AS Biology Unit 3

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 1,2,3,4,5,6,7,8 & 9
• Additional Practicals

Question Type
Short-open, open-response and calculation
(Minimum 5 marks on calculation)
 1.Benedict's Test for glucose & Iodine Test for
Starch (Quantitative test)
 
Benedict’s Test for Glucose
A test with Benedict’s reagent can not only reveal if it is a reducing sugar or not, but
also how strong of a reducing agent it is.

Method
1. Pipette 5 cm3 of the solution being tested into the test tube
followed by 2 cm3 of
Benedict’s reagent.
2. Place the test tube into a test tube rack and then into the water
bath and leave it for exactly
2 minutes.
3. Remove the test tube and observe its colour.
 
This test is semi-quantitative because by observing the colour
change on a scale from blue to red
it’s possible to estimate the concentration of the reducing sugar. A
blue colour is a negative
result since this is the colour of the Benedict’s reagent, while
colours closer to brick-red give a
positive result with an increasing concentration of reducing sugar.
 
 
 Independent variable: Concentration of reducing sugar
Dependent variable: Colour change of Benedict’s reagent

Other variables to be controlled

1.Temperature
Result

Iodine Test for Starch

Method
1. Pipette 2 cm³ of the test solution into a test tube and
add 2 drops of potassium iodide solution.
2. A colour change from brown to blue-black indicates the
presence of starch.

Independent Variable : Concentration of Starch

Dependent Variable: Colour Change of Iodine Solution

Other variables to be controlled

1.Temperature

Result
Use of colour standards
The concentrations of both starch and reducing sugars can be
estimated using colour standards which is when the benedict’s test
and iodine test for starch are carried out on known concentrations of
starch and reducing sugars. The colours produced are known as
colour standards to which the results of testing unknown solutions
can be compared to estimate their concentrations.

Risk Assestment
 2.Measure vitamin C content of food and drinks

This experiment can be used to estimate the concentration of Vitamin C in

fruit juices since
vitamin C is an antioxidant and DCPIP is an oxidising agent. When vitamin
C is added to DCPIP
the DCPIP is reduced which eventually causes a colour change from blue to
colourless.

Method
 Pipette 1cm^3 of blue DCPIP into a test tube.
Using a burette, add 1% vitamin C solution drop by drop.
Shake tube gently after each drop.
Continue to do this until the blue colour of the DCPIP disappears.
Record the volume of solution needed to decolourise the DCPIP.
Repeat two more times and take average. This is your control.
Repeat the experiment again with different fruit juices.
Record your findings in a table.

Independent: fruit juice.

Dependent: volume of juice required to decolourise 1cm^3 of
DCPIP
 
Other Variables to be controlled
Temperature.
Concentration of DCPIP solution.
Shake each tube same number of times.
Same end point colour.
Result:

Risk Assessment
The calculation
Since the volume and concentration of both the vitamin C solution and
DCPIP are known, the
concentration of vitamin C in each fruit juice can be calculated by
comparing it to the standard
solution.

1 cm3 of 1% vitamin C solution contains 10 mg of vitamin C. Using

this information you can calculate the mass of vitamin C needed
to decolourise a given volume of DCPIP (for instance if
1.6 cm3 were needed that would be 16 mg). From there you can
calculate the concentrations of the
fruit juices.
 3.Effect of alcohol and temperature on
membrane permeability 
Beetroot can be used to investigate the permeability of cell membranes, since when its
cell membranes are damaged a coloured pigment, that gives beetroot its purple colour,
leaks out. The higher the permeability of the membrane, the more pigment is released
outside of cells. The permeability of cell membranes is affected by a number of factors
including temperature, alcohol and solvent concentration.

Method:

1. Cut 5 equal pieces of beetroot and rinse them to remove any

pigment during cutting (use cork borer to cut)
2. Place the 5 pieces in five different test tubes, each with 5 cm^3
of water.
3. Place each test tube in a water bath at a different temperature
for the same length of time
4. Remove the pieces of beetroot from the tubes, leaving just the
coloured liquid
5. Use colorimeter – a machine that passes light through the
liquid and measures how much of that light is absorbed. The
higher the absorbance, the more pigment is released, so the
higher permeability of the membrane
Independent variable: temperature of water
Dependent variable: % transmission of light through resulting
solution

Other variables to be controlled:

1. Volume of distilled water
2. Time left in water
3. Size of beetroot piece
4. Storage conditions and age of beetroot
5. Number of beetroot discs 6. Temperature of water bath
Result

Possible evaluation issues:

1. Difficulty in maintaining temperature
2. Accurate reading of the colorimeter
3. Accurate size of beetroot
4. From the different parts of the root
5. Ensuring the same time at the different temperatures
 4.Effect of temperature, pH, concentration of
enzyme and substrate on enzyme catalysed
reactions

This experiment investigates the effects of different factors on the

rate of the digestion of milk by the enzyme trypsin. As the milk
protein called casein is digested the white, opaque milk colour
becomes more pale and translucent, eventually turning
colourless. More light passes through the transparent and lighter
solutions so a colorimeter can be used to measure the
absorbance of the solution which in turn indicates the rate of
reaction of the experiment. The faster the rate of the reaction,
the lower the initial absorbance reading will be - as paler
solutions absorb less light.

Method
Factor 1 - the effect of temperature
1. Prepare the 5 water baths and monitor their temperatures using
thermometers.
2. Use a pipette to add 2 cm3 of trypsin solution to 5 test tubes and
place one in each water
bath.
3. Then use a pipette to add 2 cm3 of milk to 5 test tubes and again
place one in each water
bath.
4. Start the stopwatch and time for 5 minutes, allowing the
contents of each test tube to
reach the temperature of the water bath.
5. Pipette 2 cm3 of trypsin and 2 cm3 of distilled water into a
cuvette and take a colorimetry
reading to set the reference absorbance of the colorimeter to zero.
6
6. Now remove the milk test tube and the trypsin test tube from the
20°C water bath and add
the contents of each to a single cuvette, shaking it and then
immediately placing it in the
colorimeter.
7. Take an initial reading and then record the absorbance reading at
15 second intervals for 5
minutes or until there is only a slight change in absorbance between
readings.
8. Repeat steps 6-7 for the remaining 4 temperatures, being sure to
rinse the cuvettes
between each temperature and setting the absorbance to zero with
the water and
trypsin cuvette each time.

Factor 2 - pH
1. Prepare the reference cuvette by adding 1 cm3 of buffer solution,
1 cm3 of trypsin and 2 cm3
of water to it and setting the colorimeter absorbance of this to zero.
2. Prepare 5 cuvettes each containing 1 cm3 of a different pH buffer
solution and 1 cm3 of
trypsin solution. Prepare a further 5 cuvettes all containing 2 cm3 of
milk solution.
3. Add the contents of 1 of the milk cuvettes to the cuvette
containing the pH 5 buffer solution
then immediately place it in the colorimeter.
4. Take an initial reading and then record the absorbance reading at
15 second intervals for
5 minutes or until there is only a slight change in absorbance
between readings.
5. Repeat steps 3 and 4 with the remaining 4 pH buffer solutions.
Factor 3 - Enzyme concentration
1. Prepare these 10 cm3 solutions of different enzyme
concentrations using the following
quantities of distilled water and 1% trypsin solution:

2. Pipette 2 cm3 of trypsin and 2 cm3 of distilled water into a

cuvette and take a colorimetry
reading to set the reference absorbance of the colorimeter to zero.
3. Add 2 cm3 of milk to a cuvette, then add 2 cm3 of the 1% enzyme
solution to the cuvette.
Mix quickly then place in the colorimeter.
4. Take an initial reading and then record the absorbance reading at
15 second intervals for
5 minutes or until there is only a slight change in absorbance
between readings.
5. Repeat steps 3 and 4 with the remaining 4 enzyme solutions.
Factor 4 - substrate concentration
1. Prepare these 10 cm3 solutions of different substrate
concentrations using the following
quantities of distilled water and 2% milk solution:

2. Pipette 2 cm3 of trypsin and 2 cm3 of distilled water into a

cuvette and take a colorimetry
reading to set the reference absorbance of the colorimeter to
zero.
3. Add 2 cm3 of milk to a cuvette, then add 2 cm3 of the 1% milk
solution to the cuvette. Mix
quickly then place in the colorimeter.
4. Take an initial reading and then record the absorbance reading
at 15 second intervals for
5 minutes or until there is only a slight change in absorbance
between readings.
5. Repeat steps 3 and 4 with the remaining 4 milk substrate
solutions.
 5.Observe, label and measure (using graticule)
an animal cells under microscope 

Method
1. Begin by securing the prepared slide to the stage of the
microscope,
.. using the accompanying clip.
2. Rotate the revolving nosepiece to line up the lowest power
objective lens.
3. Look through the eyepiece and use the coarse adjustment knob
to lower and raise the stage until an image comes into focus.
4. Then use the fine adjustment knob to focus the image further.
5. In order to see and study cells
steps 3 and 4 may need to be
repeated using a higher power objective lens - 3 are usually found
on the nosepiece.
6. Produce a biological drawing of cell(s) observed.
Biological drawings
When producing biological drawings, in this case of cells being
observed under the microscope,certain rules should be followed:
● Draw in pencil only
● Use blank white paper
● Do not sketch - draw clear, unfeathered lines only
● Include labels
● Label lines should be drawn with a ruler and not cross over each
other
● Do not shade the drawing - for instance when indicating the
thickness of membranes / cell walls instead draw 2 lines
● Include the magnification of the microscope in the title
● Make diagrams as simple as possible and draw them an
appropriate size so everything can clearly be seen
● Include a scale bar where possible
Using a graticule
By using an eyepiece graticule and a stage micrometer the size of cells and
potentially their organelles can be accurately calculated. An eyepiece
graticule is a ruled line with regular intervals that appears when you look
down the microscope and a stage micrometer is a microscopic slide with an
accurate scale and is used to calibrate the eyepiece as follows:

1. Place the stage micrometer on the stage and line up its scale
with the eyepiece graticule. On the stage micrometer 1 division is
equal to 0.1mm.

2. Count how many eyepiece divisions are equal to 1 stage division.

This can be used to calculate the length of each eyepiece division at
this magnification - for instance, if 10 eyepiece divisions are equal to
1 stage division (0.1 mm) then each eyepiece division
measures 0.1 ÷ 10 = 0.01 mm.

3. The stage micrometer can now be replaced with the microscopic

slide containing the specimen. It is important not to change any
adjustments or objective lenses at this
point.

4. When observing cells now, their length or size of organelles can be

calculated accurately by counting the number of eyepiece divisions -
for instance a cell that is 3 eyepiece divisions wide would be 0.03
mm wide.
The following formula can also be used to calculate magnification and
object and image sizes
when observing specimens:

Risk assessment
This experiment is low risk however it is important to keep in mind
that microscopes can be heavy
so should be handled carefully and not put on the edges of
workbenches where they could be
knocked off. Furthermore, when preparing various samples to
observe there is a risk of someone
being allergic, for instance where some species of plant are being
handled in the lab before being
cut to use as specimens.
 6.Observe the stages of mitosis using root tip
squash.

Method
1. The ends of a root or shoot are where growth and therefore
mitosis are occurring, so to observe mitosis the plant tissue to be
observed must be cut from these areas. Use a scalpel to measure
and cut the end 1 cm of the root.
2. Measure out 10 cm3 of 1 mol/dm3 hydrochloric acid into a
boiling tube and place it in a water bath at 60°C.
3. Transfer the root tip section into the boiling tube and leave it for
5 minutes.
4. Remove the tip and use a pipette filled with distilled water to
rinse it. Leave it to air dry on a paper towel.
5. Now cut the end 2mm of the root tip and place this on a
microscopic slide. Use a mounted
needle to spread the specimen out forming a thin layer that will
allow light to pass through
when observing it under a light microscope.
6. Use a pipette to apply a couple drops of stain to the specimen,
the stain binds to
chromosomes making them easier to see. There are several stains
that can be used
including Toluidine Blue O and Feulgen Stain.
7. Finally, place a cover slip on top of the specimen on the
microscopic slide. Press down
firmly, further spreading and thinning the specimen for observing.
The slide can now be
viewed under an optical microscope.
 7.Observe, draw and label transverse sections of
roots, stems, leaves, cells of plant tissues. Identify
.. location of sclerenchyma fibers, phloem, sieve
tubes and xylem vessels and their location.
 
Method
1. Take the plant stem and place it on a white tile and fill a petri
dish with a shallow level of
distilled water.
2. Lightly run the razor edge down the length of the stem several
times to give several thin specimens, using tweezers to place them
in the petri dish. Select the thinnest specimen and place it on a
microscopic slide.
3. Use a paper towel to dab the edges of the specimen to absorb
any excess water.
4. Put on the eye protection and gloves and use a pipette to add 2-
3 drops of the stain to the specimen. Leave it to soak in for 3
minutes and then again use a paper towel to absorb
any excess liquid.
5. Lay the coverslip onto the slide, pressing firmly to thin the
specimen and remove any air bubbles between the coverslip and
the slide.
6. Observe the specimen under the light microscope:
○ Rotate the revolving nosepiece to line up the
lowest power objective lens.
○ Look through the eyepiece and use the coarse
adjustment knob to lower and raise the stage
until an image comes into focus.
○ Then use the fine adjustment knob to focus
the image further.
○ Produce a biological drawing using either the low
or medium objective lens.Repeat steps 1-6
using the root section leaf.
Result
 8.Determine tensile strength of plant fibers

Method
1. Use the scalpel to remove 9 fibrous strings from your plant
sample, examining each one to
check there are no breakages along its length and that its diameter
is even.
2. On the chopping board or white tile use a ruler and scalpel to cut
the 9 strings into three 10
cm, three 15 cm and three 20 cm lengths.
3. Set up the clamps and retorts as shown in the diagram with the
first 10 cm string.
4. Ensure the string is properly secured with the cotton wool
cushioning directly beneath it,
then begin to add weights to the string, 10g at a time until the string
breaks. Record the
mass added in your results table.
5. Repeat step 4 with each of the other two 10 cm strings and
calculate a mean mass added
before the string breaks. Then repeat for the 15 cm and 20 cm
lengths.
6. Plot a graph of your results.
Conclusion
Different factors affecting the tensile strength of plants can also be
investigated, such as comparing
the strengths of plant fibres which have the same length but are
from different species of plant. The strength of plant fibres is
down to their chemical composition, such as the strong cellulose
chains
and links that form plant cell walls and the addition of chemicals
like lignin which add strength and
support to vessels in the vascular bundle.
 9.Investigate the antimicrobial properties
of plants 

Method
1.Prepare an agar and bacteria medium in a Petri dish.
Incubate at 25 C.
2.Crush garlic mixed with 1 cm^3 of ethanol to release extract.
3.Place garlic extract on discs of filter paper.
4.Place the discs in the Petri dish.
5.Tape the Petri dish closed in a way to allow gas exchange.
6.Make sure not to tape all the way around as anaerobic bacteria
will start to grow due to lack of oxygen.
7.Leave for two-three days
8.Observe clear zone and measure diameter.
9.Repeat for different extracts.

Independent: presence of material

Dependent: diameter of inhibition zone.

Controlled variables:
volume of material used
Volume of ethanol.
Size of paper discs.
Incubation temperature.
Result
Students will be assessed on their ability
to:

• identify the apparatus required

• identify the dependent and independent
variables, standardised or controlled variables
• describe how to measure relevant variables using
the most appropriate instrument and correct
measuring techniques
• identify and state how to control all other relevant
variables to make it a fair test
• discuss whether repeat readings are appropriate
• identify health and safety issues and discuss how
they may be dealt with
• discuss how the data collected will be used
• identify possible sources of uncertainty and/or
systematic error and explain how they may be
reduced or eliminated
• comment on the implications of biology (for
example benefits/risks) and on its context (for
example social/environmental/historical)