Dehydrogenase Activity in Chloroplasts

Equipment List

• Leaf sample
• Isolation solution
• Ice water bath
• Distilled water
• DCPIP (redox indicator dye)
• Pestle and mortar
• Test tubes
• Test tube rack
• Syringes
• Pipettes
• Lamp
• Timer
• Tape measure
• Muslin cloth
• Funnel
• Beaker
• Centrifuge
• Centrifuge tubes
• Colorimeter
• Cuvettes

Method

1. Preparation of chloroplast extract:

• Remove stalks from the leaf sample and grind the leaf tissue with a pestle and mortar in chilled isolation solution.
• Filter the homogenised mixture through muslin cloth into a beaker placed in an ice water bath.

2. Centrifugation:

• Pour the filtrate into centrifuge tubes and centrifuge at high speed for 10 minutes. This separates chloroplasts into a pellet.
• Discard the supernatant and resuspend the pellet in fresh isolation solution. Store the chloroplast suspension on ice.

3. Setting up the colorimeter:

• Set the colorimeter to the red filter and zero it using a cuvette filled with chloroplast extract and distilled water.

4. Investigation of light intensity:

• Place the test tube rack 30 cm from the light source and add DCPIP to the chloroplast suspension in the test tube.
• Immediately take a sample from the test tube and transfer it to a cuvette. Measure its absorbance using the colorimeter.

5. Data collection:

• Record the absorbance every 2 minutes for 10 minutes.
• Repeat the experiment at increasing distances from the light source (e.g., 50 cm, 70 cm, 100 cm) to investigate the effect of light intensity on dehydrogenase activity.

Risk Assessment

Hazard	Risk	Safety Precaution	In Emergency	Risk Level
DCPIP	Irritant to eyes and skin; stains	Wear eye protection; avoid contact with skin	Wash with cold water immediately	Low
Biohazard	Contamination from plant material	Wash hands thoroughly after handling plant material	Seek assistance	Low
Lamps	Temporary eye damage from bright light	Do not look directly at the lamp	Rest eyes; seek assistance if needed	Low
Electrical appliances	Liquids near electrical equipment	Keep hands dry; ensure wires and plugs are clear of liquids	Seek assistance for equipment failure	Low

Analysis

• Graph: Plot a graph of absorbance against time for each distance from the light source.
• As the light intensity decreases (greater distance from the light source), the rate of photosynthesis will also decrease, resulting in slower decolourisation of DCPIP.