CP 1 - Benedict's test for reducing sugars and iodine test for starch

• Benedict’s test for reducing sugars

o Variables:
▪ Independent variable: The concentration of the reducing sugar
solutions
▪ Dependent variable: The colour of the solution after the
addition of Benedict’s reagent
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Test tubes, test tube rack, tongs
▪ Water bath at 80°C
▪ Timer
▪ Benedict’s reagent
▪ Distilled water
▪ Stock solution of a reducing sugar
o Procedure:
▪ Plan various concentrations of a reducing sugar from a stock
solution with the help of dilutions planning up to 10 cm³ each.
▪ Pipette only 5 cm³ of the solutions into a separate test tube.
▪ Add 2 cm³ of Benedict’s reagent into the test tubes.
▪ Place all of the test tubes gently and carefully into the water
bath.
▪ Start a timer for 2 minutes.
▪ After 2 minutes, carefully remove the test tubes with the help of
tongs and place them on a test tube rack.
▪ Observe the colour of the solution in each test tube.
o Observations and analysis:
▪ Precipitate is formed when Benedict’s reagent as it contains
copper ions which gets reduced from Cu²⁺ to Cu⁺, causing the
colour change.
▪ The colours of the solution can be compared to an existing
colour scale from blue (which contains no reducing sugar at all)
to brick red (which contains a very high concentration of
reducing sugars) in order to estimate the concentration of the
 reducing sugars in the original solution.

• Iodine test for starch

o Variables:
▪ Independent variable: The concentration of the starch solutions
▪ Dependent variable: The colour of the solution after the
addition of the Iodine solution
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Test tubes, test tube rack, tongs
▪ Iodine solution
▪ Distilled water
▪ Stock solution of starch
o Procedure:
▪ Plan various concentrations of starch from a stock solution with
the help of dilutions planning up to 10 cm³ each.
▪ Pipette only 5 cm³ of the solutions into a separate test tube.
▪ Add 2 cm³ of Iodine solution into the test tubes.
▪ Observe the colour of the solution in each test tube.
o Observations and analysis:
▪ Negative result: Brown colour (No starch present)
▪ Positive result: Blue colour (Moderate amount present) to Black
colour (High concentration present)
CP 2 - Investigating the vitamin C content of food and drink
• Variables:
o Independent variable: The fruit juices
o Dependent variable: The volume required to decolourise the 1% DCPIP
solution.
• Equipment:
o Laboratory safety wear: Lab coats, eye goggles, latex gloves.
o 1% DCPIP solution
o 1% Vitamin C solution
o Different types of fruit juices
o Test tubes
o 1 cm³ and 5 cm³ syringes
• Procedure:
o Using a syringe, take 1 cm³ of 1% DCPIP solution and place into a test
tube.
o Using a syringe, take 5 cm³ of 1% Vitamin C solution and release it into
the test tube dropwise (drop by drop).
o Make sure the 1% Vitamin C solution drops into the DCPIP solution
directly—do not let it stick to the side of the test tube.
o Continue to do this until the DCPIP decolourises (turns colourless).
o Record the volume of 1% Vitamin C solution used to decolourise.
o Repeat this experiment for different types of fruit juices.

• Observations and analysis:

 o The DCPIP solution is blue in colour.
o Vitamin C is an antioxidant.
o The vitamin C found in fruit juices can be used to reduce the DCPIP
solution, causing the decolourisation.
o 1 cm³ of the vitamin C solution contains 10 mg of vitamin C. With this,
it is possible to calculate the concentration of Vitamin C required to
decolourise a solution of DCPIP by using the following formula:
▪ concentration of vitamin C in fruit juice=volume of standard
solutionvolume of fruit juice×concentration of standard
solutionconcentration of vitamin C in fruit juice=volume of fruit
juicevolume of standard solution ×concentration of standard
solution
▪ The standard solution would be the experiment using 1%
Vitamin C solution.
o Example:
▪ In an investigation, the volume of 1% vitamin C solution needed
to decolourise the DCPIP solution was 1.4 cm³ and the volume
of apple juice solution needed was 3.2 cm³.
CP 3 - Investigating membrane properties including the effect of temperature
and alcohol
• Introduction:
o Beetroot cells contain a pigment called betalain which gives the
vegetable its distinct red/pink colour which found inside each cells’
vacuoles.
o Betalain is too large to pass through the cell membrane which makes
beetroot perfect for experimenting on membrane permeability, using
temperature and alcohol.
• Testing the effect of temperature on membrane permeability using
Beetroot:
o Variables:
▪ Independent variable: The temperature
▪ Dependent variable: The percentage transmission of light
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
 ▪ Test tubes, test tube racks and tongs
▪ Scalpel
▪ Cork borer
▪ Ruler
▪ Distilled water
▪ Water bath
▪ 10 cm³ syringe
▪ Forceps
▪ Colorimeter
▪ Cuvette
▪ Timer
o Procedure:
▪ A set of 8 different temperature water baths is prepared e.g.
(0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C ,70°C)
▪ Beetroot skin is peeled and cut into a cylindrical shape in equal
lengths using a cork borer, scalpel and ruler.
▪ Ensure all sizes of samples of beetroot are equal
▪ Wash the beetroots with distilled water
▪ Add 10 cm³ of water to all beakers in the water bath
▪ Place each sample into a different water bath for 15 minutes
▪ After 15 minutes, using forceps carefully remove the beetroot
without destroying/damaging it
▪ Place a blue/green filter into the colorimeter.
▪ Zero the colorimeter using a cuvette filled with only distilled
water.
▪ Place each test tubes liquid into the cuvette and place in a
colorimeter and record the percentage transmission of light.
▪ The colorimeter must be zeroed with distilled water in between
each time the different test tube liquids are tested.
o Observation and analysis:
▪ < 0°C: ice forms between the membrane which causes holes in
the membrane that are large enough to allow the pigment to
leak out.
 ▪ Between 0°C to 40°C: the phospholipids in the cell membrane
gain kinetic energy, allowing it to move more to allow larger
gaps that allow the pigment to leak out.
▪ 40°C: the proteins on the cell membrane start to denature,
changing shape and causing gaps in the cell membrane, causing
pigment to leak out.

• Testing the effect of alcohol on membrane permeability using Beetroot:

o Variables:
▪ Independent variable: The concentration of alcohol
▪ Dependent variable: The percentage transmission of light
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Test tubes, test tube racks and tongs
▪ Scalpel
▪ Cork borer
 ▪ Ruler
▪ Distilled water
▪ Ethanol
▪ 10 cm³ syringe
▪ Colorimeter
▪ Cuvette
▪ Timer
o Procedure:
▪ Using a boiling water bath, set each piece in a different test
tube. Each test tube contains different concentrations of alcohol
e.g. (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%), but 10 cm³.
▪ Beetroot skin is peeled and cut into a cylindrical shape in equal
lengths using a cork borer, scalpel and ruler.
▪ Ensure all sizes of samples of beetroot are equal
▪ Wash the beetroots with distilled water
▪ Place each beetroot pieces into the different test tubes and
leave for 15 minutes.
▪ After 15 minutes, using forceps carefully remove the beetroot
without destroying/damaging it
▪ Place a blue/green filter into the colorimeter.
▪ Zero the colorimeter using a cuvette filled with only distilled
water.
▪ Place each test tubes liquid into the cuvette and place in a
colorimeter and record the percentage transmission of light.
▪ The colorimeter must be zeroed with distilled water in between
each time the different test tube liquids are tested.
o Observation and analysis:
▪ Ethanol and the phospholipids on the cell membrane are both
non-polar, allowing the ethanol to dissolve the phospholipids,
 which leads to gaps that allow the pigment to leak out.

CP - 4 Investigating the effect of temperature, pH, enzyme and substrate

concentration on the initial rate of reaction

• Introduction:
o Four factors are being investigated in this enzyme-controlled
experiments: Temperature, pH, enzyme and substrate concentration.
o Milk is used as it contains a protein called casein that gets digested by
enzymes like trypsin.
o Milk is white and opaque and as the protein is digested, the solution
gets more translucent which allows for light to pass through more
easily.
o A colorimeter is used to see how much light has passed through the
solution, so that we can measure the rate of reaction of the
 experiment.

• Experiment with temperature:

o Variables:
▪ Independent variable: The temperature
▪ Dependent variable: The absorbance value
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Test tubes, test tube rack and tongs
▪ Water bath
▪ Timer
▪ Thermometer
▪ Pipette
▪ Distilled water
▪ Colorimeter
▪ Cuvette
▪ 2% Skimmed milk
▪ 1% trypsin solution
o Procedure:
▪ Set the separate water baths to different temperatures on your
chosen range of temperatures. (eg. 0°C, 10°C, 20°C, 30°C, 40°C)
▪ Pipette 2 cm³ of 1% trypsin solution into 5 test tubes and place
in each water bath
▪ Pipette 2 cm³ 2% skimmed milk into 5 different, clean tubes and
place in each water bath
 ▪ Leave them for 5 minutes, so that they equilibrate (reach the
required temperature)
▪ Pipette 2 cm³ of 1% trypsin and 2 cm³ distilled water into a
cuvette to zero the colorimeter
▪ Remove the 2 cm³ of milk at the first temperature and pour it
into a second cuvette.
▪ Pour the 2 cm³ of 1% trypsin at the first temperature into the
second cuvette.
▪ Quick and gently shake the mixture and then place it into the
colorimeter.
▪ Record the absorbance immediately and then follow by 15-
second intervals of recording for 5 minutes or until there is little
change.
▪ Repeat from the step at which the colorimeter is zeroed for
different temperatures.
o Observation and analysis:
▪ As the temperature increases, the initial rate of reaction also
increases as the particles gain energy, so that the substrate and
enzyme collide and bind more frequently to form enzyme-
substrate complexes.
▪ At its optimum temperature, it is the maximum point of rate of
reaction and increasing the temperature beyond this causes its
rate of reaction to decrease as more kinetic energy breaks the
bonds in the secondary and tertiary structure of the enzyme.
▪ This changes the shape of the enzyme and it’s active site and
causes the substrate to no longer fit.
 ▪ The enzyme is denatured.

• Experiment with pH:

o Variables:
▪ Independent variable: The pH value
▪ Dependent variable: The absorbance value
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Timer
▪ Pipette
▪ Distilled water
▪ Colorimeter
▪ Cuvette
▪ 2% Skimmed milk
▪ 1% trypsin solution
▪ Buffer solutions
o Procedure:
▪ Pick your chosen range of pH and then buffer solutions for
them
▪ Pipette 1 cm³ of 1% trypsin, 1 cm³ of buffer solution and 2 cm³
distilled water into a cuvette to zero the colorimeter
 ▪ Pipette 1 cm³ of 1% trypsin and 1 cm³ of the first buffer solution
into a second cuvette.
▪ Pipette 2 cm³ of 2% skimmed milk into a third cuvette.
▪ Combine the second and third cuvette.
▪ Quickly and gently shake the mixture and place it into the
colorimeter.
▪ Record the absorbance immediately and then follow by 15-
second intervals of recording for 5 minutes or until there is little
change.
▪ Repeat from the step at which the colorimeter is zeroed for
different pH values.
o Observation and analysis:
▪ Any change in the pH value of the medium around the enzyme
will cause the bonds to be damaged.
▪ This will change the 3-D shape of the enzyme and deform the
active site. Therefore, the substrate will not be able to fit into
active site thus the reaction slows down or stops.
▪ The enzyme gets denatured.

• Experiment with enzyme concentration:

o Variables:
▪ Independent variable: The enzyme concentration
▪ Dependent variable: The absorbance value
o Equipment:
 ▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Timer
▪ Pipette
▪ Distilled water
▪ Colorimeter
▪ Cuvette
▪ 2% Skimmed milk
▪ 1% trypsin solution
o Procedure:
▪ Take the 1% trypsin solution as a stock solution and create
different concentrations using dilutions planning up to your
chosing (eg. 1%, 0.8%, 0.6%, 0.4%, 0.2%)
▪ Pipette 2 cm³ of trypsin solution and 2 cm³ of distilled water into
a cuvette to zero the colorimeter
▪ Pipette 2 cm³ of 2% skimmed milk into a second cuvette.
▪ Pipette 2 cm³ of the first trypsin solution into the second
cuvette.
▪ Quickly and gently shake the mixture and place it into the
colorimeter.
▪ Record the absorbance immediately and then follow by 15-
second intervals of recording for 5 minutes or until there is little
change.
▪ Repeat from the step at which the colorimeter is zeroed for
different concentrations.
o Observation and analysis:
▪ As the concentration of enzymes is increased, there are more
available active sites for substrates to fit into.
▪ More enzyme-substrate complexes are formed, more products
are formed and the rate of reaction is increased.
▪ At the beginning, the limiting factor is the enzyme
concentration.
▪ Once all substrates have formed enzyme-substrate complexes,
a further increase in enzyme concentration will have no effect
on the rate of reaction.
 ▪ At this point, the limiting factor is the substrate concentration.
• Experiment with substrate concentration:
o Variables:
▪ Independent variable: The substrate concentration
▪ Dependent variable: The absorbance value
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Timer
▪ Pipette
▪ Distilled water
▪ Colorimeter
▪ Cuvette
▪ 2% Skimmed milk
▪ 1% trypsin solution
o Procedure:
▪ Take the 2% skimmed milk as a stock solution and create
different concentrations using dilutions planning up to your
chosing (eg. 2%, 1%, 0.75%, 0.5% 0.25%)
▪ Pipette 2 cm³ of 1% trypsin solution and 2 cm³ of distilled water
into a cuvette to zero the colorimeter
▪ Pipette 2 cm³ of the first skimmed milk solution into a second
cuvette.
▪ Pipette 2 cm³ of 1% trypsin solution into the second cuvette.
▪ Quickly and gently shake the mixture and place it into the
colorimeter.
▪ Record the absorbance immediately and then follow by 15-
second intervals of recording for 5 minutes or until there is little
change.
▪ Repeat from the step at which the colorimeter is zeroed for
different concentrations.
o Observation and analysis:
▪ As the concentration of substrate increases, the greater the
chance of collision with enzymes, the more enzyme-substrate
 complexes formed, the more products formed and the greater
the rate of reaction.
▪ At the beginning, the limiting factor is the substrate
concentration.
▪ Once all the enzymes are occupied and working at their
maximum rate (𝑉𝑚𝑎𝑥Vmax ), a further increase in substrate
concentration will have no effect on the rate of reaction.
▪ At this point, the limiting factor is the enzyme concentration.
• Why do we measure the initial rate?
o There are enough substrate molecules present to ensure that
substrate concentration is not a limiting reactant
o The substrate concentration is only known at the beginning of the
reaction
CP 5 - Using a light microscope to observe and make labelled drawing of animal
cells
• Introduction:
o Eyepiece graticule is a transparent scale with 100 divisions, which is
placed in the microscope eyepiece.
o Stage micrometer is a transparent ruler which is used to calibrate the
eyepiece graticule.
o Each division on the stage micrometer is 0.01mm.
o You must also be familiar with parts of the microscope.
 o Stains are used to make cell structures visible when viewed under a
microscope like Methylene blue.
o In this experiment, we will be use cheek cells which can be easily
gathered and observed in a school setting.
• Calibration of eye piece graticule
o Procedure:
▪ Place a micrometer slide on the stage of the microscope
▪ Focus on the micrometer scale using the low-power objective
lens.
▪ Move the slide and rotate the eyepiece to align the scales of the
eyepiece graticule and the micrometer scale in the field of view
▪ Count the number of divisions on the eyepiece graticule and
compare them to a known length on the micrometer scale to
figure out the length of one eyepiece unit
▪ Repeat steps 1-3 with the medium-power and high-power
objective lens.
 ▪ Magnification: number of times larger an image is compared to
the real size of the object.
• Depends on the power of the objective and eyepiece lens
used.
▪ Calculating magnification:

• Observing and drawing cells:

o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Microscope
▪ Eyepiece graticule
▪ Stage micrometer
▪ Microscope Slide
▪ Coverslip
▪ Pipette
▪ Cotton bud
▪ Disinfectant solution
▪ Pen
▪ Paper
o Procedure:
 ▪ Wash your hands
▪ Take a cotton bud and swab the inside of the your cheeks.
▪ Take the cotton bud and rub it against a microscope slide.
▪ Place the cotton bud into a disinfectant solution.
▪ Add a few drops of the stain (eg. Methylene blue) to the slide.
▪ Place a coverslip on it.
▪ Turn on the microscope and set it to low power.
▪ Bring the lens as close to the slide as possible.
▪ Using the coarse adjustment on the microscope, slowly move
away from the slide.
▪ After, use the fine adjustment to focus until cells are clearly
visible.
▪ Sketch the cell membrane, using a pen and paper.
▪ Set the microscope to high power.
▪ Focus on the indivual cell and make and then draw the
organelles.
CP 6 - Preparing a root tip squash to observe the stages of mitosis
• Equipment:
o Laboratory safety wear: Lab coats, eye goggles, latex gloves.
o Garlic clove
o Distilled water
o 1 mol of hydrochloric acid (HCl)
o Water bath
o Pipette
o Scalpel
o Forceps
o Timer
o Microscope slide
o Coverslip
o Microscope
o Paper towel
o Acetic orein or Toluidene blue
• Procedure:
 o Suspend a garlic clove over water for a few days until its root start to
grow
o After roots start to grow, use a scalpel to carefully to cut the root tip
o Set the water bath to 60°C.
o Add 10 cm³ of 1 mol of HCl to a test tube and place it into the water
bath for 5 minutes
o After 5 minutes, add the freshly cut root tip into the test tube for 5
minutes
o After 5 minutes, remove the test tube and use forceps to carefully
extract the root tip.
o Bring the root tip under a tap and wash with distilled water
o Place the root tip on a paper towel to dry
o Place the root tip on a microscope slide and add a few drops of the
stain: Acetic orein or Toluidene blue
o Use forceps to macerate the root tip and place a coverslip and cover
the whole thing in paper towel.
o Press on the coverslip in order to squash the root.
o Remove the coverslip and place the slide into the microscope for
viewing.
• Observation and analysis:
o The stages of mitosis can be seen.
o In the plain of view, the mitotic index can also be calculated.
CP 7 - Using a light microscope to observe tranverse sections of roots, stems and
leaves, plant tissues and identify parts and their location
• Equipment:
o Laboratory safety wear: Lab coats, eye goggles, latex gloves.
o White tile
o Razor blade
o Tuloidene blue
o Distilled water
o Microscope slide
o Coverslip
o Microscope
o Plant stem, root and leaf
o Tweezer
• Procedure for stem, root and leaf:
o Place the specimen on a white tile.
o Wet the specimen, the tile and razor blade.
o Hold the specimen firmly and cut transerve sections sections of the
specimen as thinly as possible.
o Use tweezers to pick them up carefully and place on a microscope
slide.
o Add a few drops of the stain Tuloidene blue.
o Place a coverslip on it gently.
o View the specimen under a microscope in low power.
o Bring the lens as close to the slide as possible.
o Using the coarse adjustment on the microscope, slowly move away
from the slide.
o After, use the fine adjustment to focus until cells are clearly visible.
o Sketch a plan diagram of the plane of view using a pen and paper and
label each part.
CP 8 - Determining the tensile strength of plant fibres
• Introduction:
o Tensile strength is the maximum amount of force a fiber can take
before failing.
o Factors affecting tensile strength:
 ▪ Length of fibre
▪ Humidity of fibre
▪ Cross-sectional Area
▪ Type of plant fibre
o In this experiment, the factor that is being tested the type of the fibre,
but all the aforementioned factors can be tested in this experiment.
• Measuring tensile strength:
o Variables:
▪ Independent variable: The type of plant fibre
▪ Dependent variable: The tensile strength
o Equipment:
▪ Laboratory safety wear: Lab coats, eye goggles, latex gloves.
▪ Different types of plant fibre
▪ Scalpel
▪ Ruler
▪ Retort stand
▪ Cushioning
▪ Hook and mass
▪ Box
o Procedure:
▪ Prepare at least 3 fibrous strings per plant type.
▪ Cut them, so that they’re all the same size. (eg. 15 cm)
▪ Set up retort clamps, so that the prongs facing each other and
place a box with cushioning inside between the two reort
clamps.
▪ Tie a fibrous string between each other and place a hook in the
middle.
▪ Continuously add 10g masses until the fibrous string breaks and
record the mass.
▪ Repeat for the same fibrous strings until they run out and then
continue the same steps for the other types of plant fibres.
▪ The experiment is conducted similarly to test other factors that
affect tensile strength of plant fibres.
o Observation and analysis:
 ▪ Most plant fibre have very strong tensile strength due to their
composition.
▪ Plant stems from which fibrous strings are extracted have
sclerenchmya, which is what gives plant stems its strength, so
that they can handle situations in nature like strong winds or
other environmental events.
▪ Plant cells found in the plant stem have cell wall which is made
of cellulose that are arranged at angles and held together by
hydrogen bonds, adding to its strength.
▪ Certain plant cells have secondary cell walls that contain lignin
in it, which provides additional strength to the plant stem.
CP 9 - Investigating the antimicrobial properties of plants, including the aseptic
techniques for safe handling of bacteria
• Introduction:
o Most plants have some chemical compounds in them that have been
found to cause an antimicrobial affect to a certain or multiple type of
bacteria.
o In this experiment, different plants are being investigated for their
antimicrobial properties to see which is the most effective against a
type of bacteria.
o The area of zone of inhibition will be observed in the experiment,
which is the area around which there are no bacterial growth from
where the plant specimen was placed.
o This meant that the plant specimen had diffused from its origin to the
surrounding and has killed or inhibited the growth of bacteria in the
area.
o This experiment can also be repeated, but using different solvents and
using the same plant to see which solvent works best on a type of
bacteria.
• Experiment on the antimicrobial properties of different plants:
o Variables:
▪ Independent variable: The type of plant
▪ Dependent variable: The area of zone of inhibition
o Equipment:
 ▪ Mint
▪ Garlic
▪ Mortar and pestle
▪ Forceps
▪ Paper disk
▪ Ethanol
▪ Petri dish with agar jelly
▪ Marker pen
▪ Bacterial culture
▪ Incubator
▪ Tape
▪ Beaker
▪ Timer
o Procedure:
▪ Take mint and garlic and pound them in separate mortars and
pestles.
▪ Add ethanol and mix it into the pounded mash.
▪ Place paper disks of the same size and thickness into the
mixture and let it soak for 5 minutes.
▪ Prepare a beaker full of alcohol and soak 2 paper disks
separately for 5 minutes.
▪ Take the cover of the petri dish and divide it into quarters and
label each with letters A-D.
▪ Assign which letters are for one mint, one garlic and two control.
▪ Spread the bacterial culture onto the agar jelly and close the lid
quickly.
▪ Remove the paper disks and open the lid and place it in the
centre of the assigned quarter and quickly close it again.
▪ Tape the lid securely, but allow enough gap to allow air flow for
aerobic respiration.
▪ Place in an incubator upside down at 25°C for 24-48 hours.
▪ After the period is over, remove the Petri dish.
▪ Use a ruler and measure the diameter zone of inhibtion.
 ▪ Make sure that all the equipment and work area are thoroughly
cleaned using disinfectant solution before and after the
experiment.
▪ Calculate the area of zone of inhibition using the radius (half of
the diameter) by this formula:
• Area of zone of inhibition=𝜋×𝑟2Area of zone of
inhibition=π×r2
o Observation and analysis:
▪ Allowing air flow for aerobic respiration is a safety feature as if
there is no airflow then anaerobic bacteria can grow which can
be harmful to us.
▪ Setting the incubation temperature to safe temperature like
25°C disallows harmful bacteria to grow inside the Petri dish
▪ Placing the Petri dish upside down disallows condensation to
form, which could invalidate the experiment if it starts dripping
down on the agar jelly if it were the right side up.
▪ The greater the area of zone of inhibition, the more effective the
plant is against this type of bacteria.