Part A. Isolation of chloroplasts

Materials and Methods

Small pieces of the lettuce leaves were obtained using the scissors. Any major veins of the leaf were discarded. After placing the lettuce leaf into a cold mortar with 20 ml of cold isolation medium, it was vigorously and quickly grinded. Two layers of muslin were put on the funnel and moistened with a cold medium for isolation. The homogenate was filtered through a funnel. The resulting filtrate was collected in cooled centrifuge tubes placed in a water bath with ice and salt. The edges of the muslin were picked and remaining liquid was added to the test tubes by careful squeezing. Next, an amount of water, equal to the volume of the filtrate, was added to another centrifuge tube and .

Spectrophotometer was prepared with 2 ml of methanol.

Results

Part A. Isolation of chloroplasts

Tube

Leaf extract / cm³

Supernatant / cm³

Isolation medium / cm³

Distilled water / cm³

DCPIP solution / cm³

0.5

-

-

-

-

-

0.5

-

0.5

-

-

-

0.5

-

-

-

-

0.5

-

-

 

Part B. Investigation using the chloroplasts  

Time / min

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5

5.0

5.0

5.0

5.0

5.0

4.0

 

5.1

 

3.7

2.2

 

4.9

 

2.3

0.5

 

5.2

 

1.0

0.3

6.2

5.4

4.9

0.3

Table 1: Results from an investigation of chloroplasts activity by following the decolourization of DCPIP over time. Absorbance – 665 nm.

Discussion

The 3^rd tube demonstrates that light is essentially required for DCPIP reduction. Instead of placing the tube in front of the 100W bright light, it was located in the darkness to prevent chloroplasts of getting any light energy. Obtained results prove that DCPIP does not reduce in the absence of light. Small deviations can be attributed to ??????

The 4^th tube was designed to prove

Conclusion

In this laboratory practical, at first we

Reference list

Karp, Gerald. Cell Biology. Hoboken (NJ): J. Wiley & Sons, 2010.

Reece, Jane B., Martha R. Taylor, Eric J. Simon, Jean L. Dickey, and Neil A. Cambell. Campbell biology: concepts & connections. 7^th edition. San Francisco, CA.: Benjamin Cummings, 2009.

Wayne, Randy. Plant Cell Biology from Astronomy to Zoology. Amsterdam: Academic Press, 2010.