Emma Braithwaite
Required Practical 8 – The effect of ammonium hydroxide on the time taken for chloroplasts to
decolourise DCPIP
In this investigation you will use a chloroplast suspension and a blue dye called DCPIP to monitor the
rate of dehydrogenase activity. DCPIP goes from blue to colourless when it accepts electrons
released by the chlorophyll.
METHOD
1. Put about 50 cm3 of isolation medium into a beaker.
2. Tear 8 spinach leaves into small pieces and put the pieces into the isolation medium in the
beaker. We did not put pieces of the midrib or the leaf stalk into the beaker as these do not
contain chloroplasts.
3. Half fill a large beaker with ice and place a small beaker on top of the ice.
4. Put 3 layers of muslin over the top of the filter funnel and wet it with the isolation medium. Rest
the filter funnel in the small beaker on the ice, this kept the isolation medium cold to prevent
enzyme action that could break down any organelles, we also kept this isotonic to ensure there
was no net movement of water that could cause lysis of the cell.
5. Pour the spinach and isolation medium into the blender and blend for about 15 seconds. Pour
the blended mixture back into the beaker. We blended this mixture to ensure the chloroplasts
were separated from the other leaf structures.
6. Pour a little of your blended mixture through the muslin in the filter funnel. Carefully fold and
squeeze the muslin to assist the filtering process. Repeat until most of the blended mixture has
been filtered. Label this filtrate which is in the small beaker on ice as ‘chloroplast suspension’.
7. Label five test tubes A, B, C, X and Y. Stand these five tubes in the ice in the large beaker.
Position the lamp about 10 cm from the beaker so that all tubes are illuminated. Turn on the
lamp.
8. Set up tubes A and B as follows
Tube A
Put 5 cm3 DCPIP solution + 1 cm3 water + 1 cm3 chloroplast suspension in the tube. Immediately
wrap the tube completely in aluminium foil to exclude light.
Tube B
Put 5 cm3 DCPIP solution + 1 cm3 water + 1 cm3 isolation medium in the tube.
Tubes A and B are control experiments. Leave both tubes until the end of your investigation.
9. Set up tube C as follows
Tube C
Put 6 cm3 water + 1 cm3 chloroplast suspension in the tube.
Tube C is for you to use as a standard to help you to determine when any colour change is
complete.
10. Set up tube X as follows
Tube X
Put 5 cm3 DCPIP solution + 1 cm3 water in the tube.
Add 1 cm3 chloroplast suspension to tube X, quickly mix the contents and start the timer. Record
in seconds how long it takes for the contents of tube X to change colour from blue green to
green. This is when all signs of blue have disappeared. Use tube C to help you determine when
the colour change is complete.
Required Practical 8 – The effect of ammonium hydroxide on the time taken for chloroplasts to
decolourise DCPIP
In this investigation you will use a chloroplast suspension and a blue dye called DCPIP to monitor the
rate of dehydrogenase activity. DCPIP goes from blue to colourless when it accepts electrons
released by the chlorophyll.
METHOD
1. Put about 50 cm3 of isolation medium into a beaker.
2. Tear 8 spinach leaves into small pieces and put the pieces into the isolation medium in the
beaker. We did not put pieces of the midrib or the leaf stalk into the beaker as these do not
contain chloroplasts.
3. Half fill a large beaker with ice and place a small beaker on top of the ice.
4. Put 3 layers of muslin over the top of the filter funnel and wet it with the isolation medium. Rest
the filter funnel in the small beaker on the ice, this kept the isolation medium cold to prevent
enzyme action that could break down any organelles, we also kept this isotonic to ensure there
was no net movement of water that could cause lysis of the cell.
5. Pour the spinach and isolation medium into the blender and blend for about 15 seconds. Pour
the blended mixture back into the beaker. We blended this mixture to ensure the chloroplasts
were separated from the other leaf structures.
6. Pour a little of your blended mixture through the muslin in the filter funnel. Carefully fold and
squeeze the muslin to assist the filtering process. Repeat until most of the blended mixture has
been filtered. Label this filtrate which is in the small beaker on ice as ‘chloroplast suspension’.
7. Label five test tubes A, B, C, X and Y. Stand these five tubes in the ice in the large beaker.
Position the lamp about 10 cm from the beaker so that all tubes are illuminated. Turn on the
lamp.
8. Set up tubes A and B as follows
Tube A
Put 5 cm3 DCPIP solution + 1 cm3 water + 1 cm3 chloroplast suspension in the tube. Immediately
wrap the tube completely in aluminium foil to exclude light.
Tube B
Put 5 cm3 DCPIP solution + 1 cm3 water + 1 cm3 isolation medium in the tube.
Tubes A and B are control experiments. Leave both tubes until the end of your investigation.
9. Set up tube C as follows
Tube C
Put 6 cm3 water + 1 cm3 chloroplast suspension in the tube.
Tube C is for you to use as a standard to help you to determine when any colour change is
complete.
10. Set up tube X as follows
Tube X
Put 5 cm3 DCPIP solution + 1 cm3 water in the tube.
Add 1 cm3 chloroplast suspension to tube X, quickly mix the contents and start the timer. Record
in seconds how long it takes for the contents of tube X to change colour from blue green to
green. This is when all signs of blue have disappeared. Use tube C to help you determine when
the colour change is complete.
