Practicals

Question 1

What is the method for the practical: ‘effect of a named variable on the rate of an enzyme controlled reaction’?

Answer 1

1. Make 2 control samples by:
   -2 tubes
   -Add 5cm3 of milk suspension to each tube.
   -Add 5cm3 of distilled water to one tube- this control will indicate the
   absence of enzyme activity.
   - Add 5cm3 of hydrochloric acid to the other- this control indicates the
   colour of a completely hydrolysed sample.
2. Take three test tubes and measure 5cm3 milk into each. Place in water bath at 10 degrees for 5 mins.
3. Add 5cm3
   trypsin to each test tube simultaneously and start the timer
   immediately.
4. Record how long it takes for the milk samples to completely hydrolyse and become colourless.
5. Repeat steps 2-3 at temperatures of 20°C, 30°C, 40°C and 50°C.
6. Find the mean time for the milk to be hydrolysed at each temperature and use to work out rate of reaction
   1/mean time

Question 2

What can we conclude from the rate of enzyme reaction practical?

Answer 2

Milk contains a protein called casein which, when broken down, causes the milk
to turn colourless. Trypsin is a protease enzyme which hydrolyses the casein protein.

Question 3

Explain the method of the ‘root tip squash’ practical for the calculation of mitotic index

Answer 3

1. Heat 1 mol dm-3 HCl at 60°C in a water bath.
2. Cut a small sample of the root tip using a scalpel.
3. Transfer root tip to HCl and incubate for 5 minutes.
4. Remove from HCl and wash sample in cold distilled water and remove the very
   tip using a scalpel.
5. Place tip on a microscope slide and add a few drops of stain (e.g. toluidine blue
   O). This makes the chromosomes visible and will therefore show which cells
   are undergoing mitosis.
6. Lower the cover slip down carefully onto the slide. Make sure there are no air
   bubbles in the slide which may distort the image, and that the coverslip doesn’t
   slide sideways which could damage the chromosomes.
7. Place under a microscope and set the objective lens on the lowest
   magnification.
8. Use the coarse adjustment knob to move the lens down to just above the slide.
9. Use the fine adjustment knob to carefully re-adjust the focus until the image is
   clear (you can use a higher magnification if needed).
10. To calculate mitotic index, cells undergoing mitosis must be counted (cells
    with chromosomes visible), as well as the total number of cells.

Question 4

How do you calculate mitotic index?

Answer 4

Number of cells with visible chromosomes /
total number of all cells in sample

Question 5

Explain the method for ‘investigating water potential’

Answer 5

1. Make a series of dilutions of 1M sucrose solution. These should be at 0.0, 0.2,
   0.4, 0.6, 0.8 and 1.0M sucrose. Dilute using distilled water.
2. Measure 5cm3 of each dilution into separate test tubes.
3. Use a cork borer to cut out six potato chips and cut down the sections into
   identically sized chips. Dry each chip using a paper towel to remove excess
   water but do not squeeze.
4. Weigh each before the start of the experiment.
5. Place a potato chip in each test tube (one per sucrose concentration) and leave
   for 20 minutes.
6. Remove each potato chip, dry gently using paper towel, and weigh them in turn.
7. Calculate the percentage change in mass for each sucrose solution.

Question 6

What is the conclusion for the ‘investigating water potential’ practical?

Answer 6

● Potato chips in lower concentrations of glucose solution will increase in mass,
whilst those in the higher concentrations of glucose solution will decrease in
mass.

● In the dilute glucose solutions, the solution has a higher water potential than
the potato, so water passively moves via osmosis to the area of lower water
potential (the potato). This causes the potato to increase in mass.

● In concentrated glucose solutions, water will move out of the potato, thus the potato will decrease in mass.

Question 7

Outline the practical for ‘investigating cell membrane permeability’

Answer 7

1. Cut beetroot into 6-10 identical cubes using a scalpel.
2. Wipe/rinse to clean off any pigment released as a result.
3. If investigating temperature: place each of the cubes of beetroot in an equal
   volume of distilled water (5-15ml).
4. Place each test tube in a water bath at a range of temperatures (30-80°C)
5. If investigating concentration of solvents: create a dilution series of ethanol
   using distilled water. Ethanol concentrations should range from 0-100% ethanol.
6. Leave the samples for 20 minutes - the pigment will leak out of the beetroot.
7. Set the colorimeter to a blue filter and zero using a cuvette with distilled water.
8. Filter each sample into a cuvette using filter paper.
9. Measure the absorbance for each solution. A higher absorbance indicates
   higher pigment concentration, and hence a more permeable membrane.

Question 8

What can we conclude form the ‘investigating cell membrane permeability’ practical?

Answer 8

• As temp increases, permeability increases, membranes denature

-As conc of ethanol increases, permeability increases because it causes the membrane to rupture.

Question 9

Dissections come with risks, what are they and what should we do to prevent them?

Answer 9

Biohazard- disinfect surfaces
Disinfectant- keep away from naked flame
Scalpel- cut away from fingers and used forceps to help

Wear gloves, goggles, lab coat

Question 10

List some aseptic techniques

Answer 10

● Wipe down surfaces with antibacterial cleaner, both before and after
experiment.
● Use a Bunsen burner in the work space so that convection currents draw
microbes away from the culture.
● Flame the wire hoop before using it to transfer bacteria.
● Flame the neck of any bottles before using them to prevent any bacteria
entering the vessel (air moves out so unwanted organisms don’t move in).
● Keep all vessels containing bacteria open for the minimum amount of time.
● Close windows and doors to limit air currents.

Question 11

Describe the method for the ‘aseptic techniques’ practical

Answer 11

1. Carry out aseptic techniques detailed above.
2. Use a sterile pipette or wire hoop to transfer bacteria from broth (distilled
   water, bacterial culture, nutrients) to agar plate (petri dish containing agar jelly).
3. Spread bacteria evenly over plate using a sterile plastic spreader.
4. Use sterile forceps to place a multi disc antibiotic ring on the plate. Ring
   should only be moved by holding the centre, NOT the arms.
5. Lightly tape a lid on, invert and incubate at 25°C for 48 hours. DO NOT tape
   around the entire dish as this prevents oxygen entering and so promotes the
   growth of more harmful anaerobic bacteria.
6. Sterilise equipment used to handle bacteria and disinfect work surfaces.

AFTER INCUBATION:

7. Measure the diameter of the inhibition zone (clear circle) for each antibiotic.
   DO NOT remove the lid from the agar plate.
8. Work out the area of the inhibition zone using the formula:

area= pi x diameter^2
divided by 4

Question 12

Why can’t the petri dishes be incubated over 25 degrees?

Answer 12

Could enable pathogens to grow and become harmful to humans