think about what you expected if carbon dioxide becomes limiting

and examine the data in light of your hypothesis (Step 1). Then, answer the following questions in the Lab

Report Template.

Question C: Is there a correlation between the independent variable and dependent variables? Describe the

pattern and interpret its possible meaning.

Question D: Does the data support your hypothesis? Describe what conclusions can be logically made

regarding the effect of carbon dioxide concentration on leaf disk photosynthesis.

Question E: How might the experiment be done better with a view to testing your hypothesis?

Clean up Don’t discard anything from Part 2 yet. You will be using the solutions and leaf disks in Part 3; so, immediately

proceed with the next part of the lab.

Part 3: Evidence of respiration

Overview In this part, we want to demonstrate evidence that our plant disks are respiring as well. Even when they are in

the light, respiration is occurring; plant cells need the ATP generated by respiration to fuel cellular processes

just like animal cells. Respiration uses oxygen and it’s the oxygen that was produced during photosynthesis

that is making some of your leaf disks to float; so, if we can shut down photosynthesis, we should see the leaf

disks lose their buoyancy as the oxygen within the plant tissue is used up for respiration.

Also, in this part, we are going to do a fun exercise to connect the two processes- photosynthesis and

respiration.

Safety See the Part 1 Safety section for a reminder of the hazards associated with these experiments. In addition, you

will be blowing through a straw for around 2 minutes; therefore if you have respiratory problems or are having

difficulties with this, consider having a friend or family member help you. If there is no one to help you and

you are not wanting to do this, you can skip step 3 listed below.

Materials and equipment Use the materials and solutions prepared in Part 2. In addition, you will also need a clean drinking straw for

Part 3.

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Directions Follow these steps:

Step 1: Prepare leaf disks for a respiration trial- Typically, all the disks will have floated in Cup A and that will

be the cup used for this respiration trial. First add a drop or two of liquid dish detergent to the cup. Detergent

is made of chemicals that act as surfactants and will reduce the surface tension of water; thus, allowing the

leaf disks to sink when they become denser than water again and lose their buoyancy. Gently mix in the

detergent with a clean spoon. Place Cup A under the light again for three minutes to fully charge up the leaf

disks with oxygen.

Step 2: Conduct the respiration trial- Place Cup A into a completely dark chamber. This could be a kitchen

drawer or cabinet that closes tightly. It’s important that there is no light at all. Start the timer and check the

cup for sunk leaf disks at 0 minutes (should be 0), 3 minutes, 5 minutes, 10 minutes, 20 minutes, and 30

minutes. Record your data in Table 7. In between the time intervals, you can start working on Step 3.

Step 3: Connecting respiration and photosynthesis- Typically, Cup F will not be having any floating leaf disks

after by 30 minutes under the light (Part 2). Use the forceps to carefully remove the leaf disks from this cup

and place them in the barrel of the syringe and insert the plunger to the 2 mL mark. Take a clean drinking

straw and blow through it into the “F” cup. Blow slow and steady for 2 minutes. (Safety note: If you have

respiratory problems or are having difficulties with this, consider having a friend or family member help you. If

there is no one to help you and you are not wanting to do this, you can skip doing step 3 altogether.) After

bubbling your breath into the cup for 2 minutes, use the syringe to draw up the solution to the 9 mL mark and

de-gas the leaf disks as you did before. Pour the leaf disks back into the cup and place under the light with the

stop watch going. Record the time when the first leaf disk rises and the leaf counts at 5 minutes, 7 minutes, 9

minutes, 11 minutes, 13 minutes, 15 minutes, and 30 minutes. (Table 8)

Step 4: Answer these questions pertaining to Part 3-

Question F: Is the experiment where Cup A is put into a dark chamber (steps 1 and 2) a convincing

demonstration of respiration? Review Module 26 of the OLI curriculum and describe what the plant cells are

doing with the oxygen molecules that had been trapped in the plant tissues and were causing the leaf disks to

float.

Question G: Do a quick internet search to find out what the % concentration is for carbon dioxide in the air

and in exhaled breath. Report those numbers and explain why we would expect the leaf disks to float in Cup F

after blowing into the cup and putting it under the light even though it didn’t have leaf disks floating

previously when put under the light.

Question H: In light of your observations of this part of the lab and the chemical reactions explained in Module

26 of the OLI curriculum, describe how photosynthesis and respiration are tied together.

Clean up Leaf disks should be thrown away in the trash (or composted if you do that at home) and the cups, syringe,

and graduated cylinder should be washed thoroughly, dried, and returned to your lab kit.

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Lab Discussion Forum Here is what you should post for this discussion assignment: (a) a description of the experimental system that

you used for Part 2 (type of leaf, type of light, etc.) along with your data from Part 2 as well; and (b) a

description of how you controlled for leaf variability; i.e., how did you make sure that each cup for Part 2 had

comparable leaf disks? Complete directions and a checklist/rubric are provided at the discussion page labeled

“Lab Assignment #5 Discussion” in Canvas.

Taking it further For those wanting to take these concepts further, apply what you have learned about photosynthesis to

examine the effects of increased carbon dioxide levels in the atmosphere. Investigate these questions:

What are the different ways that various plant species fix carbon dioxide? Explore the major types called C3,

C4, and CAM plants by viewing this Khan Academy video series.

How will the increased carbon dioxide level in the atmosphere and warmer conditions at many places on Earth

affect plant growth? Reflect on the potential effects by reading this article.