Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis. Lab report

Chloroplast preparation Tear 35g of spinach leaves and place in a blender with 100 mL of homogenizing medium Blend at top speed, 3x 10 seconds Part i:

Chloroplast preparation Filter with 3 layers of cheesecloth, squeezing to collect as much liquid as possible.

Split into two 50mL centrifuge tubes Part i:

Chloroplast preparation Spin 500x g for 2 minutes.

Supernatant Keep for next step Pellet Discard Part i:

Chloroplast preparation Centrifuge supernatants 2000x g for 10 minutes.

Supernatant Discard Pellet Chloroplasts! Part i:

Chloroplast preparation ● Gently resuspend chloroplasts in 6 mL 0.01M NaCl.

● Incubate on ice for 10 minutes. Results in swelling of outer membrane.

● Centrifuge 10,000x g 5 minutes.

● Resuspend in 2 mL 0.01M NaCl. Part ii:

Chlorophyll determination ● Add 0.1 mL of chloroplasts to 19.9 mL acetone, cover with Parafilm TM and shake ● Pour into cuvette and read at 654 nm (use acetone as a blank) Part ii:

Chlorophyll determination ● Multiply absorbance by 5.6 to obtain the concentration in mg/mL ● Use C 1 V 1 = C 2 V 2 to obtain a 15 mL solution (diluted with 0.01M NaCl) of 0.5 mg chlorophyll/mL:

chloroplast suspension for your assay! Part iii:

pH Assay ● Combine 1.1 mL 0.048 M NaCl, 0.6 mL PMS, and 3.6 mL distilled water in each of two 10mL beakers with “flea” stir bars ● Position probe in beaker Part iii:

pH Assay ● Add 3.7 mL of prepared suspension in beaker with the stir bar on.

● Turn on the light and monitor pH every 10 seconds for 2 minutes ● Continue another 2 minutes with the light off and cover in place ● Repeat assay, but add CCCP instead of turning the light off Data Analysis ● Simply determine the average and standard deviation for each time point during the two trials ● Create graph with both trials using curved connecting lines:

– include error bars – clearly indicate when the light was turned off/CCCP was added Photosynthesis: Hill Reactions Protons are pumped into the thylakoid during light conditions. Protons diffuse out through ATP synthase in both light and dark conditions! Lab Report ● Lab 4 is to be submitted as a formal lab report (see the General Lab Report Guidelines AND the Lab 4 Report Requirements ) ● Your lab report must be submitted through Nexus by 11:59 PM on November 12 th as a pdf file ● It is worth 10% of your final grade! Lab Report ● Results : no word limit – All results will be available by Thursday midday – Follow the Lab 4 Report Requirements!

● Discussion : 1000 words max – As per Guidelines and Requirements – Focus on agreement between theory and results obtained – Improvements? New hypotheses to test? Lab Report ● Abstract : 250 words max – Don’t forget to include specifics ● References :

– Need minimum of 7 references (including lab manual) – Use acceptable format – Ensure you reference all information appropriately