CALCULATIONS

To develop the skills needed to plan an investigation using dilutions.

Example 1: Plan how you will use the stock 2% starch solution to make 5cm3 of the following
five concentrations of starch solution 2%, 1%, 0.5%, 0.2% and 0.1%
To make 5cm3 of 2% starch solution- take a clean pipette and take 5cm3 of the stock starch
solution
To make different concentrations of starch solution, use the equation:
C1V1=C2V2
Where,
C1 – concentration of the given stock solution
V1 – volume of the stock solution
C2- concentration of the solution to be made
V2- volume of the solution to be made
So, to make 5cm3 of 1% starch solution
C1=2%
V1=x
C2= 1%
V2= 5cm3
2× x = 1×5 x = 5/2= 2.5
Use a clean pipette to take 2.5cm3 of stock solution and add 2.5cm3 of distilled water.
Example 2:
Explain how you could use serial dilution to make a range of concentrations from a stock
solution of 10% fructose.
Stock is 10 ml of 10% fructose solution
To make 8% fructose solution, C1V1=C2V2
10 × x = 8% × 10
So, x is 8ml. Take 8ml of stock solution using a clean pipette and add 2ml of distilled water.
To make 6% fructose solution
10 × x = 6% × 10
So, x is 6ml. Take 6ml of stock solution using a clean pipette and add 4ml of distilled water.
Example 3: The horticulturist needed to prepare a vitamin C solution of 10mg cm -3 solution for
her investigation. However, she had no vitamin C powder or tablets available. There was a
stock vitamin C solution of 50 mg cm-3 available in the laboratory.

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Describe how 200ml of 10mg cm-3 vitamin C solution can be prepared from the 50mg cm-3
solution.

Take 40ml of stock solution.

Add 160 ml of water.
Use a pipette/burette/measuring cylinder to measure volumes.
Standard deviation
Standard deviation can be calculated using the equation given below

s = standard deviation
Σ = sum of
x = each value in the data set
x = mean of all values in the data set
n = number of values in the data set

(Ʃ𝑥)2
Ʃ𝑥 2 − 𝑛
S=√
𝑛−1

(In a data set of 1,2,3,4,5,6 …Ʃx2 is 12+22+32+42+52+62 and (Ʃx)2 is (1+2+3+4+5+6)2)

Explain why standard deviation is used for analysing the data. ( 3)

S.D. shows variation from the mean (1)
a larger S.D. means greater spread, smaller S.D. is more reliable representation of the data (1)
therefore, although ranges of A and B are the same, the S.D. shows there is more variation in
the data for method B (1)
Example 1:

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The student calculated the standard deviation for method A using the following formula:
Numerator = 219.34 (1)
Denominator = 5 (1)
Standard deviation = 6.6 (1)

Example 2: Calculate the missing standard deviation (SD) using the following formula. [3]

Answer : Ans: 17.5

Example 3: using second equation calculate the standard deviation for 60 minutes
Time kept in Absorbance/a.u Standard
allicin/minute Trial 1 Trial 2 Trial 3 Mean deviation
0 0.00 0.00 0.00 0.00 0.00
10 0.17 0.15 0.19 0.17 0.02
Answer = 0.043
20 0.30 0.27 0.29 0.29 0.02
30 0.39 0.25 0.37 0.34 0.06
40 0.40 0.37 0.39 0.39 0.02
50 0.39 0.45 0.43 0.42 0.03
60 o.48 0.40 0.46 0.45

Magnification

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Calibration of eye piece graticule
Place a micrometer slide on the stage of the microscope and focus on the micrometer scale,
using the low power objective.
Move the slide and rotate the eye piece to align the scales of the eye piece graticule and the
stage micrometer in the field of view (two lines should coincide with each other).
Count the number of divisions on the eye piece graticule that are equivalent to a known length
on the micrometer slide and work out the length of one eye piece unit.
(if 100 μm is equivalent to 2 epu, then 1 epu is 100/2=50 μm)
Repeat this with medium and high-power objectives.

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Example 1:

Calculate the eye piece scale using the stage micrometer scale. Each division of the stage
micrometer is equal to 10µm. show your working
Answer
One division on the stage micrometer is 10 µm
5 division = 50 µm = 20 eyepiece units
So, one graticule unit is 50÷20= 2.5 µm
Shown below is a cell at the same magnification that the eyepiece scale was calibrated.
Use the value you calculated, to calculate the actual length of
the cell shown.
Answer: the cell is 6 units wide
Each unit is 2.5 µm
So, the length of the cell is 2.5× 6= 15 µm

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6
 UNIT 3
1.Use a semi-quantitative method with Benedict’s reagent to estimate the concentrations of reducing
sugars and with iodine solution to estimate the concentrations of starch, using colour standards.
Objectives:
o To understand what is meant by a semi-quantitative test
(by observing colour change on a scale it is possible to estimate the concentration of the reducing
sugar)
o To be able to estimate concentrations of reducing sugars using Benedict’s reagent.
o To be able to estimate concentrations of starch using iodine solution.
o To develop the skills needed to plan an investigation using dilutions.
Example: Describe how a valid and reliable investigation can be carried out by the nutritionist to
determine the reducing sugar content of the milk brands
o Reducing sugar content of the milk brands can be determined by using Benedict’s reagent.
o Take 5cm3 of milk each from different brands in different test tubes and add 1cm 3 of Benedict’s
reagent to each of this.
o Place these test tubes in a water bath kept at 700C and start the stopwatch.
o After 2 minutes take the test out from the water bath and observe the colour change from blue
to green to red.
o Use a standard colour chart to identify concentration of reducing sugar in each milk product.
o Repeat at least three times for each milk brand and find the mean concentration.
o Throughout the experiment control the variables such as temperature, using temperature-
controlled water bath and volume of Benedict’s reagent used using graduated pipette.

Reducing sugar test is carried out using Benedict’s test. Add Benedict reagent and heat the sample in
a water bath kept at 700C

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Example 2: Describe how to carry out an investigation to determine which part of the Canna lily plant
contains most starch
o Take different parts of the Canna lily plant such as leaf, stem and root.
o Take 50g of each part of the plant and crush it using a mortar and pistil and add same volume
of distilled water, sieve it and extract.
o Take 5cm3 from each of the extract in a test tube and add 0.5cm3 of iodine solution into it.
o Observe the colour change from brown to blue-black.
o The part with darkest blue-black colour has most starch
o Repeat at least three times for each part of the plant.
o Throughout the experiment control the variables such as volume of the plant extract used,
using a graduated pipette and mass of the plant part used using an electronic balance.
o Use same plant for repetition as well

A qualitative test tells you whether something is present or absent.

A quantitative test enables you to determine exactly how much of the substance is present.
A semi-quantitative test enables you to estimate roughly how much of the substance is present.
Benedict’s test is suitable only for reducing sugar. Non reducing sugar does not react with Benedict’s
reagent.
Copper ions react with reducing sugar. The more the reducing sugar present, the more the copper ion
react with reducing sugar. So, the solution will be least blue.
To make the investigation more reliable:
Use larger quantities of each sample, so, repeats readings can be taken to find the average.
To make the investigation more accurate:
Compare the result with the reference values on the nutritional label, which will show the glucose
content.
Calculate the concentration of glucose in the sample and then compare with the estimate generated
experimentally.

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Dilutions
Example 1: Plan how you will use the stock 2% starch solution to make 5cm 3 of the following five
concentrations of starch solution 2%, 1%, 0.5%, 0.2% and 0.1%
To make 5cm3 of 2% starch solution- take a clean pipette and take 5cm3 of the stock starch solution
To make different concentrations of starch solution, use the equation:
C1V1=C2V2
Where,
C1 – concentration of the given stock solution
V1 – volume of the stock solution
C2- concentration of the solution to be made
V2- volume of the solution to be made
So, to make 5cm3 of 1% starch solution
C1=2%
V1=x
C2= 1%
V2= 5cm3
2× x = 1×5 x = 5/2= 2.5
Use a clean pipette to take 2.5cm3 of stock solution and add 2.5cm3 of distilled water.
Example 2:
Explain how you could use serial dilution to make a range of concentrations from a stock solution of 10%
fructose.
Stock is 10 ml of 10% fructose solution
To make 8% fructose solution, C1V1=C2V2
10 × x = 8% × 10
So, x is 8ml. Take 8ml of stock solution using a clean pipette and add 2ml of distilled water.
To make 6% fructose solution
10 × x = 6% × 10
So, x is 6ml. Take 6ml of stock solution using a clean pipette and add 4ml of distilled water.

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Practice question
The reagent used to test for reducing sugars is a blue solution containing soluble copper ions. Reducing sugars
react with the copper to form an insoluble precipitate.
After testing, the precipitate formed can be removed. This leaves a solution that is less blue.
Devise a procedure to determine the concentration of reducing sugars in a sweet potato.
• Take 5cm3 of sweet potato extract in a test tube and carry out reducing sugar test, Benedict test.
• add 1cm3 of Benedict’s reagent to the test tube containing the extract
o Place the test tube in a water bath kept at 700C and start the stopwatch.
o After 2 minutes take the test out from the water bath and filter the solution.
o Measure the intensity of blue color in filtrate
o Use a standard colour chart to identify concentration of reducing sugar in.
o Repeat at least three times and find the mean concentration.
o Throughout the experiment control the variables such as temperature, using temperature-controlled
water bath and volume of Benedict’s reagent used using graduated pipette.

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 CORE PRACTICAL 2: INVESTIGATING THE VITAMIN C CONTENT OF FOOD AND DRINK
Objectives:
To be able to calculate the vitamin C concentration of fruit juices using the titration method.
To solve the problems set in practical contexts.
To process and analyze data using appropriate mathematical skills.
Vitamin C is an antioxidant. So, it reduces the DCPIP causing the colour change from blue to colourless.
Acid fruit juices will not completely decolourise the DCPIP. So, the solution will turn pink
Vitamin C content changes when a vegetable is boiled
Boiling damages cell membranes and increases permeability of membranes. So, vitamin C moves out of
the cells by diffusion. Thereby reducing the Vitamin C content
Procedure
Pipette 2cm3 blue DCPIP into a conical flask.
Using a burette add 1% vitamin C solution drop by drop.
Shake the conical flask gently after each drop.
Continue the titration until the colour of DCPIP changes
from blue to colourless.
Record volume of solution needed to decolourise the DCPIP.
Repeat further 2 times and calculate mean volume of vitamin C needed to decolourise DCPIP
Repeat the procedure with different fruit juices.
Find out the concentration of juice using the equation

Describe how the vitamin C content of broccoli could be measured

Extract juice from the broccoli.
Take 2ml of 1% DCPIP in a conical flask
Take standard vitamin C solution in a burette and titrate against DCPIP.
Record the volume of Vitamin C needed to decolourise DCPIP.
Repeat the titration and calculate the mean volume of vitamin C solution needed
Titrate the juice against DCPIP until the colour changes from blue to colourless.
Record the volume of juice added
Repeat the procedure three times and find out the mean volume of juice added.

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Describe an investigation that the student could carry out to compare these two methods of cooking
on the vitamin C content of carrots.
Take 2carrots of the same mass and gene.
Some carrots need to be boiled in water and some cooked in microwave
Take juice out of this carrots/ cooking water being used
Take 2ml of 1% DCPIP in a conical flask and titrate the juice against DCPIP
Titrate until colour of DCPIP changes from blue to colourless.
Repeat the process and find out the mean volume of juice added.
Use a 1% vitamin C solution and repeat the titration.
Find out the mean volume of Vitamin C solution needed to decolourise DCPIP
Find out the vitamin C content
Concentration of vitamin C in carrot juice =
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑽𝒊𝒕𝒂𝒎𝒊𝒏 𝑪 𝒔𝒐𝒍𝒖𝒕𝒊𝒐𝒏
𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝑽𝒊𝒕𝒂𝒎𝒊𝒏 𝑪
𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒄𝒂𝒓𝒓𝒐𝒕 𝒋𝒖𝒊𝒄𝒆 𝒖𝒔𝒆𝒅

Variables to be controlled and how to control and what will happen if it is not controlled
(Controlling variables produce a valid result)
Temperature – using temperature-controlled water bath
Concentration of DCPIP solution-use same mass of DCPIP in same volume of water (1%)
(change in concentration leads to change in the volume of juice needed to decolourise DCPIP)
Shaking time- Shake each tube same number of times after adding each drop of juice into the DCPIP.
(too much adds oxygen which will slightly restore the DCPIP to blue)
Same end point colour. i.e. until the blue colour of DCPIP just disappears
Mass of the fruit used- take same mass of fruit using an electronic balance. (if mass is greater vitamin c
content will be higher)
Safety and ethical issues
DCPIP may be an irritant so wear gloves
Burns when handling hot objects. So, use heat resistant gloves.
Limitation
End-point difficult to judge as needs to be just when blue colour disappears especially in highly
coloured juices
Calculation 1
A standard vitamin C solution is required to determine the Vitamin C content of the citrus fruits.
Describe how 100ml of a vitamin C solution with 10mg cm -3 can be prepared using a 50mg cm-3 stock
vitamin C solution.
(C1 V1 = C2 V2)
50*x =10*100, so, x= 20 ml. So, take 20ml of stock solution and add 80ml of distilled water using a
pipette/ a measuring cylinder

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Calculation 2
In an investigation, a student compared the vitamin C concentration of freshly squeezed fruit juice and
fruit juice stored in a carton. The table shows the results of this investigation.

The standard solution contained 10 mg of vitamin C per cm3.

1.5 cm3 of this standard solution decolourised 1 cm3 of 1% DCPIP.
Calculate the concentration of vitamin C in carton lemon juice.
Equivalence of DCPIP calculated (1.5 x 10 OR 15 OR ÷26.1)
(In 10mg 1 cm3. So, its percentage is 10)
Concentration of vitamin C calculated and quoted to 1 d.p. (15 ÷ 26.1 = 0.6 (mg cm-3))
concentration of vitamin C in carton lemon juice= (volume of standard vitamin C used÷ volume of
carton lemon used) × concentration of standard vitamin C used
= (1.5 ÷ 26.1) × 10= 0.6 mg cm-3

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 CORE PRACTICAL: 3 INVESTIGATE MEMBRANE PROPERTIES INCLUDING THE EFFECT OF
ALCOHOL AND TEMPERATURE ON MEMBRANE PERMEABILITY
Objectives:
To know how the effect of temperature and alcohol on membranes can be determined
To be able to recognize quantitative variables that should be controlled in an investigation
The effect of temperature on cell membranes
Independent variable: temperature of water
Dependent variable: % transmission of light (through resulting solution)

Procedure
Cut equal sized pieces of tissue

Rinse under running water to

remove all betalin pigment
released by cutting

Place the pieces in equal volumes

of distilled water:
• Use a range of temperatures
• Leave for equal time

Remove pieces carefully and shake

each solution gently to disperse the
pigment

Assess amount of pigment lost

using a colorimeter to measure the
absorbance or transmission value
of the solution

Plot values on graph:

• Temperature on X axis
• Absorbance or transmission
on Y axis
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It is necessary to rinse the beetroot discs before they were added
Some pigments might have leaked during cutting.
To remove the red pigment released by cells during preparation.
If it is not removed it will change the transmission or absorbance value

It is important to use equal sized cylinders of beetroot in this experiment

Equal sized cylinders contain same volume of pigment and they are of the same surface area.
This will give a valid result.

The discs should be removed carefully

In order to avoid (physical/more) damage to the discs/membranes/cells which would cause more
pigment release causing the results to be invalid

Effect of a decrease in size of beetroot cube

This reduces the surface area. So, less pigments would be leaked, and the solution will be less dark.

Variables to be controlled and how

• Volume of water in the boiling tube - Use a pipette to take 5ml of distilled water
• species/age /variety of beetroot –take the samples from the same beetroot
• Rinsing time – Rinse it for a fixed time for each
• The part of beetroot – take the beetroot disc from middle part of the beetroot.
• Time left in water- use stop-watch and kept in water bath for 20 minutes
• Size of beetroot piece- use same cork-borer and ruler to get the same size (2cm).
(as these variables affect the membrane permeability, if it is not controlled it will increase/decrease
the absorbance measured and the result will be invalid)
How the degree of redness can be measured
Degree of redness can be measured by using a colorimeter with a blue filter in it.
Add 2 cm3 of distilled water in a clean cuvette and set up the absorbance as zero (zero the
colorimeter).
Take the coloured solution and shake well.
Transfer the coloured solution into a clean cuvette and measure the absorbance.

Before measuring the absorbance of each coloured solution, the colorimeter was set to zero using
distilled water/ alcohol only.
To find out the absorbance of distilled water/ alcohol.This ensures that the absorbance measured is
due to betalain / the pigment only and make the result valid.
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Describe an experiment you have carried out to investigate the effect of temperature on the
permeability of cell membranes
Take 15 beetroot discs of same size, 2cm in size.
Wash the beetroot discs thoroughly to remove the leaked pigments and wipe it well with tissue paper.
Set up water bath for 25°C, 30°C, 35°C, 40°C and 45°C
Take 5ml of distilled water in a boiling tube and place it in water bath for 10 minutes so that the
distilled water gets water bath temperature.
After 10 minutes place the beet root disc in the boiling tube and leave for 25 minutes.
After 25 minutes take out the boiling tube shake it well and remove the disc.
Transfer the coloured solution to a clean cuvette.
Find out the absorbance using a colorimeter with blue filter in it.
Standardise the colorimeter using distilled water.
Repeat the procedure three times at each temperature and find out the mean absorbance.

The tubes were placed in the water baths for 1o minutes before the cylinders were added
This make sure that the distilled water and the water bath temperature are equilibrated. So that the
effect of correct temperature can be assessed.

Effect of an increase in temperature on the colour of the solution

Increase in temperature increases the rate of diffusion / permeability. So, more pigments would be
leaked, and the solution may be darker
At lower temperatures, the tonoplast and cell membrane are intact. The pigment molecules are too
large, and it cannot pass through the membrane easily. So, the transmission is high.
Increase in temperature increases the movement of phospholipid molecules of the membrane and the
membrane become more fluid. Increase in kinetic energy of the molecules increases the diffusion of
pigment molecules. So, more pigment passes through the membrane and the percentage transmission
decreases.
At higher temperatures, the protein molecules in the membrane become completely denatured and
the membrane develops gaps through which the pigments can flood out. So, percentage transmission
again decreases and levels out as the concentration of pigment is the same inside and outside the cells.

Repeating the procedure (using 5 discs) at each temperature

To find out the mean absorbance/ transmission and to calculate standard deviation / measure
reliability. By calculating the standard deviation, variability in the result can be analysed.
This also help to identify anomalies

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Describe an experiment you have carried out to investigate the effect of alcohol concentration on the
permeability of cell membranes
Take 15 beetroot discs of same size, 2cm in size.
Wash the beetroot discs thoroughly to remove the leaked pigments and wipe it well with tissue paper.
Take five different concentrations of ethanol, 0%,10%,20%,30% and 40% in five different test tubes.
Take 10cm3 of 10% ethanol in a boiling tube and place a beet root disc in the boiling tube and leave it
for 25 minutes.
After 25 minutes shake the boiling tube once and remove the disc.
Transfer the coloured solution to a clean cuvette.
Find out the absorbance using a colorimeter with blue filter in it.
Standardise the colorimeter using distilled water.
Repeat the procedure three times and calculate the mean absorbance.
Repeat the same procedure under the same condition for each ethanol concentration.
The effect of ethanol on the permeability of beetroot cell membranes
Ethanol causes the membrane to be disrupted as phospholipids dissolve in ethanol.
Membrane proteins are denatured by ethanol.
This also disrupt the vacuole membrane.
So, the pigment can escape from the cell / vacuole when the membrane is disrupted.

Safety issues
Use of sharp items to cut the beetroot discs. Always cut away from yourselves
Ethanol is highly flammable. So, keep it away from naked flames.

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 CORE PRACTICAL 4: INVESTIGATE THE EFFECT OF TEMPERATURE, Ph, ENZYME
CONCENTRATION AND SUBSTRATE CONCENTRATION ON THE INITIAL RATE OF ENZYME-
CATALYSED REACTIONS
Objectives: to be able to measure the initial rate of enzyme activity
To understand why measuring initial rate is important
To understand the variables that can affect the rate of an enzyme-catalysed reaction and the
result of changing each variable
To be able to calculate the rate of a reaction using the gradient of a line
The effect of changing enzyme concentration on rate of enzyme-catalysed reaction.
• Make up 5 different concentrations of enzyme (0%,1%,2%,3%,4%) using distilled water.
• Set up water bath for temperature to keep constant at 30ᴼC.
• Place a test tube containing 2cm3 casein (milk protein) solution into water bath alongside
second tube containing 2cm3 of 2% trypsin.
• Allow to acclimatise for 5 minutes so that they will be at same temperature. Then add
trypsin to casein, start stop clock and place them back in the water bath. (while mixing it
may warm up or cool down. So, if it is not placed back in the same water bath the rate
would change)
• Find out the time taken for casein solution to turn transparent. (mark a ‘X’ on the other side
of tube, as soon as seen through solution stop clock).
• Repeat a further 2 times to find out the mean time taken. Then repeat for other
concentrations as well.
• Calculation: rate = 1/time taken for the solution to become clear

OR
• Make up 5 different concentrations of enzyme (0%,1%,2%,3%,4%) using distilled water.
• Set the absorbance of colorimeter to zero using 2cm3 of 2% trypsin and 2cm3 of the distilled
water in a cuvette.
• Take 2cm3 of the 0.2% milk suspension (casein) in a cuvette.
• Add 2cm3 of 2% trypsin solution to the milk in the cuvette and mix them quickly.
• Place the solution into the colorimeter and measure the absorbance immediately and then
at 15 seconds intervals for 5 minutes.
• Rinse the cuvette with distilled water and repeat the procedure for other concentrations of
trypsin (enzyme) as well.
• Record the data collected in a table.

1
The effect of changing substrate concentration on rate of enzyme-catalysed reaction.
• Make up 5 different concentrations of casein (0%,0.2%,0.4%,0.6% and 0.8%) using distilled
water.
• Set the absorbance of colorimeter to zero using 2cm3 of 2% trypsin and 2cm3 of the distilled
water in a cuvette.
• Take 2cm3 of the 0.2% milk suspension (casein) in a cuvette.
• Add 2cm3 of 2% trypsin solution to the milk in the cuvette and mix them quickly.
• Place the solution into the colorimeter and measure the absorbance immediately and then
at 15 seconds intervals for 5 minutes.
• Rinse the cuvette with distilled water and repeat the procedure for each concentration.
• Record the data collected in a table.
The effect of changing temperature on rate of enzyme-catalysed reaction.
• Set up five different water baths of temperatures 20ᴼC, 25ᴼC, 30ᴼC, 35ᴼC and 40ᴼC
• Take five test tubes an add 2cm3 of 1% trypsin solution to each of the test tube and place
them in each of the water bath.
• Take five clean test tubes and add 2cm3 of 0.2% casein to each of the test tube and place
them in each of the water bath and leave them in the respective water bath for 5 minutes
so they reach the water bath temperature.
• Set the absorbance of colorimeter to zero using 2cm3 of 2% trypsin and 2cm3 of the distilled
water in a cuvette.
• After 5 minutes pour the 2cm3 casein from the water bath set at 20ᴼC into a cuvette and
add 2cm3 of trypsin from the same water bath into the cuvette containing casein.
• Mix them by shaking gently and place the cuvette into the colorimeter.
• Measure the absorbance immediately and then at 15 second interval for 5 minutes.
• Repeat the same for trypsin and casein from other temperatures as well.
• Record the data collected in a table.
The effect of changing pH on rate of enzyme-catalysed reaction.
• Take buffer solution of five different pH values, 5,6,7,8, and 9
• Set the absorbance of colorimeter to zero using 1cm3 of trypsin and 2cm3 of the distilled
water and 1cm3 of buffer solution in a cuvette.
• Add 1cm3 of trypsin solution and 1cm3 of buffer solution with a pH 5 into a cuvette and add
this into 2cm3 of the milk suspension in another cuvette.
• Mix the solution and the milk by shaking gently and place the cuvette into the colorimeter.
• Measure the absorbance immediately and then at 15 second interval for 5 minutes.
• Repeat the same each buffer solution and record the data collected in a table.

2
Using a colorimeter
Protein is broken down by enzyme and the solution become transparent. Light passes more easily
through the final solution. So, the reaction can be monitored using a colorimeter.
Calculating initial rate of reaction using a tangent
Draw a tangent to the line at the steepest point.
Turn this line into a right-angled triangle.
Divide the change in the value on the Y-axis by the change in the value on the X-axis
OR
Draw a tangent on the curve from the origin
Calculate the gradient, y/x
Express the initial rate of the reaction using the units given(cm3s-1)
Variables to be controlled and how to control them
Temperature – using temperature-controlled water bath set at 30ᴼC.
Volume of enzyme – using a measuring cylinder and take same volume of enzyme
Volume of substrate – use measuring cylinder/ take same mass using a balance
Concentration of substrate – dissolve the same volume /mass of substrate in equal volume of
water
pH of the medium- use a buffer solution of pH 7
suggestions of the risks and how to minimise them.
• Respiration or breathing problems; so, use coated enzymes or wear mask
• There is possible skin reaction to enzyme; so, wear gloves or wash with copious water
• Getting enzymes into the blood through skin or cuts; so, wear plaster or gloves
• High level of alkali damages skin/eyes; so, wear gloves/goggles
• Enzymes could damage the eyes; so,wear gloves
Effect of catalase concentration on the initial rate of hydrogen peroxide breakdown
Independent variable: Catalase concentration
Dependent variable: Initial rate of volume of oxygen produced per time
• Set up apparatus to collect oxygen over water with the collecting tube filled with water and
the screw clip closed
• Cut 10 discs of potatoes each 0.2 mm thick.
• Place one disc in a boiling tube with 5 cm3 of buffer solution of pH 7.
• Add 5 cm3 of 1 % hydrogen peroxide solution and immediately place bung and delivery tube
firmly into boiling tube.
• Place the other end of the delivery tube under the collecting tube.

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 • Start stop-watch as soon as the first oxygen bubble enter the collecting tube. Collect any
gas produced in a 3-minute period.
• Repeat the procedure three times and find the mean volume of gas produced.
• Initial rate of reaction= (volume of oxygen produced)/ time.
• Repeat the same procedure under same condition using different numbers of potato disc
(2,3,4 and 5 and no disc as the control)
Variables to be controlled and how
species/age of potato – Take potato disc from the same potato
pH of the medium- use a buffer solution of pH 7
temperature- use water bath set at 30ᴼC
volume of hydrogen peroxide- use a syringe to take the same volume
concentration of hydrogen peroxide- mix same volume of hydrogen peroxide in equal volumes of
water
Determining suitable concentrations of substrate and enzyme to use in any investigation
Run the experiment with a range of different concentrations
Choose a combination in which the rate of change is not too {high / fast} / too {low / slow} / time
taken is not too {long / short}
Change in pH affect rate of reaction
Change in pH changes the shape of active site of the enzyme as it affects the ionic bond formed
between the R-groups.
This affect the formation of enzyme substrate complex.

4
Describe an experiment that could be carried out to investigate the effect of enzyme concentration on
the initial rate of reaction.
1. idea of a range of concentrations of enzyme (at least 5) ;
2. idea of substrate concentration not limiting ;
3. reference to mixing ;
4. description of how to measure dependent variable with time ; 4. and 5. Must relate to reaction /
enzyme named
5. description of how to measure the initial rate of reaction ; ACCEPT clear indication of rate measured
soon after mixing, plot and calculate rate from linear part of graph
NOT time taken for all substrate to be converted but could get Mp4
6. reference to an appropriate named controlled variable ; ACCEPT e.g. pH, temperature, volume,
concentration of substrate
7. reference to {replicates / repeats} at each enzyme concentration ; IGNORE repeat for other
concentrations
ACCEPT repeat whole experiment
8. control {described / used as comparison}; ACCEPT control used is with {no enzyme
/ distilled water}
an investigation into the effect of enzyme concentration on the initial rate of this reaction.
In this investigation, the substrate concentration was a factor that was kept constant.
Suggest two other factors that should be kept constant. For each factor, state how it can be kept constant
pH ;
buffer ;
temperature ;
water bath ; not room temperature
time of reaction ;
stopwatch ;
volume of {enzyme / substrate} ; not amount
measuring cylinder / pipette ;
type of enzyme ;
same batch of enzyme ;

5
CORE PRACTICAL: 5 (i) USE A LIGHT MICROSCOPE TO MAKE OBSERVATIONS AND LABELLED DRAWINGS OF
SUITABLE ANIMAL CELLS; (ii)USE A GRATICULE WITH A MICROSCOPE TO MAKE MEASUREMENTS AND
UNDERSTAND THE CONCEPT OF SCALE
Learning tips: to be able to answer questions about the magnification of images, make sure you learn the
following equation:
𝐬𝐢𝐳𝐞 𝐨𝐟 𝐢𝐦𝐚𝐠𝐞
Magnification = 𝐬𝐢𝐳𝐞 𝐨𝐟 𝐫𝐞𝐚𝐥 𝐨𝐛𝐣𝐞𝐜𝐭
You should know how to rearrange the magnification formula to calculate any of the values.
Remember to convert all lengths to the same unit, usually µm
Procedure: making observations
Wash hands with soap and water
Take a cotton bud and gently rub it on the inside of the cheek, then rub the cotton bud in the
centre of a glass slide.
Place the cotton bud in a beaker of disinfectant.
Add a few drops of methylene blue to the sample and then cover it with a cover slip.
Turn the objective lens to low power and examine the stained slide under the microscope.
Carefully sketch a few of the cells.
Use the eyepiece graticule to measure the cell’s diameter.
Add a scale bar to the diagram drawn and adda a title and include the magnification at which you
made the observation.
(Total magnification is eyepiece lens magnification × objective lens magnification)
Then turn the objective disc to the medium-power lens and focus until the cells are clear and
distinct.
Finally turn the objective disc to the high-power lens and focus using the fine-focusing knob only.
Draw and label the details of the cells accurately.
Measure the length and breadth of two cells.
Place the glass slide in a jar of disinfectant.
Measuring a cell using a microscope
Calibrate the eyepiece graticule with the objective lens that will be used. Rotate the eyepiece so
that the eyepiece scale aligns with stage micrometer scale.
Count the number of divisions on the eyepiece graticule that are equivalent to a known length on
the stage micrometer slide and calculate the length of one eyepiece unit.
Now measure the size (length) of the cell using the eyepiece scale, multiply the total number of
divisions on the eyepiece scale covering the cell with length of one eyepiece unit.
CORE PRACTICAL 7: USE A LIGHT MICROSCOPE TO: TO MAKE OBSERVATIONS OF (i) TRANSVERSE
SECTIONS OF ROOTS, STEMS, LEAVES; (ii) PLANT TISSUES; (iii) IDENTIFY SCLERENCHYMA FIBRES,
PHLOEM, SIEVE TUBES, XYLEM VESSELS AND THEIR LOCATION

Objectives: to be able to use a microscope competently to observe biological specimens

To learn how to draw and label diagrams accurately
To be able to identify sclerenchyma fibre, phloem, sieve tubes and xylem vessels
Procedure:
Collect a piece of plant stem.
Cut several thin transverse sections of the stem using a sharp razor into a watch glass containing
water.
Select the thinnest section and place it on a slide and add a drop of water.
Add two drops of stain, toluidine blue on the section and leave it for 5 minutes.
Then place a coverslip and gently remove excess stain.
Examine the slide under the low power of microscope.
Bring the lens as close to the slide as possible while watching it from the side.
Then look through the eyepiece, focus using coarse focusing knob, moving the lens away from the
stage. (this avoids damage to the slide and the lens)
Use the fine focus until a clear view of the section is established.
Draw and label a simple outline plan of the section.

Repeat the same using a piece of root and leaf

Size of the tissues can be compared by using a micrometer slide and eyepiece graticule after
calibration.
Cross section of leaf

Stain is used to observe the cell

The thin sections of the plant stem and root do not absorb much light, so they are difficult to see
using a light microscope. The stain absorbs more light and making it easier to see the structures.
And all the cells are not stained. So, it is easier to differentiate them.
If too much stain is used it will absorb more light and it will be difficult to find out the differences
between cells.
Safety:
Take care when using razor: cut the section by keeping fingers away from the edge of the razor
Toluidine blue is corrosive: avoid skin contact with the stain. So, use gloves
CORE PRACTICAL: 6 PREPARE AND STAIN A ROOT TIP SQUASH TO OBSERVE THE STAGES OF MITOSIS

Objectives: to know how to prepare a temporary slide of a root tip to observe mitosis
To recognize the stages of mitosis in dividing cells
To identify hazards, associated risks and control measures for the procedure

Describe how to prepare a root tip squash so that chromosomes can be seen

o Remove last 5-10mm of root tips as tip contain meristematic cells.

o Place it in hydrochloric acid to soften tissue (breaks down the pectin holding cellulose cell
wall together).
o Transfer the root tip on to a slide.
o Add a drop of stain, orcein/ acetocarmine (to make the chromosome visible).
o Chop it with a sharp scalpel for better staining.
o Cover it with a coverslip
o Heat the slide to intensify the colour.
o Give vertical pressure with thumb to separate the cells (This makes it easier to see the
chromosomes and to identify the stages of division).
o Examine under the microscope on low power to identify the area of dividing cells.
o Then move to high power and identify the stages of the cell cycle in the field of view.
o Count the number of cells in undergoing mitosis and calculate the mitotic index using the
formula:
𝒏𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒄𝒐𝒏𝒕𝒂𝒊𝒏𝒊𝒏𝒈 𝒗𝒊𝒔𝒊𝒃𝒍𝒆 𝒄𝒉𝒓𝒐𝒎𝒐𝒔𝒐𝒎𝒆𝒔
Mitotic index= × 𝟏𝟎𝟎
𝒕𝒐𝒕𝒂𝒍 𝒏𝒖𝒎𝒃𝒆𝒓 𝒐𝒇 𝒄𝒆𝒍𝒍𝒔 𝒊𝒏 𝒕𝒉𝒆 𝒗𝒊𝒆𝒘

1
The information the cell counts give about the stage of mitosis
The cell counts show the relative duration of each stage in the cell cycle. The longer a phase, the
more cells are likely to be going through that phase at any point in time.
Safety
1M HCl is corrosive. Wear eye protection, lab coats and disposable gloves, to avoid contact.
Acetocarmine is corrosive. Wear goggles, lab coats and gloves, to avoid direct contact.
Take care with scalpels and always carry them in a tray. Always cut away from you.
Be careful while warming the slide, so as to not get burned. Could use a slide warmer to avoid
burning.
Suggest two variables that should be controlled when the pieces of plant root tissue are selected
Age of plant- fewer cells in mitosis in older plants
Tissue taken from tip of a growing root / same part of the plant- tip region contain meristematic
cells
Soil type / growth medium of plants – lack of minerals, phosphate affect DNA replication. Fewer cells
in mitosis in plants growing in poor soil
Role of mitosis in the life of an organism
Mitosis produces identical daughter cells for replacement of damaged cells and repairing damaged
tissues.
Also increase in cell number leads to growth.
Stages of mitosis

2
 CORE PRACTICAL: 8 DETERMINE THE TENSILE STRENGTH OF PLANT FIBRES

Tensile strength
The force / weight / strain / stress / tension a fibre can take without breaking
OR
The force / weight / strain / stress required to make a fibre break
Procedure to compare the tensile strength of a plant fibres

o Take same length of plant fibre.

o Measure diameter with micrometre screw gauge.
o Find out the area of the fibre using the formula πd2÷4
o Control environmental variable such as temperature and humidity
o Add masses until fibre breaks /measure the mass that breaks the fibre
o Repeat and find the mean /average mass needed to break the fibre.
o Repeat the procedure in case of anomalous result.
o Calculate tensile strength using the formula breaking force(N)/cross-sectional Area (m2),
where F=mass × area
o Repeat the same procedure using other fibres as well and compare the result.
How stem cross-sectional area could be determined
A transverse section of the fibre is cut using a sharp blade and ensure that the section is flat
Place the fibre on a microscope slide and cover it with coverslip and mount it on the stage.
Eye piece graticule is calibrated with stage micrometer.
Using the calibrated data calculate the diameter of the fibre
Using this area is calculated using πr2
OR
Measure the diameter of the fibre using a micrometer screw gauge.
Quote area of a circle formula πd2÷4 or πr2

1
Safety measures
Use a container with soft fabric in it so that the weight cannot land on foot /cannot cause injury
Safety glasses should be worn to protect eyes from fibre when it breaks
Variables to be kept constant and How
Length (of fibre)- use ruler so all the fibre will be of the same length
Diameter (of fibre)- select after measuring with micrometer screw gauge
Temperature- thermostatically controlled room/ thermostatically controlled water bath.
Nature of fibre- use fibre from the same species/ same plant
Soaking time- using stop clock for a stated time.
Humidity- air conditioning / beaker of water in sealed chamber to keep the atmosphere
saturated.

2
CORE PRACTICAL 9 : INVESTIGATE THE ANTIMICROBIAL PROPERTIES OF PLANTS, INCLUDING ASEPTIC
TECHNIQUES FOR THE SAFE HANDLING OF BACTERIA

OBJECTIVES: To successfully compare the effect of plants on microbial growth

To understand the safety issues of microbiological work and how to apply good aseptic
techniques
Antimicrobial properties: Ability to kill / slow down growth of Bacteria or micro-organisms
Compare the antimicrobial property of plant
o wash hands with soap and water and disinfect the bench area
o Take same mass of plant material, garlic, and mint (2gm)
o place them in a mortar and grind into a paste with a pestle separately
o Add 10cm3 of alcohol/ distilled water into the paste.
o Take same sized sterile paper discs for all the extracts and soak them in respective plant
extract.
o One paper disc dipped in alcohol/ distilled water is used as a control.
o Take sterile agar medium in a petri dish and allow it to cool.
o Spread the microorganism evenly on the agar plate using sterile spreader.
o Place the paper discs dipped in plant extract and the control disc in the agar plate using
sterile forceps.
o Lid should be taped cross way.
o Incubate it upside down at 20 to 30°C for 24 hours.
o Measure the diameter/ area of zone of inhibition using a ruler or a graph paper.
o Compare the result. (the larger the zone of inhibition the greater the antimicrobial effect)
Making plant extract

Crush 3gm of plant material.

Add 10cm3 of denatured ethanol and shake it well.

Describe how the bacteria should be added to the Petri dish.

o Use sterile equipment, such as a pipette and spreader, to

avoid contamination.
o Spread bacteria uniformly over agar and mixed in with
molten agar

1
Explain why the lid should be secured cross way.

This reduces contamination.

Also allows aerobic conditions and reduces the growth of
anaerobic bacteria being cultured.

Incubation temperature used

Between 20°C to 30°C
Temperatures above 30 °C are closer to human body temperature, so there is a risk of incubating
human pathogens which could leads to infection.
Suitably prepared petri dish
Distribute bacteria on to agar using sterile spreader.
Use single bacterial strain.
Agar and petri dishes should be sterilised.
Why the discs should be sterilised before being soaked in the extract
This prevents contamination by microbes.
That could be harmful / pathogenic
It could compete with those on the plate and affect its growth.
Using sterilised disc making the investigation valid.
Why clear zones are found around discs

Anti-microbial solution diffused out of disc.

This killed the bacteria / inhibits bacterial growth

Finding out the size of clear zone

By graph paper tracing
Count the squares and calculate area. Then, subtract the area of paper disc.
OR
Measure mean diameter using a ruler. Subtract the diameter of the paper disc. Using the radius
calculate the diameter using the formula πr2.

2
How you would calculate the mean diameter of the clear zone which is not a circle.
Record several measurements
Divide the total diameter obtained by number of measurements to get mean diameter.
Explain why measuring the area of the clear zone around each disc is more accurate than measuring
the diameter of the clear zone
The shape of the clear zone may be irregular, so the diameter may not represent an accurate
measurement.
Measuring the area of the clear zone enables a fair comparison to be made between discs.
Variables to be controlled and How
o Temperature (of incubation) - use incubator set at 25°C
o pH of agar - by using a buffer solution of pH 7
o Mass / source of material- use a balance and take 2g of material/ use same plant
o Volume of extract- use a graduated pipette and take same volume (0.1cm3)
o Size of filter paper disc – use same hole punch
o Extraction method – grind the material for 2 mins.
Suggest a reason for including the control discs in this investigation
To allow comparison and to check that the paper discs themselves were not responsible for the
inhibition of growth. The control discs enable us to be sure that the only factor that was different
was the independent variable.
Sterile technique is important in microbiology. Explain why this is the case even in this activity,
where the bacterial species have been selected because they are harmless to humans.
Aseptic techniques are vital in microbiology to ensure there is no contamination of cultures by
microorganisms from the environment, and that people and the environment are not contaminated
by the microorganisms being handled. Even cultures thought to be low risk should be treated with
caution because:
o bacteria may mutate to form pathogenic strains
o our knowledge of the hazards may be incomplete
o the culture may have become contaminated.