Part A. Isolation of chloroplasts.

Aim

The overall aim of this laboratory practical is to show the link between light-dependent and light-independent reactions through the investigation of electron movement by addition of artificial electron acceptor to the isolated chloroplasts and compare the rate of chloroplast activity by measuring absorbance in different conditions.

Materials and Methods

Part A. Isolation of chloroplasts.

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 tube and they were centrifuged for 10 minutes. Supernatant was gathered and remaining was resuspended with the isolation medium.

Part B. Investigation using the chloroplasts.

5 tubes were prepared and investigated according to the protocol.

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

-

-

Results

Part A. Isolation of chloroplasts

In the first part of the laboratory the chloroplasts were previously isolated from lettuce leaves in order to be used later in the experiment.