Put 50cm^3 of isolation medium into the medium beaker.
Tear the spinach into small pieces and, omitting the stalk and midrib, put the pieces into the isolation medium.
Fill a large beaker with partially crushed ice and put a small beaker in so that it is surrounded by ice.
Wet 3 layers of muslin with isolation medium and place them over the top of the funnel.
Put the funnel into the small beaker. Blend the leaf-isolation medium mixture for 15 seconds and then pour a small amount through the muslin.
Gently fold and squeeze the muslin. Repeat until most of the mixture has been filtered. Label the small beaker 'chloroplast suspension'.
Label a clean test tube 'A' and add 5cm^3 of DCPIP and 1cm^3 of distilled water to it. Wrap in aluminium foil immediately and stand in the other large beaker after filling it with ice.
Label another clean test tube 'B'. Add 5cm^3 of DCPIP, 1cm^3 of distilled water and 1cm^3 ammonium hydroxide to it. Place it in the beaker with 'A'.
Label another clean test tube 'C'. Add 6cm^3 of distilled water to it and put it into the large beaker.
Label 2 test tubes 'X1' and 'X2' respectively. Add 5cm^3 of DCPIP and 1cm^3of water to both. Put them in the large beaker.
Label 2 test tubes 'Y1' and 'Y2' respectively. Add 5cm^3 of DCPIP and 1cm^3of ammonium hydroxide to both. Put them in the large beaker.
Place a lamp 10cm away from the large beaker containing the test tubes and switch it on.
Add 1cm^3 of chloroplast suspension to each tube. Shake 'X1', 'X2', 'Y1' and 'Y2' thoroughly and start the timer. (If 'X1' to 'Y2' have the chloroplast suspension added at different times, record this and account for the difference later in Step 14.)
After 5 minutes, use the colorimeter to test the absorbances of the mixture of each test tube. Record these values. Use 'C' as the standard.
Repeat Steps 10-14, this time labelling with 'X3', 'X4', 'Y1' and 'Y2'.

	Created by Eleanor H almost 8 years ago

Methodology and applications of an example of Required Practical 8 for AQA A-Level Biology. Investigation into the effect of a named factor on the rate of dehydrogenase activity in extracts of chloroplasts.