Required Practicals

Question 1

Give a general method for investigating the effect of temperature on an enzyme-controlled reaction.

Answer 1

1- Draw an ‘x’ on 3 test tubes.
2- Add (named volume cm3) of a testing solution (e.g: milk powder).
3- Add (named smaller volume cm3) of your enzyme solution into (same named volume cm3) of buffer solution in another 3 test tubes.
4- Stand all test tubes in a hot water bath of (first temperature).
5- Leave until all test tube solution have reached correct temperature (check with thermometer).
6- Add the enzyme and buffer solutions into each testing solution test tubes and place a bung, then put back into the water bath.
7- Place test tubes back into water and time how long it takes for the milky solution to turn colourless (so you can see the ‘x’).
8- Repeat all previous steps for the different temperature being measured.
9- Record all times and calculate means.
10- Calculate rate of reaction and record in summary table.
11- Draw a graph of summarised results.

Question 2

What would be appropriate control variables for investigating the effect of temperature on an enzyme-controlled reaction?
How would they be controlled?

Answer 2

• Same volume of solutions each time - Using an appropriate measuring cylinder or syringe, measure the same volume of each solution respectively for each test/repeat (e.g: 10cm3 of testing solution, and 2cm3 of enzyme and buffer solution each.).
• Same concentration of enzyme solution - Use the same one large beaker of enzyme solution for each test, and mix the solution before measuring a volume out of it, to ensure concentration is kept the same.
• Same pH of solutions - Use a buffer solution of the same volume to neutralise the separate solutions to equal pHs for each test.
• Same temperature for each repeat - Use a thermometer to constantly check the temperature of the water baths and ensure they are kept the same/ as close as possible for each repeat at each temperature.
• Same size test tubes - measure each test tube you use to ensure they are the same size.
• Same height of ‘x’ - use a ruler to accurately measure the ‘x’ height to ensure they are drawn on at the same height and size on each test tube.

Question 3

Describe how you would prepare amend stain the root tips for an experiment to investigate cell division by mitosis.

Answer 3

Preparing the root tips:
- Use a plastic pipette to half fill a bijou bottle with 1M HCl, and stand the bottle in a 250cm3 glass beaker containing 1c, depth of water.
- Place the beaker containing the bijou bottle of acid into a 40 degrees water bath and leave for 15mins to allow the acid to reach the water bath temperature.
- Transfer a garlic clove (using a cocktail stick) to the bottle of acid so that the roots are submerged in the acid, and leave this in the beaker in the water bath at 40 degrees for 5 minutes.
- After 5 minutes, remove the garlic clove using the cocktail stick from the acid, replaced the lid on the bijou bottle and gently rinse the roots in the tap water.
Staining the root tips:
- Using scissors, cut of the end 3mm of the root tips and allow them to fall onto a small watch glass. Add one drop of 1% toluidine blue stain.
- Leave the garlic in the stain at room temp for 2 mins, then remove the excess stain and rinse the roots tips in the watch glass with a plastic pipette and tap water.

Question 4

Why do you add 1M HCl to the bijou bottle when preparing the root tips in a root tip mitosis experiment?
Why rinse the root tips off after leaving them in acid in the water bath for 5 minutes?

Answer 4

• To denature the enzymes to prevent reactions and to soften the cell walls to help flatten the root tips.
• To prevent the acid from damaging the cells.

Question 5

Describe how you would prepare the root tip squash for an experiment investigating root tip mitosis.

Answer 5

• Use a scalpel to transfer the root tips to a clean microscope slide. Add a drop of water to the root tips and gently spread the root tips with a mounted needle, ensuring they are not overlapping.
• Carefully place a coverslip on top, minimising the introduction of air bubbles.
• Cover the slide and coverslip with a paper towel by wrapping the towel around the slide several times.
• Gently press on the coverslip with your thumb to squash the root tips to spread them out.

Question 6

How would you calculate mitotic index?

Answer 6

Cells in mitosis / total number of cells