Resuspend the chloroplast pellet in 5.0 ml of cold suspension buffer. Use a glass stirring rod to gently disrupt the packed pellet. This is the chloroplast suspension for use in subsequent procedures. Store the suspension in the dark at 4˚C. Be sure the suspension is well mixed before removing samples from it.
Determine the concentration of chloroplasts using a hemocytometer. Record the results.
Determine the concentration of chlorophyll in your chloroplast solution. Use suspension buffer to blank the spectrophotometer at a wavelength of 652 nm. You may need to dilute the solution to get a reading in the linear range of the spectrophotometer . The conversion factor is: (35 mg chlorophyll / ml) / . Be sure to account for any dilutions you performed.
Calculate the average amount of chlorophyll per chloroplast (the average chloroplast volume is 0.6 fl). Record your results on the board so the values for the entire class can be compiled.
Grinding solution: 0.33 M sorbitol, 10 mM sodium pyrophosphate (NaP2O7), 4 mM MgCl2, 2 mM ascorbic acid, pH 6.5
Suspension solution : 0.33 M sorbitol, 2 mM EDTA, 1 mM MgCl2, 50 mM HEPES, pH 7.6
Homework Questions
1. What would happen to the chloroplasts if you resuspended them in water instead of in suspension buffer?
2. Would the amount of chlorophyll be affected by resuspending the chloroplasts in water? If so, would the measured amount increase or decrease?
3. Which type of tissue contains more chlorophyll per gram of tissue ?
4. We do not know how many chloroplasts are in an individual cell in either tissue. What are the two possible explanations for why one tissue has more chlorophyll than the other?
5. What was the concentration of chloroplasts in both of your suspensions?
6. What was the concentration of chlorophyll in both of your suspensions?
7. What was the average amount of chlorophyll per chloroplast in each of your suspensions?
8. Using your results only, why do you think one of your samples was darker green than the other ?
9. Using the results of the entire class perform a t-test to see if the amount of chlorophyll per chloroplast is different between the two tissue types. Report your answer in an appropriate format.
10. Which answer to question 4 is supported by this t-test?
