biology lab report
Protocol for Lab 5 – Aerobic Respiration
Part 1 Isolation of Mitochondria from Cauliflower
- Weigh 50g of rosettes cut from fresh cauliflower head.
- Cut rosettes and place it on ice
- Prepare juice extractor by placing ice and an empty 150 ml beaker into the right compartment.
- Collect pulp from left compartment and record total volume of the extract. Approx. 20ml
- Filter the pulp using six layered cheese cloth and collect it in a beaker sitting on ice.
- Place two 50 ml test tubes in the ice bucket A and B.
- Add 10 ml of cold isolation buffer and 10 ml of extract to tube A. Mix thoroughly and centrifuge at 3000 RPM for 10 min. (Make sure tubes are balanced before running centrifuge. Weight- balance other tube with water to make it equal to tube A).
- Collect supernatant and add to tube B.
- Pellet in tube A contains cell debris, cells, cell wall material, nuclei and mitochondria. Add 1ml buffer to pellet and mix.
- Prepare slide, place 1 drop of suspension plus methylene blue and place cover slip on it.
- Centrifuge tube B at 12000 RPM for 30 min to get mitochondrial pellet and a supernatant. (Balance test tubes before centrifugation).
- Resuspend the pellet (to avoid clumps) using cold stirring glass rod. Add one drop of assay buffer. Continue breaking clumps and adding assay buffer until 5ml buffer is added.
- Mix the suspension and transfer to a precool 15 ml test tube M.
- Prepare slide using suspension from M tube and adding Janus Green B dye to it. Cover using cover slip.
- Add 1 ml of mitochondrial suspension to a 15 ml test tube P. Add 1 ml of Neatetrazoleum chloride(NTC). Note the color. Incubate the test tube at 37 deg water bath for 45 min. Observe for color change every 15 min.
Part 2 Electron Transport Chain: Enzyme Assay
- Label 6 (13x100) test tubes 1 to 6.
- Add substances in proportions given in below table.
Tube #
Assay Buffer (ml)
Starch (ml)
DPIP 200 UM (ml)
H2O (ml)
UM DPIP
1 (Blank)
2.5
2.0
Part 1 Isolation of Mitochondria from Cauliflower
- Weigh 50g of rosettes cut from fresh cauliflower head.
- Cut rosettes and place it on ice
- Prepare juice extractor by placing ice and an empty 150 ml beaker into the right compartment.
- Collect pulp from left compartment and record total volume of the extract. Approx. 20ml
- Filter the pulp using six layered cheese cloth and collect it in a beaker sitting on ice.
- Place two 50 ml test tubes in the ice bucket A and B.
- Add 10 ml of cold isolation buffer and 10 ml of extract to tube A. Mix thoroughly and centrifuge at 3000 RPM for 10 min. (Make sure tubes are balanced before running centrifuge. Weight- balance other tube with water to make it equal to tube A).
- Collect supernatant and add to tube B.
- Pellet in tube A contains cell debris, cells, cell wall material, nuclei and mitochondria. Add 1ml buffer to pellet and mix.
- Prepare slide, place 1 drop of suspension plus methylene blue and place cover slip on it.
- Centrifuge tube B at 12000 RPM for 30 min to get mitochondrial pellet and a supernatant. (Balance test tubes before centrifugation).
- Resuspend the pellet (to avoid clumps) using cold stirring glass rod. Add one drop of assay buffer. Continue breaking clumps and adding assay buffer until 5ml buffer is added.
- Mix the suspension and transfer to a precool 15 ml test tube M.
- Prepare slide using suspension from M tube and adding Janus Green B dye to it. Cover using cover slip.
- Add 1 ml of mitochondrial suspension to a 15 ml test tube P. Add 1 ml of Neatetrazoleum chloride(NTC). Note the color. Incubate the test tube at 37 deg water bath for 45 min. Observe for color change every 15 min.
Part 2 Electron Transport Chain: Enzyme Assay
- Label 6 (13x100) test tubes 1 to 6.
- Add substances in proportions given in below table.
Tube #
Assay Buffer (ml)
Starch (ml)
DPIP 200 UM (ml)
H2O (ml)
UM DPIP
1 (Blank)
2.5
2.0