Why use 0. To prevent osmotic shock and lysis of chloroplasts. Chloroplasts are removed from 0. Students are not explicitly told to add unboiled chloroplasts to cuvette 1 the blank , although this is indicated in Table 4.
If unboiled chloroplasts are not in the blank cuvette, all the other readings will be nonsense. Set the Spec 20 to nanometers use the wavelength control knob, on top of the instrument. Some older versions of this exercise suggested using nm, but we have found that nm works better. Adjust the 'zero' setting with no cuvette in the sample holder. Then insert each of the other tubes to read the absorption of the DPIP. Question: "It says to set the spectrophotometer to nm.
Why that wavelength, and how important is it to be exact? Would it work at other wavelengths as long as you were consistent? Looking in the teachers' guide, I see spinach extract has its lowest absorbance around nm.
So I'm thinking maybe we set it there in order to let as much light as possible initially hit the DPIP? Is this correct? Also, can you calibrate using distilled water, or should you always use ethanol? Any 'light' you can shed on this whole part of the lab would help.
Answer 1: "In choosing the wavelength setting for the spectrophotometer in the photosynthesis lab, you are trying to find a wavelength at which you can most easily see changes in absorbance by DCPIP against a background of absorbance by chlorophyll. So you are not necessarily choosing the wavelength at which DCPIP absorbance is greatest, or the wavelength at which chlorophyll absorbance is lowest.
Instead, you are choosing the wavelength at which the 'difference' between the absorbances is greatest. In other words, you compare the entire absorption spectrum of DCPIP with the entire absorption spectrum of chlorophyll, and then choose the wavelength at which the spectra are furthest apart show the greatest difference.
This wavelength is nm. Of course, in making this choice, you must pick a wavelength that is strongly absorbed by reduced DCPIP. So it won't work to pick a wavelength down in the blue region of the visible spectrum—DCPIP doesn't absorb blue much at all.
Instead it reflects blue wavelengths, which is why it appears blue to our eyes. Other wavelengths will work, but the sensitivity of the measurement is less at these other wavelengths. You are correct—consistency is important, and as long as you use the same wavelength that is, any wavelength which DCPIP absorbs throughout the experiment, you should be able to get results. But it just makes the whole procedure so much easier if you choose the best wavelength to begin with.
It is not necessary to be 'very exact'—the setting could be nm or nm but, again, it just makes things easier if you make an effort to set it at the best wavelength when you begin. I am a little unsure what you mean about calibrating with water versus ethanol. You set the zero on the spec with nothing in the holder—no cuvette, no liquid, nothing. Then you set the percent transmittance with a cuvette containing phosphate buffer, distilled water, and unboiled chloroplasts reaction tube 1 in the edition D of the lab manual.
Unfortunately, edition D is not very clear on how to set the percent transmittance. It is indicated in Table 4. It is absolutely necessary to include the correct volume of water, the correct volume of phosphate buffer, and the correct amount three drops of unboiled chloroplasts in the cuvette used for setting percent transmittance setting the blank.
I don't see any way that ethanol would work at all. But maybe I am misunderstanding the question. Answer 2: "You set the wavelength at nm because that is the wavelength for which DPIP has its greatest absorbance darkest color. As for the filter setting, I have two specs that don't have a filter setting and one that does. Mine has two settings only, and When you are using the Vernier probes and the colorimeter, you use the red setting. Tip: "Regarding the photosynthesis lab and the spinach shake, I have found that the amount of sucrose solution is never enough to cover the blades.
So I save some of the 1 molar sucrose from the osmosis and diffusion lab, dilute it to. Tip: "Since we only have one Spec 20, I had to get really creative. I had each group of three students do a separate part of the lab and plan an assembly line.
One kid would calibrate with the blank cuvette, another would put the next cuvette in, etc. It went quickly and although we did not get large amounts of data, it exposed the kids to the procedure.
They felt very confident answering the free-response question on photosynthesis and scored well on it, so I guess it worked. Tip: " For the boiled chloroplasts, you must vigorously re-suspend with pipette Pasteur or disposable until the clumps are broken up. The labs included are:. Question: "We just finished the photosynthesis lab and unfortunately it didn't work even though the spec was set right and the kids appeared to measure everything correctly.
Is there a trick to making the unboiled chloroplasts in light photosynthesize? If the sucrose solution isn't absolutely cold, the heat of the blender will deactivate the chloroplasts.
I have done this lab with no problems for several years as long as I can find at least one working Spec 20 in the department. Everything has to be kept as cold as possible until you are ready to use it. Also, did you use a heat sink and activate the spinach with the light for a couple of hours?
Even without the Spec 20 results, my students could see that a change was taking place because the chloroplast cuvette kept getting lighter and lighter in color. If you see this happening and it still doesn't register on the Spec 20, there is something wrong with the machine. Those things can get really finicky sometimes. Also, fingerprints on the cuvette and any of a number of other things can get you. Question: "For the second year in a row measuring the rate of photosynthesis using spinach leaves, DPIP and a Spec 20 have failed to show any activity with spinach chloroplasts.
Since I followed the directions exactly for producing a chloroplast solution and knowing my Spec 20s work properly, I am left to wonder about DPIP. Should this be diluted further? I am at a loss for this lab. Answer: "Mine has never failed to work. There is an error in the AP manual I remember and the College Board has it listed on their site it may be out of date. It involves one of the controls if I remember correctly. I made up my DPIP about 10 years ago according to directions in the original teacher's manual and I store it in a brown bottle and I'm still using it years later.
You can see the change occurring easily It fades quite dramatically in the 'exposed to light' batch. I put my spinach under the grow light overnight before the lab and blend them in sucrose solution and filter the puree through cheesecloth. I have always had textbook-perfect results unless I had clumps in the boiled ones. Then that one had some erratic points.
I have had groups who will reach for the wavelength knob and turn it instead of the knobs on the front of the machine. That will really skew the data if you don't catch it. We usually have to start over if I don't catch that, so now I put a piece of masking tape over that knob after we choose the correct wavelength to prevent inadvertent resetting of the wavelength. Tip: "I use the Spec 20 and it does well even though the groups stack up waiting a turn; I got a second one this year and it helped.
One thing I did this year was make my chloroplast suspension too concentrated I assume. The DPIP was clear by the end of the first five-minute reading and then the graphs flattened out.
I assume the rxn was running so fast that it completely reduced all the available DPIP. If one looks at only the first five minutes of data, the results were as predicted.
After that, they were all over the place. Any suggestions? Answer: "Don't dispose of it, keep it for next year! Tightly cap the bottle and store it with your other organic solvents. I have used the same bottle of solvent for two to three years with excellent results.
Bell , St. Mary's Hall, San Antonio, Texas. Students were able to collect decent data. The only catch is that it only runs at three wavelengths. That's not a problem for the DPIP.
However, you cannot use it for making an absorption spectrum unless you're satisfied with only seeing the absorption at three wavelengths. We also have two Spec 20s that groups take turns using in order to determine the absorption spectrum.
As Doug pointed out, when you get to the longer red wavelengths you lose some accuracy. I'm not sure if that is true with the newer Spec 20s. Tip: "I used the new oxygen gas probe for a photosynthesis lab and in less than six minutes using three leaves snipped off a house plant I had the neatest curve you have ever seen. I have also used the CO2 probe from Vernier with excellent results in photosynthesis and other labs.
Answer 4: "If you had time to make full use of the spectrophotometers within your department, that might be more worthwhile. However, having enough colorimeters and LabPros to allow smaller student groupings, and maybe even to be able to purchase some other probes as well would make more sense to me.
To maximize each student's experience with the equipment and procedure, I like having students work in pairs as much as possible. Answer 5: "I love spectrophotometers—but, if I was starting out, I'd consider buying colorimeters rather than a spec for biology. You can buy about colorimeters that work with graphing calculators and data collection devices CBLs, etc.
If your school is utilizing these data collection devices, it is a more student friendly way to go! Additionally, if I did decide to buy colorimeters, I'd definitely consult the appendix of a college chemistry book for the condensed lesson on the full-blown spectrophotometer so that my students knew how the 'real deal' worked.
The data you collect with the colorimeter will be as if not more accurate than the full-blown spectrophotometer. I have quit using the Spec 20s and gone to doing all my AP Chemistry experiments with these. They are cheap enough that I have 12 of them and can have students working in pairs.
Answer 7: "I had a spectrophotometer from Fischer that interfaced with the PC. It never worked right. Last year I had a great deal of money to start the AP Biology course and I liked the versatility of the Vernier product line. I already had familiarity with their Serial Box interface and the probes are very reliable. This year I purchased six DataLogger interfaces and two colorimeters.
I didn't need more since the AP Chemistry teacher had four colorimeters already. I recently finished the photosynthesis lab using the colorimeters and got great results on my first time three sections of AP Biology. We had our specs tuned up and the company wouldn't even touch them.
The PC interface is very intuitive and the support from Vernier is fantastic. I highly recommend the Vernier line of products. PASCO also puts out a similar line of interfaces and probes. Answer 8: "I would concur with those who have had good data from the Vernier colorimeters.
Even better, with a LabPro they can be interfaced to a computer OR calculator. My class results for the AP photosynthesis lab are beautiful. I do have some Spec 20s, but the Vernier colorimeters give nearly equally reliable data.
The only disadvantage is that with three wavelengths you cannot do a meaningful absorption spectrum of a sample.
I use Vernier colorimeters for some other experiments though, and they are great. I would be wary of some of the Carolina products, which I feel are marked up substantially above their value.
Answer 9: "I see uses for both pieces of equipment. My vote IMHO is to use the spec since I also like to work a little bit with absorption, transmission, and emission ideas.
I tie them into light, photosynthesis, and vision; not too much time here, but enough that the spec seems like a natural extension. So, if you might have a grant, that would be my vote if you're thinking of including ancillary topics. Since you can't adjust the 'black box' colorimeter and peek inside beyond a few set wavelengths, then it's tougher to get the students to understand it. Now that I've stuck my neck out, it might not surprise you to know that of the specs, I even like the 'older' style with a dial rather than the digital read out.
For me IMHO there is value to seeing the dial move, and to seeing the inverse relationship between absorbance and transmission labeled on the dial. On the other hand, I certainly see the value of the colorimeter; it might be fine, if your real goal is to get data, or if integrating data into CBLs is important to you and your school.
I find that they both give good data Vernier is the one I have used. The preset wavelengths can be used for a variety of labs, including bacterial growth by turbidity that I recently worked on. Since creating a growth curve was my goal here biology 1 , the quick colorimeter was great. Now, one political tip: If after reading all the postings and you're interested in both, you could get the expensive specs now while the money is available, and gradually get the less expensive colorimeters over a period of years if you want them for other uses.
Tip: "On the photosynthesis lab, the teacher's manual on page 22 Edition D gives a satisfactory protocol to prepare a colorimeter in place of using the Spec Eleven tubes are prepared for the students to use as comparison. This has worked well for me when my spectrophotometer quit working. I would not, however, add any food coloring. When we tried this, the results were much harder to determine than it would have been if left alone.
The color difference is minimal. In fact, their experimental tubes were more blue than blue-green. On their graphs, most students used the test tube numbers 1 through 11 on the y-axis to show the percent of transmittance, but some related 11 tube to percent transmittance and adjusted the rest accordingly. Its at least worth checking it out and letting the students try it.
There are yeast labs to show fermentation I do this as an additional lab that are much easier if you have no time to make substitute equipment aquarium rocks for glass beads, beans instead of peas, soda lime in the respirometers in place of KOH, etc. But there are also protocols for respirometers made from Gatorade bottles, and you can use crickets, or whatever, to measure cell respiration. The first year is hectic to say the least; sometimes graphing and analyzing 'ideal data' from the teacher's manual is an option rather than leaving the lab out.
In my opinion, both are quite important labs to address. Question: "I have some great new computers and Vernier colorimeters DPIP will not reduce as much as if the chloroplasts were not boiled. When chloroplasts are boiled, the enzymes that are needed for photosynthesis denature. Without this reduction reaction, the light reactions of photosynthesis cannot occur.
Chloroplast require light energy for photosynthesis, so the chloroplasts should not work well in the dark. It is expected that boiling chloroplasts would result in them no longer working. Heating them too much could cause damage just like heating MDH in a previous experiment caused the enzyme to no longer work. No, because they were not exposed to light.
Were boiled chloroplasts able to reduce DPIP? Yes, but the ones in the dark did not reduce it as much as the others because it had not sunlight. This will allow you to monitor the rate of photosynthesis. In order to allow the DPIP to come into contact with chloroplasts, the cells will need to be carefully disrupted.
What is the source of the electrons that will reduce DPIP? When the light shines on the chloroplast, the light provides enough energy to bump the electrons to a higher energy level thus reducing the DPIP. The source of the electrons can also come from the photolysis of water. The DPIP color changed only when the chloroplast solution was present. The DPIP solution is an electron acceptor, therefore, it accepts the electrons from the chloroplasts.
When it accepts the electrons, it is reduced and the color changes from a blue hue to colorless. What is the role of DPIP in this experiment? It is an electron acceptor and is reduced by electrons from chlorophyll.
The electrons come from the photolysis of water. What was measured with the spectrophotometer in this experiment? The spectrophotometer measures the percentage of light transmittance through the cuvette due to DPIP reduction. Cuvettes are designed to hold samples for spectroscopic measurement, where a beam of light is passed through the sample within the cuvette to measure the absorbance, transmittance, fluorescence intensity, fluorescence polarization, or fluorescence lifetime of the sample.
DPIP is a redox dye commonly used as a monitor of the light reactions in photosynthesis because it is an electron acceptor that is blue when oxidized and colourless when reduced.
In this investigation, DCPIP 2,6-dichlorophenol-indophenol , a blue dye, acts as an electron acceptor and becomes colourless when reduced, allowing any reducing agent produced by the chloroplasts to be detected. DPIP gains electrons, thus it was reduced.
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