Practicals Flashcards
What is the method for the practical: ‘effect of a named variable on the rate of an enzyme controlled reaction’?
- 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. - Take three test tubes and measure 5cm3 milk into each. Place in water bath at 10 degrees for 5 mins.
- Add 5cm3
trypsin to each test tube simultaneously and start the timer
immediately. - Record how long it takes for the milk samples to completely hydrolyse and become colourless.
- Repeat steps 2-3 at temperatures of 20°C, 30°C, 40°C and 50°C.
- Find the mean time for the milk to be hydrolysed at each temperature and use to work out rate of reaction
1/mean time
What can we conclude from the rate of enzyme reaction practical?
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.
Explain the method of the ‘root tip squash’ practical for the calculation of mitotic index
- Heat 1 mol dm-3 HCl at 60°C in a water bath.
- Cut a small sample of the root tip using a scalpel.
- Transfer root tip to HCl and incubate for 5 minutes.
- Remove from HCl and wash sample in cold distilled water and remove the very
tip using a scalpel. - 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. - 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. - Place under a microscope and set the objective lens on the lowest
magnification. - Use the coarse adjustment knob to move the lens down to just above the slide.
- Use the fine adjustment knob to carefully re-adjust the focus until the image is
clear (you can use a higher magnification if needed). - To calculate mitotic index, cells undergoing mitosis must be counted (cells
with chromosomes visible), as well as the total number of cells.
How do you calculate mitotic index?
Number of cells with visible chromosomes /
total number of all cells in sample
Explain the method for ‘investigating water potential’
- 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. - Measure 5cm3 of each dilution into separate test tubes.
- 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. - Weigh each before the start of the experiment.
- Place a potato chip in each test tube (one per sucrose concentration) and leave
for 20 minutes. - Remove each potato chip, dry gently using paper towel, and weigh them in turn.
- Calculate the percentage change in mass for each sucrose solution.
What is the conclusion for the ‘investigating water potential’ practical?
● 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.
Outline the practical for ‘investigating cell membrane permeability’
- Cut beetroot into 6-10 identical cubes using a scalpel.
- Wipe/rinse to clean off any pigment released as a result.
- If investigating temperature: place each of the cubes of beetroot in an equal
volume of distilled water (5-15ml). - Place each test tube in a water bath at a range of temperatures (30-80°C)
- If investigating concentration of solvents: create a dilution series of ethanol
using distilled water. Ethanol concentrations should range from 0-100% ethanol. - Leave the samples for 20 minutes - the pigment will leak out of the beetroot.
- Set the colorimeter to a blue filter and zero using a cuvette with distilled water.
- Filter each sample into a cuvette using filter paper.
- Measure the absorbance for each solution. A higher absorbance indicates
higher pigment concentration, and hence a more permeable membrane.
What can we conclude form the ‘investigating cell membrane permeability’ practical?
- As temp increases, permeability increases, membranes denature
-As conc of ethanol increases, permeability increases because it causes the membrane to rupture.
Dissections come with risks, what are they and what should we do to prevent them?
Biohazard- disinfect surfaces
Disinfectant- keep away from naked flame
Scalpel- cut away from fingers and used forceps to help
Wear gloves, goggles, lab coat
List some aseptic techniques
● 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.
Describe the method for the ‘aseptic techniques’ practical
- Carry out aseptic techniques detailed above.
- 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). - Spread bacteria evenly over plate using a sterile plastic spreader.
- 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. - 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. - Sterilise equipment used to handle bacteria and disinfect work surfaces.
AFTER INCUBATION:
- Measure the diameter of the inhibition zone (clear circle) for each antibiotic.
DO NOT remove the lid from the agar plate. - Work out the area of the inhibition zone using the formula:
area= pi x diameter^2
divided by 4
Why can’t the petri dishes be incubated over 25 degrees?
Could enable pathogens to grow and become harmful to humans
What is the test for starch?
Iodine
Orange to Blue/black
Test for sugar?
Benedict’s Solution
Blue to yellow, green or brick-red
Test for lipid?
Ethanol
Colourless to cloudy emulsion
Test for protein?
Biuret
Blue to purple
What are some tips for doing biological drawings?
-Use a sharp pencil
-No shading
-Single and continuous lines
-Do not cross labelled lines
-Lines should not have arrows
-Labels should be drawn with a ruler
-Do not use colour
-Include a scale
Describe the chromatography of leaf pigments practical
- Draw a straight line in pencil approximately 1cm above the bottom of the filter paper being used. Do not use a pen as the ink will obscure the results.
- Cut a section of leaf and place it in a mortar. Add 20 drops of acetone and use the pestle to grind up the leaf sample and release the pigments.
- Use a capillary tube to extract some of the pigment and blot it onto the centre of the pencil line you have drawn.
- Suspend the paper in the solvent so that the level of the liquid does not lie above the pencil line and leave the paper until the solvent has run up the paper to near the top.
- Remove the paper from the solvent and draw a pencil line marking where the solvent moved up to. The pigment should have separated out and there should be different spots on the paper at different heights above the pencil line
. - Calculate the Rf value for each spot (distance travelled by solute/distance travelled by solvent). Always measure to the centre of each spot.
What factors affect rate of mobility? (chromatography)
Affinity:
pigments have different affinities to the chromatography paper, those with lower affinities will travel further up the paper.
Solubility:
pigments that are more soluble travel faster up the paper and will end up closer to the top at the solvent front.
Describe the chloroplast and dehydrogenase practical
In this method the named variable is light intensity
- Remove stalks from leaf samples. Grind sample using a pestle and mortar and place into a chilled isolation solution.
- Use a muslin cloth and funnel to filter the sample into a beaker. Suspend the beaker in an ice water bath to keep sample chilled.
- Transfer to centrifuge tubes and centrifuge at high speed for 10 minutes. This will separate chloroplasts into the pellet.
- Remove supernatant and add pellet to the fresh isolation medium. Store isolation solution on ice.
- Set the colorimeter to the red filter. Zero using a cuvette containing chloroplast extract and distilled water.
- Place test tube in the rack 30cm from light source and add DCPIP. Immediately take a sample and add to cuvette. Measure the absorbance of the sample using the colorimeter.
- Take a sample and measure its absorbance every 2 minutes for 10 minutes.
- Repeat for different distances from lamp up to 100 cm. This will vary the light intensity.
NB: This experiment should be done in a darkened room to make results more reliable. The sample should not be put too close to the lamp as temperature may affect results.
What can we conclude from the dehydrogenase and chloroplast practical?
● As the light intensity decreases, the rate of photosynthesis also decreases. This is because the lowered light intensity will slow the rate of photoionisation of the chlorophyll pigment, so the overall rate of the light dependent reaction will be slower.
● This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons. This means that it will take longer to turn from blue to colourless.
● When the DCPIP is blue, the absorbance is higher. The rate at which the absorbance decreases can therefore be used to determine the activity of the dehydrogenase enzyme. A higher rate of decrease, shown by a steep gradient on the graph, indicates that the dehydrogenase is highly active.
Describe the temperature and respiration of yeast practical
In this method the named variable is temperature
- Set up a water bath at 35°C.
- Add 5cm3 of the yeast and glucose solution to three test tubes. Place test tubes
in the water bath and leave them, for the solution to equilibrate for 10 minutes. - Add 2cm3 of methylene blue to the test tubes and start the timer. Shake for 10 seconds and place test tube back in water bath. Record how long it takes for the
methylene blue to turn colourless for each test tube. - Repeat the experiment using temperatures of 40°C, 50°C, 60°C and 70°C.
- Find the mean of the results for each temperature and use to calculate the
average rate of respiration.
NB: the yeast and glucose solution should be buffered to maintain a constant pH.
What conclusion can be made from the temp and respiration of yeast practical?
● Yeast has an optimum temperature range for respiration, which is shown by the peak on the graph. As the temperature moves away from the optimum, the rate of reaction will decrease as enzyme action decreases, and at high temperatures denaturation may occur.
● As enzymes are crucial to respiration, as their activity decreases, so does the rate of respiration. This means that the methylene blue will take longer to turn colourless when the temperature is further from the optimum.
Describe the maggots and choice chambers practical
- Set up choice chamber to have four quadrants as follows: dark and dry, dark and damp, light and dry, light and damp.
- Use dark paper or cardboard to block out the light on one half. Use wet paper towels to make damp areas. Use a drying agent such as anhydrous calcium chloride to make dry areas.
- Place 10 maggots in the centre of the choice chamber using a spoon. Do not use forceps as this may cause harm to them.
- Leave for 10 minutes.
- Record how many maggots are in each quadrant.
- Repeat by moving maggots back to the centre of the choice chamber and
repeating steps 3-4.
