Lab 8: Pre-Lab
Your task in Lab 8 is to design and carryout an experiment regarding the rate of photosynthesis. You will use your research proposal to create an experimental protocol, and analyze provided your data as part of this lab. To prepare for Lab 8, please review this pre-lab page. Once you feel confident regarding the below topics, complete the corresponding LABridge in Blackboard.
Photosynthesis & The Light Reactions
CONNECTION ALERT! Cellular respiration is the topic of Chapter 10 in your BIOL 120 lecture. Please review your textbook as needed for this lab.
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Review & Connections: Energy is the currency of life: all living organisms require energy to survive and reproduce. Metabolism is the series of reactions and processes, catalyzed by enzymes, which together maintain life. These reactions fall into two types: catabolic or anabolic. These processes are the inverse of each other and in photosynthetic organisms occur in tandem as the anabolic reactions of photosynthesis create the products that are then broken down by the catabolic reactions of cellular respiration.
Photosynthesis is a light-dependent process in which radiant energy is captured, and converted to chemical energy which is then utilized to create fuel for organisms. Chlorophyll A is the primary photoreceptive pigment and is adept at absorbing wavelengths in the blue and red spectrum, but wavelengths in the green and yellow (500-600 nm) are transmitted, thus, most plants appear green. You should know about the other secondary pigments and with what colors they are associated.
There are two primary sets of reactions in photosynthesis.
Photosynthesis is a light-dependent process in which radiant energy is captured, and converted to chemical energy which is then utilized to create fuel for organisms. Chlorophyll A is the primary photoreceptive pigment and is adept at absorbing wavelengths in the blue and red spectrum, but wavelengths in the green and yellow (500-600 nm) are transmitted, thus, most plants appear green. You should know about the other secondary pigments and with what colors they are associated.
There are two primary sets of reactions in photosynthesis.
- The Light Reactions: The mechanism behind this important process starts with photoreceptive pigments within the chloroplasts of plant cells. When these pigments absorb light energy, they become excited, and along with the energy from splitting water, the process generates adenosine triphosphate (ATP) and NADPH, chemical forms of energy. *We will be measuring this process! Specifically, we will measure how quickly NADP+ is reduced to NADPH.
- The Calvin Cycle: The ATP and NADPH are then used to reduce CO2 and assemble glucose (C6H12O6), a common monosaccharide and major energy source for most living organisms. Often plants assemble glucose into the more complex polysaccharide of starch. Without the ability of photosynthesis to convert light into fuel, heterotrophs (all animals including humans) would cease to exist.
Please review the light reactions and the Calvin cycle.
Be sure you know the basic steps of photosynthesis, and the reactants and products. Could you write the full equation? Be sure you understand the reduction of NADP+ to NADPH.
Do you know enough about spectrophotometry?
Spectrophotometry is the technique we would have used in lab this week. It is a technique that can be used to identify unknown concentrations in a sample, based on its color! In our lab, samples that undergo a lot of photosynthesis will become a lighter color purple. Solutions that undergo little photosynthesis will stay a darker color purple.
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Spectophotometry uses the principles of light and color to help identify concentrations of various components in a solution.
Wavelengths of light are associated with various colors due to their frequency. When light hits an object or solution (like a plant leaf in the figure at right), wavelengths are either absorbed or reflected/transmitted. We see color based on the associated wavelengths that are transmitted or reflected. We do not see the colors associated with the wavelengths that are absorbed.
For example, view the absorption spectrum for typical plant pigments (graph in the sidebar). The graph shows that when light hits a plant, purple, blue, red, and orange wavelengths have high absorbance values, meaning they are mostly absorbed by the plant and we don not see these colors. Green and yellow wavelengths have low absorbance values- meaning they are reflected... this is what gives plants their green color! When a color-sensitive indicator is used the absorbance values are directly proportional to the concentration in the solution.
Recall that during the light reactions of photosynthesis, a molecule called NADP+ is reduced to NADPH (energy is loaded onto it in the form of electrons). So the rate at which NADP+ becomes NADPH approximates the actual rate of photosynthesis. In our lab this week, we will be using something called DIP as a substitute for NADP+. DIP = NADP+ DIPH = NADPH DIP is a dark purple color. As the light reactions of photosynthesis proceed, the DIP becomes lighter and lighter as it is reduced to DIPH (like NADP+ to NADH). BUT! We cannot see the subtle changes in color. Instead, we will use the spec to determine the value of the color, as measured by its absorbance.
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We see the color of the wavelengths that are transmitted or reflected. We do not see the colors of wavelengths that are absorbed.
Absorption spectrum for typical plant pigments
Click here for a review on spectrometry.
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Can you answer these questions? What is a spec? How does it work? How will we use it in lab? What is DIP? How can it tell use anything about the rate of photosynthesis? What is the relationship between the solution's color and the rate of photosynthesis?
If you can easily answer the questions above, you are ready to move on. If not, please review the content on this page further.
What would we have done in lab?
REMEMBER! You designed a research proposal for this lab! You have selected the independent variable you are interested in. Your options included: wavelength of light (with colored lenses), type of light, amount of light, temperature (hot, cold), addition of fertilizer, herbicide or pesticide, or concentration of chloroplasts. The procedure here is what would have been done for you to collect data on the affect of your selected independent variable on the dependent variable of "rate of photosynthesis."
Specifically, we were going use isolated chloroplasts suspended in solution and expose them to the variable(s) of your choice. We would have then measured the rate of the light reactions by determining how much NADPH (really DIPH) is produced by our active chloroplast.
Read the procedure we would have used below.
REMINDER: What the heck is DIP? Normally, the light reactions require NADP+. Photons excite electrons and they are used to reduced NADP+ to NADPH (which is then used in the Clavin Cycle). Instead of adding NADP to "feed" our chloroplasts, we are adding DIP as a stand in. This is because DIP is color reactive, meaning we can use color changes to determine how much DIP is reduced to DIPH. That is how we will determine the rate of the light reactions in our differing environments: DIP is a blue/purple color, as it is reduced to DIPH the color intensity is diminished, it gets closer to clear. |
Chloroplast diagram. Click to enlarge.
The complete photosynthesis reaction with redox labels. Click to enlarge.
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- So...after your experiments run in the test tubes, we will use the spectrometer to determine the absorbance value of the solution. You will use the solution from one of your controls to set the max absorbance on your spectrometer: the standard conditions with chloroplasts.
- View the relationship between absorbance values and rate of the light reaction below:
- You will collect the absorbance values for your control (in standard conditions) and any other experimental tests you decided to run in a data table. Similar to our last lab, you will complete data collection & analysis on your data in your Lab Notebook Entry.
Be sure you understand the relationship between absorbance values and rate of the light reaction. Review the chloroplast diagram and be certain you know the equation for photosynthesis, what gets oxidized and what gets reduced.
Once you feel confident in this material, please proceed to Blackboard and take the Lab 8 Pre-Lab Quiz.
Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!
Lab 8: ProtocolIn today's lab you will work with your lab group to review and revise your photosynthesis research proposal, conduct your experiment and analyze your data.
Exercise I. Review/revise your proposal Exercise II. Conduct a virtual lab using spectrophotometry Exercise III. Begin to analyze your data |
Lab Objectives: Following today's lab, you should be able to...
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Exercise I. Review & Revise
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Procedure
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Lab 8 Notebook Guide. Click to download.
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5. Review the slide show below (from Lab 6) for a refresher on the parameters in your experimental protocol.
6. Write up a step-by-step summary of how you might have conducted this experiment.
7. Lastly, create some type of illustration or diagram of your experimental design. You can use whatever program/software you chose.
Word and PowerPoint would suffice.
6. Write up a step-by-step summary of how you might have conducted this experiment.
7. Lastly, create some type of illustration or diagram of your experimental design. You can use whatever program/software you chose.
Word and PowerPoint would suffice.
Exercise II. Practice Spectrophotometry
Spectrophotometry is an increbly useful technique! This virtual lab is meant to give you sense of what this technique is like and how it can be used. Hopefully you'll get to use this skill later on in other classes.
1. Enter the virtual Spec-Lab (please update or install your viewing software if prompted). You will see 5 cuvettes. Cuvettes are special spec test tubes. The one labeled 0 is your “blank” and is used to reset your spec. Cuvette tubes 1-3 you will fill with your own mix. Cuvette tubes 4 and 5 are standards and cannot be changed. We will IGNORE tubes 4 and 5.
The solutions you will prepare in step 2 below will closely mirror the possible colors of the DIP solutions we would have used in lab, meaning the model we will create from these solutions to applicable to DIP and will be used in Exercise III.
2. Prepare your solutions for tubes 1-3. The arrows beside the colored squares at the top right allow preparation of solutions. Add the blue and “white” (white = water) solutions to create any shade of blue you desire. DO NOT ADD ANY RED.
SPECIAL NOTE: The concentration of blue dye in this solution is 8.0ppm.
SPECIAL NOTE: The concentration of blue dye in this solution is 8.0ppm.
- Click tube 1. Add your preferred volume of blue stock solution and water. It will show in the drop bottle. Record the final volume of the blue solution and water in your sample in your Notebook Guide in ppm. Click the dropper bottle to add the solution to tube 1.
- Repeat the above steps for tubes 2 and 3.
3. Test the absorbance of your solutions.
- Turn the bottom left knob to turn on the spec.
- Click the first top button to set the spec to read absorbance values.
- Set the wavelength to 550nm.
- Click tube 0, it should float into the spec. This is just water and used to “reset” the spec. Therefore, it is also referred to as your BLANK. Read and record the absorbance value (ABS) provided on the spec’s screen. It should be very close to zero. Click on the compartment’s lid to return the tube to the rack. If the value is NOT close to zero, press the auto-zero button just below the spec.
- Complete the above steps for tubes 1, 2 and 3. Be sure to record the ABS values in your Notebook Guide.
- Once you have these values you can leave the virtual lab.
Turn it on.
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Set to take absorbance values (ABS).
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Set wavelength for 550nm.
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4. Determine the concentration of “blue” of your solutions. Record the results in your table. Use this equation:
- Final concentration of your dilutions = what we are solving for, the concentration of “blue” of your solutions
- Volume of stock solution = volume of blue solution you added to each tube (no units)
- Original concentration of stock blue solution = 8 ppm
- Total volume after dilution = total amount you added (blue + red + water) (no units)
5. Complete the analysis in your Lab Notebook Guide. You will use your absorbance measurements and calculated concentrations to create a model of the relationship between the two variables.
- Do you think these variables are correlated?
- Create a scatterplot with these two variables in Excel. You will have 4 points (one for each tube). Be sure ABS is on your Y-axis, it's the dependent variable. Be sure concentration is on your x-axis, it is the independent variable.
- Be sure to trim your axes to your values so the graph takes up the entire space. You don;t want a bunch of points scrunch in one corner.
- Add a line of best-fit (linear trend line) to your scatterplot: Select your graph >> hit plus (+) >> check "trendline"
- Add the equation of the line: Click on the new line >> scroll to the bottom on sidebar at right >> check "display equation on chart"
6. Copy/paste your graph into your Lab Notebook Guide. Now you can use this model to make some predictions. Complete the questions in your Lab Notebook Guide.
AnalysisProcedure
Remember: Statistics solve the problem of determining if "more" or "higher" or "different" than is actually enough to be important and biological relevant. Using the principles of probability, they help us parse what we observe from randomness (chance alone) as meaning (a real difference, or a real relationship). Statistics tell us how likely we would be to make the same observations we have made, if chance and randomness were the only drivers. If the probability is very low (<5%), we refer to these patterns as significant.
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Original photosynthesis data in Excel. Click to download.
The data generator used to create the original photosynthesis data sheet contained an error. If you feel your results are counter intuitive and you'd like to give it another shot, please use these "new data" for analysis.
Visit the stats section of our research library.
T-test Online Calculator.
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Get Started on your Posters!
Each team member MUST submit your poster individually in blackboard before the due date/time. You can turn in a completed draft for feedback before you final is due is you would like. Final posters will not be accepted late, even if others from your group already turned them in. Currently, your final poster is due the week of Apr 13 - 17. The template on the right has detailed directions for each section.
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This is the template for your poster. It is a PowerPoint file and can only be opened in PowerPoint. It is properly sized and includes specific directions for each section. Click here to download and open this file.
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You already have a lot of what you need!
Your research proposal has research that gets your introduction started. Use the research library for more! Your proposal and the protocol you developed in your lab notebook will provide the bulk of the methods section.
In your methods section, be sure to detail what you WOULD have done in lab had we met, DO NOT include the methods for the virtual labs we used as substitutions.
Your Lab Notebook also has all the results you need plus some good points to include in your discussion!
Your research proposal has research that gets your introduction started. Use the research library for more! Your proposal and the protocol you developed in your lab notebook will provide the bulk of the methods section.
In your methods section, be sure to detail what you WOULD have done in lab had we met, DO NOT include the methods for the virtual labs we used as substitutions.
Your Lab Notebook also has all the results you need plus some good points to include in your discussion!
SOME SUGGESTIONS FOR A GREAT POSTER
- Studies show that you have only 11 seconds to grab and retain your audience's attention so make the punchline prominent and brief.
- Most of your audience is going to absorb only the punch line. Those who are directly involved in related research will seek you out anyway and chat with you at length so you can afford to leave out much of the details and tell those who are really interested the "nitty-gritty" later.
- Graphic materials should be easily visible from a minimum distance of 6 feet. For general text, 18 point is readable.
- For section headings (Introduction, Methods, etc.) use Helvetica or Arial, Boldface, 36 point. Keep in mind that san serif fonts (having characters without curlicues or other embellishments) are easiest to read, particularly from a distance.
- Use left-justification; text with even left sides and jagged right sides is much easier to read.
- Edit Ruthlessly! There ALWAYS is way too much text in most posters. Posters primarily are visual presentations; the text materials serve to support the graphic materials. On average the total amount of space should include about 30% text, 50% graphics and 20% empty space.
- Spatial organization makes the difference between reaching 95% rather than just 5% of your audience. Time spent hunting for the next idea or piece of data is time taken away from thinking about the science. Arrange the contents in a series of 3, 4, or 5 columns. Columns facilitate the flow of traffic past the poster remembering that people read from the upper left downward.
- What to include? Images of crayfish or chloroplasts, important equations, important molecules, your own pictures from lab, flow charts or illustrations...think outside the box
Help us build a better research library. Please find at least one reference that would be relevant to add to our on-line library for this experiment.
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Lab 8 BIOL 120 CONNECTIONS Section 1.6: Doing Biology Big Picture 1: How to Think Like a Scientist BioSkillls 2: Reading & Making Graphs BioSkillls 3: Interpreting Standard Error and Using Statistical Tests BioSkillls 4: Working with Probabilities Chapter 10: Photosynthesis |
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