A2 Biology Unit 6

Duration Total Marks No. of

1 hr 20 min 50 Questions
3 (may vary)
% of total AS % of total A2
20% 10%

Content
• Core Practical 10,11,12,13,14,15,16,17,18
. Question Pattern

Question 1: Based on one of the Core Practical

Question 2: Data based question

Types of statistical test

1. Student’s t-test
2. Spearman’s Rank Correlation Coefficient
3. Chi Square Test
Question 3: Practical Biology and Investigative skills
Core Practicals
 Content
Unit 4
1. Investigate the effects of light intensity, light wavelength, temperature and
availability of carbon dioxide on the rate of photosynthesis using a suitable
aquatic plant.

2. Carry out a study of the ecology of a habitat, such as using quadrats and
transects to determine the distribution and abundance of organisms, and
measuring abiotic factors appropriate to the habitat.

3. Investigate the effects of temperature on the development of organisms

(such as seedling growth rate or brine shrimp hatch rates), taking into
account the ethical use of organisms

4. Investigate the rate of growth of microorganisms in a liquid culture, taking

into account the safe and ethical use of organisms

5. Investigate the effect of different antibiotics on bacteria.

Unit 5
6. Use an artificial hydrogen carrier (redox indicator) to
investigate respiration in yeast.

7. Use a simple respirometer to determine the rate of respiration

and RQ of a suitable material (such as germinating seeds or
small invertebrates).

8. Investigate the effects of exercise on tidal volume, breathing

rate, respiratory minute ventilation, and oxygen consumption
using data from spirometer traces.

9. Investigate the production of amylase in germinating cereal

grains.
1. Investigate the effects of light intensity, light wavelength,
temperature and availability of carbon dioxide on the rate of
photosynthesis using a suitable aquatic plant.

Method
1. Place a piece of pondweed in a beaker of water.
2. Cover one side of the beaker with the aluminium foil to block
out the light.
3. Cover the other side of the beaker with one of the light filters.
4. Add half a spatula of sodium hydrogencarbonate to the water
to provide carbon dioxide.
5. Leave for 5 minutes.
6. Place the bench lamp a set distance from the beaker.
7. Set up the photosynthometer. Leave for 5 minutes.
8. Record the volume of gas produced during this time.
9. Replace the filter with another colour of filter and repeat the
experiment.

Graph
● Plot a bar graph of colour of filter against volume of gas
produced.

Conclusion
● Volume of gas produced is proportional to rate of
photosynthesis.
● The greatest volume of gas will be produced when there is no
filter used, because all
wavelengths of light can be absorbed.
● All filters will decrease volume of gas, but a green filter will
decrease it the most
because chloroplasts don’t absorb much green light - it is mostly
reflected, which is why
chloroplasts appear to be green.
2. Carry out a study of the ecology of a habitat, such as using quadrats
and transects to determine the distribution and abundance of
organisms, and measuring abiotic factors appropriate to the habitat.

Various abiotic factors that affect the abundance of the

distribution of a certain species can be
investigated in this ecological study. Some examples include:

● Light intensity (e.g. area in shade or in sunlight)

● Distance from a large tree / river / lake / path
● River depth
● Area - woodland, heath or grassland
● Measuring equipment depending on the factor being

Method
When carrying out an ecological study of a habitat there are 2
main methods of sampling:
1. Transects - this samples across an area and measures an
environmental gradient
2. Quadrats - this samples at 2 environmentally different areas
Sampling Methods:

- Random Sampling: Used when measuring density of a plant species or

slow moving animals.

1. Set up grid using tape measure and use random numbers to generate
points to place at least 10 quadrats
2. Count number of chosen species in each quadrat or estimate %
abundance
3. Density = Total no. of plants counted / (Area of one quadrat x Total no. of
quadrats taken)

- Systematic Sampling: A line transect is used to study changes in plant

species across an area.

1. A tape measure is laid along several zones to be looked

2. At least 10 quadrats are then placed at regular intervals
Method 1 - Quadrats
1. Draw a large grid over the images or maps of the 2/3 areas being
sampled at. Assign a
numerical scale to the grid, creating a coordinate grid.
2. Use a random number generator to generate 20 coordinates for the
first area, these will
be where the quadrats are placed.
3. Place a quadrat at the generated coordinates and count the
number of the species being
investigated in the quadrat, or the percentage cover. If a box of the
quadrat is more than
half-filled by the species being investigated, count that as a full box
and then calculate the
percentage of full boxes counted. If investigating small species, or
species that are hard
to distinguish from each other then percentage cover is the most
practical way to measure
abundance; whereas if investigating a species like daisies where they
are easy to count
then record species frequency. Record
the abundance in a suitable table.
4. Repeat step 3 with the remaining 19
quadrat sampling locations.
5. Repeat steps 2-5 in the other sampling
areas
Method 2 - transect
1. Choose an area with a clear environmental gradient in the
abiotic factor being
investigated - for instance under a tree where it is shaded, out
into the open where there is
no shade.
2. Lay the belt transect across the gradient, starting in the
shaded area. Place the quadrat at
the 0m mark and count the number of the species being
investigated in the quadrat, or the
percentage cover, recording the results in a suitable table. Also
take a measurement of the
independent variable being investigated, in this case use the
light sensor and record a
reading of the light intensity.
3. Repeat step 2, placing the quadrat every 2 metres down the
transect to take samples so
you have taken 10 across the 20m line.
4. Repeat steps 1-3 in different areas at the site with the same
environmental gradient being
investigated. This means you end up with multiple results for
each distance.
Limitations:

- Difficult to control all variables (abiotic factors affecting the

variables being investigated)

- Difficult to standardise measurement of yield

- Difficult to harvest coconuts high on palm

- Storms will cause damage to palm trees close to edge of sea

- Difficult sampling technique due to e.g. uneven arrangement

of palm tress

- Laboratory conditions may not relate to what happens in

reality/real life situation

- We assume that the species is evenly distributed throughout

the area and that the placing of the quadrats is entirely random

- Movement of organisms

- Sampling taken within a small amount of time

Ethical:

- Minimise disturbance to the habitat

- Change in food chain/food web

3. Investigate the effects of temperature on the development of
organisms (such as seedling growth rate or brine shrimp hatch
rates), taking into account the ethical use of organisms

Independent variable: Temperature

Dependent variable: Number of brine shrimp hatched OR number
of seedlings
germinated

Variables to be controlled are:

- Same age / size of organism

- Same parent organism
- Light intensity
- O2 concentration
- Mineral Concentration
- Water
- Food
- Time
- pH

Ethics
When handling living organisms for scientific research
and learning it is important to treat them with care
and to minimise potential harm. When using
organisms experimentally as a student you have a
responsibility to gain as much advancement in
knowledge and learning as possible from them. The
availability of the organisms you could use in this
experiment should be considered, since those easier to
obtain will make increase the practicality of the
research
Method 1 - Brine Shrimp

1. Use the marker pen to mark 5 of the beakers with the following temperatures: 15°C, 20°C,
25°C, 30°C and 35°C
2. Use the weighing scales and spatula to measure and weigh out 2g of sea salt into each
beaker, followed by 100 cm³ of dechlorinated water. Stir the mixture with the stirring rod in
order to dissolve the salt.
3. Dampen some paper with the salt solution and place a pinch of brine shrimp eggs on to
the
paper. Use a magnifying glass to separate and count the eggs until you have roughly 40 on
the paper.
4. Cut the paper around the eggs so the size of the paper is smaller than the area of the 5
beakers prepared, then place the piece of paper eggs-side-down into one of the beakers.
Allow 2-3 minutes for the eggs to fall off the paper and into the beaker, finally removing the
paper from the water with tweezers or forceps.
5. Repeat steps 2, 3 and 4 for the remaining 4 beakers.
6. Place the marked beakers into the water baths and refrigerators that correspond the
temperature written on them and leave them for 24 hours.
7. Prepare the sixth and last beaker as described in step 2.
8. Remove the first beaker and use a bench lamp or other light source to shine light into the
beaker, causing the brine shrimp that have hatched to swim to the surface. Use a pipette to
remove the shrimp one at a time, placing them into the empty sixth beaker as you count
them.
9. Record the number of brine shrimp that have hatched at that temperature in a results
table
and place the hatched shrimps in a location as instructed by your teacher.
10. Repeat steps 8 and 9 with the remaining 4 beakers.
Method 2 - Seedlings
1. Use a marker pen to label 5 petri dishes with the following
temperatures 15°C, 20°C, 25°C,
30°C and 35°C
2. Mould a 1 cm layer of cotton wool into the bottom of each
petri dish and place 20 seedlings
on to the petri dish, using tweezers or forceps. Spread the
seedlings out so they are evenly
distributed across the dish.
3. Use the measuring cylinder to slowly add 30 cm³ of water to
each dish and then place each
of the marked Petri dishes in their corresponding temperature
environments.
4. Every 24 hours, add more distilled water to each dish so the
seedlings and cotton wool
does not dry out. Be sure to add the same volume of water to
each dish.
5. After 5 days remove the Petri dishes from the water baths and
refrigerators and count the
number of seeds that have germinated for each temperature,
recording the results in a
suitable table.
4. Investigate the rate of growth of microorganisms in
a liquid culture, taking into account the safe and
ethical use of organisms

A colorimeter or light sensor with data logger can be used to measure the
rate of growth of microorganisms like yeast. As a yeast population grows
the broth they live in becomes more cloudy due to the presence of more
microorganisms. This increase in turbidness means less light passes
through the culture, so by monitoring the light passing through the
culture across a 24 hour period their growth can be estimated.

Method
1. Before starting the experiment disinfect the workbench,
this helps to prevent contamination
when preparing the yeast culture.

2. Fill a 500 cm³ conical flask with 250 cm³ of the glucose
solution and then weigh out 1.25g of
yeast and add it to the flask along with a magnetic stirring flea
that enables you to
constantly and evenly stir the culture.

3. Place the flask on the magnetic stirrer base and stopper the
flask with some cotton wool

secured with a covering of aluminium foil.

4. The flask can be incubated at room temperature.
Measuring growth: Method 1 – Colorimetry

1. Use a pipette to fill a cuvette with the 0.5% glucose solution

and take a colorimetry reading
to set the reference absorbance of the colorimeter to zero.
2. Once the yeast sample has been prepared, use a sterile pipette
to transfer 4 cm³ of the
culture into a cuvette and take an absorbance reading, recording
the age of the culture in
minutes and its absorbance in a table.
3. Repeat steps 1 and 2 at the following ages of the culture: 30
minutes,1 hour, 90 minutes, 2
hours, 5 hours, 8 hours, 11 hours.
 5. Investigate the effect of different antibiotics on
bacteria.

The effectiveness of different antibiotics on a specific bacteria can

be investigated in the following practical. Antibiotics kill bacteria or
slow their growth and their effectiveness and this will be shown by
the clear area produced around the sample of each antibiotic.

Independent variable: Type of antibiotic used

Dependent variable: Area of inhibition zone

Method
1. Before starting the experiment disinfect the workbench, this
helps to prevent
contamination when preparing the bacteria culture.
2. Mark on the agar plate the date, your initials and the bacteria
being grown. Also mark
evenly across the plate 6 letters A-F to show where to place the
different antibiotic discs.
3. Use a bunsen burner to flame the bottle of the neck of the
bacterial flask causing the air to
rise and carry away undesired airborne microorganisms and then
use the sterile pipette to
transfer 2 cm³ of the liquid broth to the agar plate.
4. Use the sterile plastic spreader to evenly distribute the bacteria
across the agar jelly.
5. Take the first filter paper disc and use the forceps to completely
submerge it in the first
antibiotic for 10 seconds. Slightly lift the lid of the agar plate at a
angle allowing you to
quickly transfer the disc to the agar jelly.
6. Repeat step 5 for the other 4 antibiotics as well as a control
disc soaked in sterile water,
sterilising the forceps in between each antibiotic by holding
them in the bunsen burner
flame.
7. Tape the lid on at 4 points making it secure enough but also
allowing oxygen to enter so
the bacteria may respire aerobically. Then incubate the dish at
25 °C for 48 hours.
8. Disinfect the work surface again and wash your hands.
9. After the 48 hours have passed use a ruler to measure the
diameter of the inhibition zone
(clear zone) created around each disc

Tips to minimise contamination

● Have the lid removed from the agar plate for as
little time as possible.
● Work near the bunsen burner flame as this helps
to sterilise the air by heating and raising
air containing potential contaminants such as other
microorganisms.
Conclusion
The effectiveness of each antibiotic can be compared by looking
at the area of the inhibition
zones created. The one with the largest inhibition zone has
killed the most bacteria and is
therefore the most effective. The disc dipped in water acts as a
control to show that the antibiotics
alone are causing the death of bacteria and not any other
factor.
6. Use an artificial hydrogen carrier (redox indicator) to
investigate respiration in yeast.

Independent variable: Temperature

Dependent variable: Time taken for the redox indicator to turn red
Controlled Variable
-       Mass/number of seeds
-       Age of seeds
-       pH
-       No. of organisms
-       Temperature à Water baths between 20-40ºC
-       Amount of soda lime
-       Equilibration / acclimatisation e.g. Time left before measuring

Method
Preparing the yeast -
Add 10g of dried yeast and 50g of glucose to 1000 cm³ of distilled water, mixing
thoroughly with a
stirring rod. Allow the yeast culture to stabilise for 24 hours before starting the
experiment.
1. Set up the first water bath at 35°C
2. Use a pipette to place 10 cm³ of the yeast suspension into one
test tube and 1 cm³ of
TTC into a different test tube.
3. Place both test tubes into the water bath and leave them for 5
minutes to allow them to reach the temperature of the water bath.
4. Quickly pour the TTC solution into the test tube containing the
yeast, give it a stir with the glass stirring rod and start the
stopwatch.
5. Stop timing once the solution has turned red and record the time
taken in a suitable
table.
6. Repeat steps 1-5 with the remaining 4 temperatures.
7. Use a simple respirometer to determine the rate of respiration
and RQ of a suitable material (such as germinating seeds or
small invertebrates).

Independent variable: The main purpose of this experiment is to

understand how to use a respirometer and how it works. If you only use one
species of animal or seed there will be no independent variable. However, if you
use multiple species then the independent variable is the species of organism
being used.
Dependent variable: Distance moved by coloured fluid in the
manometer.

Variables to be controlled:
-       Same person / age /
gender / time of day
-       Temperature
-       Diet before testing…
-       Standardise exercise
-       Breathing must be measured over a set time (e.g. 5 minute

Method
1. Use either the pipette and funnel to add 5 cm³ of potassium
hydroxide solution to the test tube, or the spatula and weighing
scales to add 5g of soda lime to the test tube.
2. Secure the gauze in place above the solution / powder in the test
tube.
3. Select the first species of organism and place a known mass of
them on to the gauze.
4. Set up the remainder of the respirometer as shown in the diagram
below. If the manometer does not already have a drop of coloured
fluid in it, one can be added using a dropping
pipette.
5. Use the syringe to move the coloured fluid to the end of the
manometer furthest from the test tube and mark its position with a
pen, or record its distance from the end of the manometer by
measuring with a ruler.
6. Close the 3-way tap to allow no more gas exchange to occur
between the apparatus set
up and the outside atmosphere and start the stopwatch immediately.
7. Measure the position of the coloured fluid from its starting point
every minute for 5 minutes,
recording the distance moved in a suitable table. As the organisms
respire they take in
oxygen, causing the pressure in the test tube to decrease which draws
in air from the
manometer tube; so the volume of oxygen taken in can be calculated
if the diameter of the ubing is known. Once calculated, the rate of
oxygen uptake can be calculated per gram
of organism and then compared between organisms.

8. Open the 3-way tap connection to the outside air and use the
syringe to reset the capillary fluid.
9. Repeat steps 3-8 with any other organisms being investigated.
8. Investigate the effects of exercise on tidal volume, breathing
rate, respiratory minute ventilation, and oxygen consumption
using data from spirometer traces.

Independent variable: Whether the test subject is resting or exercising.

Dependent variable: Tidal volume and breathing rate.

Method
A spirometer is a piece of equipment that can be used to calculate the air
capacity of someone’s
lungs. To use it, it must first be calibrated as follows:
● Empty the spirometer so no air remains in it, align the pen to the paper so it
can record results.
● Now add a set volume of air to the spirometer, such as 1 dm³ and again let
the pen mark on the graph. From this you can use the horizontal scale to
calculate how many squares on the graph are equivalent to the volume of air
just added to the spirometer.

You can now begin the main part of the experiment with the
calibrated spirometer.
1. Get the test subject to sit down in front of the spirometer and get
them to put a nose clip on
so they are only breathing through their mouth.
2. Insert the disinfected mouthpiece of the spirometer into the
student’s mouth and allow them
to breathe normally until they’re are accustomed to breathing into the
apparatus.
3. Set the kymograph to a rate of 1 mm per second and turn it on so it
starts rotating.
4. After an exhalation, adjust the 2-way tap so the subject is now
breathing through the
spirometer equipment instead of the normal atmosphere.
5. Record their normal, resting breathing for a minute and then ask
them to breathe deeply for
one breath, then returning to normal breathing for another minute.
6. Now ask them to breathe out as deeply as they can, again returning
to normal breathing
afterwards.
7. The effects of exercise can then be observed by switching the
spirometer to the closed
position and asking the test subject to exercise for 2 minutes - such as
running on the spot
or star jumps.
8. Then immediately reinsert the mouthpiece, turn the spirometer to
the open position and
record the results for 1 minute.
From here, the respiratory minute ventilation can be calculate for
before and after exercise using the following formula: Respiratory
minute ventilation (dm per minute) = Tidal volume (dm ) x
Breathing rate ³ ³ (breaths per minute)
9. Investigate the production of amylase in germinating
cereal grains
Cereal grains contain a store of starch which is insoluble. This needs to be transported to
the embryo so it has a supply of energy for growth. In order to be transported across the
grain, the starch needs to be made soluble which occurs when the developing embryo
releases a hormones called gibberellins which stimulate other cells causing the release of
amylase - the enzyme that digests starch. In this experiment, the cereal grains will be
soaked in different concentrations of gibberellins, thus causing different amounts of
amylase to be released and so a different amount of starch digested. The starch digestion
can be observed when the agar plates containing the soaked seeds are washed with
potassium iodine in iodide solution which turns blue black in the presence of starch.
Therefore, the more starch that is digested, the larger the clear zone around the seeds will
be created