Dehydrogenase activity in chloroplasts A Level Biology
In this required practical, students are going to investigate the effect of ammonium hydroxide and the effect on chloroplasts decolourising DCPIP Solution. Some advanced preparation and planning are required here, the experiment requires a large amount of ice, and the reagents need to be refrigerated for 24 hours before the practical session. The spinach leaves should also be left in the light for a couple of hours before the experiment without allowing the leaves to get hot.
There is also some scope to expand the skills used in this experiment by using a colorimeter to analyse the samples.
Subject: Biology | Level: A Level |
You will need:
Method
Students are provided with cold Isolation Medium and DCPIP Solution, which has been refrigerated. 50cm³ of Isolation Medium is dispensed into a labelled beaker.
10 spinach leaves are selected, and the leaf stalk and middle stem are removed, the green part of the leaves are cut up and placed into the beaker containing the Isolation Medium.
Students should half fill the 400ml beaker with ice and place an empty 100ml beaker inside.
Three pieces of muslin should be cut to size so that it fits into the filter funnel. Once inside the funnel, the muslin is moistened using the Isolation Medium and the funnel is placed inside the 100ml beaker.
The spinach and Isolation Medium is added to the 500ml plastic beaker and is blended for approximately 15 seconds using a blender. The resulting solution is then poured back into the 100ml beaker. A small amount is then filtered through the muslin lined funnel into the iced beaker.
The muslin needs to be squeezed to help with the filtration. The iced 100ml beaker is then labelled Chloroplast Solution.
Five Test Tubes are labelled with the numbers 1 to 3 and A and B and are left to stand in the 400ml beaker containing the ice. A lamp should be positioned approximately 10-15cm from the beaker, ensuring all tubes are in the light.
Three of the five test tubes should be set up as control samples and a colour standard to compare results with as follows:
Tube
Reagents
Lighting Condition
1
5cm³ DCPIP Solution
1cm³ Water
1cm³ Chloroplast Suspension
Dark (wrap in aluminium foil) – control
2
5cm³ DCPIP Solution
1cm³ Water
1cm³ Chloroplast Suspension
Exposed to Light (without wrapping) - control
3
6cm³ Water
1cm³ Chloroplast Suspension
Exposed to Light (without wrapping) – standard for colour comparison
· The remaining two test tubes, labelled A and B should be set up as follows:
Tube
Reagents
Procedure
A
5cm³ DCPIP Solution
1cm³ Water
1cm³ Chloroplast Suspension
Quickly mix the contents and start the stopwatch.
Record the time in seconds taken for the contents of this tube to change from blue-green back to green. Tube 3 can be used as the original colour comparison.
Repeat 4 times.
B
5cm³ DCPIP Solution
1cm³ Ammonium Hydroxide
1cm³ Chloroplast Suspension
Quickly mix the contents and start the stopwatch.
Record the time in seconds taken for the contents of this tube to change from blue-green back to green. Tube 3 can be used as the original colour comparison. If the decolourisation does not happen within 300 seconds (5 minutes), record the colour at this time.
Repeat 4 times.
Students record their results in a suitable table, and they should also note the end colour of the tubes 1 and 2 in their results.
Technician tips
Phosphate Buffer should be produced and kept cool until required for the dilution of the Isolation Medium and the DCPIP Solution.
Once prepared, the Isolation Medium, DCPIP Solution and the Ammonium Hydroxide Solution all need to be refrigerated.
Phosphate Buffer Recipe (1L): Wearing eye protection, dissolve 8.96g of Di-sodium Hydrogen Phosphate 12-water and 3.4g in 1 Litre of distilled water.
Isolation Medium Recipe (500mls): Wearing eye protection, dissolve 68.46g of Sucrose and 0.38g of Potassium Chloride in 500mls of Phosphate Buffer.
DCPIP Recipe (500mls): Wearing eye protection, dissolve 0.02g of DCPIP Powder and 1.86g of Potassium Chloride in 500mls of Phosphate Buffer Solution.
1M Ammonium Hydroxide Recipe (250mls): Wearing splash-proof goggles and a fume cupboard, 14mls of 35% Ammonium Hydroxide is added to 200mls of distilled water and mix well. Add the remaining 50mls of distilled water to the solution with further mixing.
Lots of ice is required per student during this practical. A large supply should be provided.
Muslin could be pre-cut into the appropriate size for the filter funnels to save a little time.
The blending of the spinach leaves and Isolation Medium could be done individually by students, or a large volume could be pre-prepared and provided on ice for students to use.
It is handy to have the Isolation Medium dispensed into water bottles before refrigeration. This will make it easier for students to dispense it and wet the muslin filter.
