Chloroplast Concentration Lab Report

Assessment of Chloroplast Concentration in Cell Fractions following Differential Centrifugation and Biochemical Assay. Subsequent Efficacy Analysis of Herbicide ‘Diuron’ on Inhibition of Electron Transport Chain Activity.

Question and Hypothesis Part I To better understand the role of chloroplast activity in plants and to provide evidence for their photosynthetic capabilities, we performed a multi-step experiment intended to highlight a component of the energy production process found within these organelles. As we were specifically interested in evaluating chloroplasts, we carried out a differential centrifugation process of pea seedling tissue in order to both concentrate and co-fractionate the sample with the chloroplast and

B prefix cuvettes containing no DCIP were used as blanks to set baseline absorbance for the following L-group--cuvettes containing 0.5ml DCIP and kept in a light apparatus to allow for photosynthetic light reactions to take place, and D-group, cuvettes containing 0.5ml DCIP and placed in the light-proof box to prevent light reactions. Set 1 (B1, L1, D1) contained no cell fraction. Set 2 (B2, L2, D2) contained 0.5 ml of P1 cell fraction. Set 3 (B3, L3, D3) contained 0.5ml of P2 cell fraction. Set 4 (B4, L4, D4) contained 0.5ml of S2 cell fraction. Initial absorbance readings were taken immediately following addition of cell fraction to prepared cuvettes. L1-4 and D1-4 samples were then placed in respective light and dark environments. Two additional absorbance readings taken at intervals of seven minutes for a total of fourteen minutes. Total cuvette volume was kept constant at 5.0mL with addition of isolation buffer. Absence of DCIP in some cuvettes was accounted for with an equal volumetric addition of

The more chloroplasts exposed to light, the more photosynthetic activity we expected to occur, and the more DCIP would be reduced- invoking color change. Cell fractions containing less chloroplasts would then show less reduction of DCIP, and thus, less color change. Cell fractions kept in the dark (D set) as a negative control were expected to exhibit no color change, like those containing no cell fraction.
Results:
Experimental data from testing indicated that L2 (P1 cell fraction) showed the largest decrease in absorbance 0.80 to 0.00, over the fourteen-minute interval (Fig. 1). This is equal to a 100% decrease in absolute change in absorbance (Fig. 2). L3 (P2) showed the next largest decrease in absorbance, at 85% absolute change. The S2 sample contained in L4 showed a small decrease in absorbance at 27% absolute change. Dark exposed cell fractions mostly exhibited an increase in absorbance (D2, D4) whereas D2 showed a 24% absolute reduction in absorbance (Fig.