Unit description

Introduction Students are expected to develop experimental skills, and a knowledge and understanding of experimental
techniques, by carrying out the core practicals and other recommended practical investigations and experiments while they
study units 1 and 2. This will require them to work safely, produce valid results and present data in the most appropriate
format. This unit will assess students’ ability to apply their knowledge and understanding of experimental design, procedures
and techniques developed throughout units 1 and 2.

General instructions:
Make sure you draw tables and graphs correctly. The independent variable should always go in the left
hand column of a results table and on the horizontal axis of a graph. Numbers in tables, including
calculations, should only be reported to the limits of the least accurate measurement. Data on graphs
should be scaled so that the graph fills more than half of the available space

1. SERIAL DILUTIONS PLANNING

In the exam, you may be asked to calculate the volume of stock solution needed to dilute it to the right
concentration.

A formula to help calculate this is as follows: C1V1=C2V2

 C₁ stands for the concentration of the stock solution

 V₁ stands for the volume of stock solution required
 C₂ stands for the concentration needed at the end
 V₁ stands for the volume of the diluted solution.

Example:

The starch stock solution is 2% concentration and student A needs 0.5% concentration and 10 cm³ of
starch solution. Calculate the volume of 2% starch stock solution and water needed to make 0.5%
concentration, 10 cm³ of starch solution.

2.5cm3 from the stock solution.

Final volume is 10cm3. What can u add

 Plan how you will use the stock 2% glucose solution to make the following five concentrations of
glucose solution: 2%, 1%, 0.5%, 1.5% and 0.25%. Write your plan in the space below.
2. BENEDICT’S REAGENT METHOD:

Benedicts test: https://www.youtube.com/watch?v=5t9PfEEgL9Y

Semi-quantitative Benedict's test: estimating the concentration of reducing sugars

Benedict’s solution can be used to carry out a semi-quantitative test on a reducing sugar solution to
determine the concentration of reducing sugar present in the sample

 It is important that an excess of Benedict’s solution is used so that there is more than enough
copper (II) sulfate present to react with any sugar present
 The intensity of any colour change seen relates to the concentration of reducing sugar present in
the sample
 A positive test is indicated along a spectrum of colour from green (low concentration) to brick-
red (high concentration of reducing sugar present)
Variables:
Independent variable: The concentration of the reducing sugar solutions

Dependent variable: The colour of the solution after the addition of Benedict’s reagent

What is Benedict’s reagent:

Benedict's reagent (often called Benedict's qualitative solution or Benedict's solution) is a chemical
reagent and complex mixture of sodium carbonate, sodium citrate, and copper(II) sulfate pentahydrate.
It is often used in place of Fehling's solution to detect the presence of reducing sugars

Significance of this test:

Benedict’s Test is used to test for simple carbohydrates. The Benedict’s test identifies reducing sugars
(monosaccharide’s and some disaccharides), which have free ketone or aldehyde functional groups.
Benedict’s solution can be used to test for the presence of glucose in urine.

Must watch: https://www.youtube.com/watch?v=d6tHWPW5WLM&list=PLPvPDSW-g-

69HJ_sjAMBe7meJy1CDWQ5N

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.

TESTING FOR NON-REDUCING SUGARS

 Some sugars don't react with Benedict's reagent; these are known as non-reducing sugars

 A few extra steps can be taken to test for non-reducing sugars using Benedict's reagent

Method

1. Add dilute hydrochloric acid to the sample and heat in a water bath that has been brought to the
boil

2. Neutralise the solution with sodium hydrogencarbonate

o Use a suitable indicator (such as red litmus paper) to identify when the solution has been
neutralised, and then add a little more sodium hydrogencarbonate as the conditions need
to be slightly alkaline for Benedict’s test to work

3. Then carry out Benedict’s test as normal

o Add Benedict’s reagent to the sample and heat in a water bath that has been boiled – if a
colour change occurs, a reducing sugar is present

Results and analysis

 The addition of acid will hydrolyse any glycosidic bonds present in any carbohydrate molecules

 The resulting monosaccharides left will have an aldehyde or ketone functional group that
can donate electrons to copper (II) sulfate (reducing the copper), allowing a precipitate to form\
3. IODINE TEST FOR STARCH

 Iodine solution can be used to carry out a semi-quantitative test on a food sample to determine
the concentration of starch present in the sample
 The intensity of any colour change seen relates to the concentration of starch present in the
sample
 A positive test is indicated along a spectrum of colour from dark brown (low concentration) to
blue-black (high concentration of starch present)

Variables:

Independent variable: The concentration of the starch solutions

Dependent variable: The colour of the solution after the addition of the Iodine solution

Observations and analysis:

Negative result: Brown colour (No starch present)

Positive result: Blue colour (Moderate amount present) to Black colour (High concentration present)

Review: https://youtu.be/xFLuYKy3m1g?si=SD_ZWwt_NYZSTTSt
 Vitamin C is found in green vegetables, fruits, and potatoes

 It is essential for a healthy diet

 The chemical name for vitamin C is ascorbic acid

o Ascorbic acid is a good reducing agent and therefore it is easily oxidised

 Methods for the detection of vitamin C involve titrating it against a solution of an oxidising
agent called DCPIP

o DCPIP is a blue dye that turns colourless in the presence of vitamin C

o Titration is a method of chemical analysis that involves determining the quantity of a

substance present by gradually adding another substance; in this case the concentration of
vitamin C is determined by gradual addition of a vitamin C solution to DCPIP
2,6-dichlorophenolindophenol (DCPIP) can be used to estimate the concentration of vitamin C in food.
DCPIP is blue when dissolved in water, is red in acid conditions, and is reduced by ascorbic acid (vitamin
C) to a colourless compound.

Variables:

Independent variable: The fruit juices

Dependent variable: The volume required to decolourise the 1% DCPIP solution.

DCPIP test: https://www.youtube.com/watch?v=fHCcf6ZH4LQ

Observations and analysis:

 The DCPIP solution is blue in colour.

 Vitamin C is an antioxidant.

 The vitamin C found in fruit juices can be used to reduce the DCPIP solution, causing the
decolourisation.

 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:
 o The standard solution would be the experiment using 1% Vitamin C solution.

 The volume of vitamin C solution required to decolourise DCPIP should decrease as the
concentration of the vitamin C solution increases

 The results of the experiment can be plotted on a graph of volume of vitamin C needed to
decolourise DCPIP against the concentration of vitamin C

 The line of best fit for such a graph is known as a calibration curve; unknown substances can be
compared to it to gain an estimate of their vitamin C concentration

 The calibration curve produced from this experiment can be used to estimate the concentration
of vitamin C in fruit juices
 Beetroots are root vegetables that appear red because the vacuoles in their cells contain a water
soluble red pigment called betalains. These pigment molecules are too large to pass through cell
membranes. The effect of either temperature or alcohol on membranes can be investigated.

Colorimeter: The amount of pigment in a sample can be measured using a piece of equipment known as
a colorimeter

 A colorimeter is a machine that passes light through a coloured liquid sample and measures how
much of that light is absorbed by or transmitted through the sample
 This gives an indication of how much of the colour is present in the solution, e.g. more absorbance
or less transmission both indicate a darker coloured solution

Must watch: https://www.youtube.com/watch?v=UzwOfFvLtJ0

Testing the effect of temperature on membrane permeability using Beetroot

Variables:

Independent variable: The temperature/ concentration of alcohol,

Dependent variable: The percentage transmission of light

Watch: https://www.youtube.com/watch?v=HDigBCZJMwQ
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

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.
Results

 The general pattern you would expect to see is that as temperature increases, membrane
permeability also increases
 As temperature increases, the phospholipids within the cell membrane move more because they
have more kinetic energy; increased movement means the phospholipids are not as tightly packed
together, increasing the permeability of the membrane
 Temperature affects the 3D shape of proteins; at high temperatures membrane proteins
denature, increasing the permeability of the membrane
 The volume of water inside the cells expands, putting pressure on the membrane and damaging
membrane components; this can increase membrane permeability
 If experimenting with temperatures below 0 ℃, membrane permeability may be increased once
the cells have thawed again
 Ice crystals that form can pierce the cell membrane, making it highly permeable
 Limitations

 Cuvettes may differ in thickness; a thicker cuvette will absorb slightly more light than a thinner
cuvette
 Solution: use the same cuvette for every reading, or repeat the investigation many times and find
a mean
 Note that scratched cuvettes can have the same impact on absorbance as thicker cuvettes
 The beetroot pieces may not be identical in size and shape, meaning some test tubes could contain
slightly more beetroot tissue than others
 Solution: cut the discs as accurately as possible using a scalpel and ruler, and repeat each
investigation several times to find a mean
 Some parts of beetroot tissue have more pigment in their cells than others
 Solution: conduct several repeats, using different parts of the beetroot and find a mean

Variations

 The impact of alcohol on membrane permeability could also be investigated

 The expected trend is
 As alcohol concentration increases the permeability of the membrane increases
 This is because alcohol dissolves lipids in the cell surface membrane so the membrane loses its
structure and the beetroot pigment can leak out

Review of enzymes: https://www.youtube.com/watch?v=rlH1ym916Fo

Temperature

 Enzymes have a specific optimum temperature – the temperature at which they catalyse a
reaction at the maximum rate
 Lower temperatures either prevent reactions from proceeding or slow them down:
 Molecules move relatively slow
 Lower frequency of successful collisions between substrate molecules and active site of enzyme
 Less frequent enzyme-substrate complex formation
 Substrate and enzyme collide with less energy, making it less likely for bonds to be formed or
broken (stopping the reaction from occurring)

 Higher temperatures speed up reactions:

o Molecules move more quickly

o Higher frequency successful collisions between substrate molecules and active site of
enzyme

o More frequent enzyme-substrate complex formation

o Substrate and enzyme collide with more energy, making it more likely for bonds to be
formed or broken (allowing the reaction to occur)

 However, as temperatures continue to increase, the rate at which an enzyme catalyses a

reaction drops sharply, as the enzyme begins to denature:

o Bonds (eg. hydrogen bonds) holding the enzyme molecule in its precise shape start
to break

o This causes the tertiary structure of the protein (ie. the enzyme) to change

o This permanently damages the active site, preventing the substrate from binding

o Denaturation has occurred if the substrate can no longer bind

o Very few human enzymes can function at temperatures above 50°C

  This is because humans maintain a body temperature of about 37°C, therefore even temperatures exceeding 40°C
will cause the denaturation of enzymes

 High temperatures causes the hydrogen bonds between amino acids to break, changing the conformation of the
enzyme

Must watch : https://www.youtube.com/watch?v=5bIOwLlT3IA

Must watch: https://www.youtube.com/watch?v=A8Ts4V_osvo

Investigating the effect of temperature, pH, enzyme and substrate concentration on the initial rate
of reaction
 Four factors are being investigated in this enzyme-controlled experiments: Temperature, pH, enzyme and substrate concentration.

Milk is used as it contains a protein called casein that gets digested by enzymes like trypsin.

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.

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.

What is colorimeter: https://www.youtube.com/watch?v=DgFfWn6PssE

1) EXPERIMENT WITH TEMPERATURE: 2) EXPERIMENT WITH pH

Variables: All enzymes have an optimum pH or a pH at which they operate best
Independent variable: The temperature Enzymes are denatured at extremes of pH
Dependent variable: The absorbance value Hydrogen and ionic bonds hold the tertiary structure of the protein
(ie. the enzyme) together
Observation and analysis: Below and above the optimum pH of an enzyme, solutions with an
As the temperature increases, the initial rate of reaction also increases excess of H+ ions (acidic solutions) and OH- ions (alkaline solutions) can
as the particles gain energy, so that the substrate and enzyme collide cause these bonds to break
and bind more frequently to form enzyme-substrate complexes. This alters the shape of the active site, which means enzyme-
At its optimum temperature, it is the maximum point of rate of reaction substrate complexes form less easily
and increasing the temperature beyond this causes its rate of reaction Eventually, enzyme-substrate complexes can no longer form at all
to decrease as more kinetic energy breaks the bonds in the secondary At this point, complete denaturation of the enzyme has occurred
and tertiary structure of the enzyme. Where an enzyme functions can be an indicator of its optimal
This changes the shape of the enzyme and it’s active site and causes environment:
the substrate to no longer fit. Eg. pepsin is found in the stomach, an acidic environment at pH 2 (due
The enzyme is denatured. to the presence of hydrochloric acid in the stomach’s gastric juice)
Pepsin’s optimum pH, not surprisingly, is pH 2.
 When investigating the effect of pH on the rate of an enzyme-
catalysed reaction, you can use buffer solutions to measure the
rate of reaction at different pH values:
o Buffer solutions each have a specific pH
o Buffer solutions maintain this specific pH, even if the
reaction taking place would otherwise cause the pH of
the reaction mixture to change
o A measured volume of the buffer solution is added to
the reaction mixture
 o This same volume (of each buffer solution being used)
should be added for each pH value that is being
investigated

3) EXPERIMENT WITH ENZYME CONCENTRATION 4) EXPERIMENT WITH SUBSTRATE CONCENTRATION

 Enzyme concentration affects the rate of reaction  Substrate concentration affects the rate of reaction
 The higher the enzyme concentration in a reaction mixture,  The higher the substrate concentration the faster the rate
the greater the number of active sites available and of reaction
the greater the likelihood of enzyme-substrate complex  More substrate molecules means more collision between
formation enzyme and substrate so the more likely an active site will be
 As long as there is sufficient substrate available, the initial used by a substrate
rate of reaction increases linearly with enzyme concentration  The is only the case up until a certain concentration of
 If the amount of substrate is limited, at a certain point any substrate, at which point a saturation point is said to have been
further increase in enzyme concentration will not increase the reached
reaction rate as the amount of substrate becomes a limiting o At this point all active sites are occupied and
factor increasing the substrate concentration will not affect
the rate of the reaction
 Substrate concentration will decrease over time (if no new
substrate is added)
 The rate of reaction will therefore decrease over time
 This means the initial rate of reaction will be
fastest throughout the reaction
Practical: Investigating the rate of enzyme reactions
 The progress of enzyme-catalysed reactions can be investigated by:

o Measuring the rate of formation of a product

o Measuring the rate of disappearance of a substrate

 There are many enzymes that can be used in this practical; some common examples are catalase, amylase and protease

 The initial rate of reaction can be calculated to determine the effect of changing enzyme or substrate concentrations
o The initial rate of reaction is at the start of the reaction

o You can calculate the initial rate of reaction using a graph of results showing volume of product/substrate against time

o Draw a tangent to the graph through the origin

o Calculate the gradient of the tangent - this is the initial rate of reaction
 Many biological structures are too small to be seen by the naked eye

 Optical, or light, microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and
studied

o Light is directed through a thin layer of biological material that is supported on a glass slide

o This light is focused through several lenses so that an image is visible through the eyepiece

o The magnifying power of the microscope can be increased by rotating the higher power objective lens into place
Video tutorial of a light microscope: https://www.youtube.com/watch?v=tVcEEw6qbBQ&t=17s

1. Using an eye piece graticule with a microscope to make measurements and understand
the concept of scale
 Eyepiece graticule and stage
micrometer
https://www.youtube.com/watch?v=P
W4s42K-zio&t=156s

Using an eyepiece graticule & stage micrometer

 A graticule is a small disc that has an engraved ruler

 It can be placed into the eyepiece of a microscope to act as a ruler in the field of view, so is sometimes known as an eyepiece
graticule

 As an eyepiece graticule has no fixed units it must be calibrated for the objective lens that is in use

o The graticule in the eyepiece remains the same size when the magnification of the microscope is altered, so recalibration
is needed at each viewing magnification

 Calibration of the eyepiece graticule is done a microscope slide with an engraved scale known as a stage micrometer

 By using the eyepiece graticule and the stage micrometer together, the size of each graticule unit can be calculated

o After this is known the graticule can be used as a ruler to measure objects in the field of view
*Calculate the size of the units of the eyepiece graticule in the image below.

Note that the large divisions in the top half of the image show the stage micrometer and that each stage micrometer division is 1 mm
across.

Step 1: Observe the number of eyepiece unit divisions per micrometer unit

In the image, the stage micrometer has three lines

Each micrometer division has 40 eyepiece graticule divisions within it

Step 2: Calculate the size of each eyepiece graticule unit

40 graticule divisions = 1 mm (1000 µm)

1 graticule unit = 1000 ÷ 40 = 25 µm

o An object that spanned five eyepiece graticule units could therefore be measured as follows

5 x 25 µm = 125 µm
Practice:
2. Using a light microscope to make observations and labelled drawings of suitable
animal cells;

Watch: https://www.youtube.com/watch?v=uEkMcKl_3_8

Drawing cells
 To record the observations seen under the microscope, or from photomicrographs taken, a labelled biological drawing is often
made

o Biological drawings are line drawings that show specific features that have been observed when the specimen was viewed

 There are a number of rules or conventions that are followed when making a biological drawing

o The drawing must have a title

o The magnification under which the observations shown by the drawing are made must be recorded

o A sharp pencil should be used

o Drawings should be on plain white paper

o Lines should be clear, single lines with no sketching

o No shading

o The drawing should take up as much of the space on the page as possible

o Well-defined structures should be drawn

o The drawing should be made with proper proportions

o Label lines should not cross or have arrowheads and should connect directly to the part of the drawing being labelled
 o Label lines should ideally be kept to one side of the drawing in parallel to the top of the page, and should be drawn with
a ruler

o Only visible structures should be drawn; not structures that the viewer thinks they should be able to see!

 Drawings of cells are typically made when visualizing cells at a higher magnification power

 Plan drawings that show the arrangement of cells within a tissue or organ are typically made using samples viewed under lower
magnifications

 Individual cells are never drawn in a plan diagram

Practice:

Identify structure labelled as 1,2,3,4,6,7,10and 13.

 CORE PRACTICAL 6

Prepare and stain a root tip squash to observe the stages of mitosis.
Learning tip
●● Before starting this practical, you may need to remind yourself about
parts of the microscope and how to use the instrument
Procedure

To see mitosis in action, you need to look at living cells. Garlic bulbs grow

roots that have actively dividing cells in their tips, in a region called the

meristem (see figure A). Each cell has only eight chromosomes, so it is

relatively easy to see the chromosomes once they have condensed. In order

to see the chromosomes inside the cells, you need to separate the cells and

spread them out into a layer that is ideally just one cell thick. Plant cells are

glued together by a middle lamella of pectins. Hydrochloric acid will break

down the pectins that hold the cell together. Acetic orcein will stain the

chromosomes dark red and fix the cells, stopping mitosis.

You should examine your preparation carefully, looking for cells undergoing

different stages of mitosis. Identify the different stages by comparing your

preparation with labelled pictures or photographs of cells during mitosis. Bear

in mind that mitosis is a dynamic process, so cells may have been fixed in

transition from one stage to the next. You will have to interpret what you see.

Make sure you follow all necessary safety precautions. You should also

complete your own risk assessment before starting this practical.

 Growth in plants occurs in specific regions called meristems

 The root tip meristem can be used to study mitosis

 The root tip meristem can be found just behind the protective root cap

 In the root tip meristem, there is a zone of cell division that contains cells
undergoing mitosis

 Pre-prepared slides of root tips can be studied or temporary slides can be prepared
using the squash technique (root tips are stained and then gently squashed, spreading
the cells out into a thin sheet and allowing individual cells undergoing mitosis to be
clearly seen)
Apparatus

 Roots of onion or garlic

 Microscope slide

 Cover skip

 1 M hydrochloric acid

 Stain (acetic orcein, toluidine blue for example)

  Paper towel

 Scalpel blade

 Cutting tile

 Water bath at 60oC

 Boiling tube

 Optical microscope

Method

1. Garlic or onion (Allium cepa) root tips are most commonly used (the bulbs can be
encouraged to grow roots by suspending them over water for a week or two)

2. Prepare a boiling tube of 1M hydrochloric acid and place in a water bath at 60oC for 10
minutes

3. Remove the tips of the roots (about 1cm) and place in the warmed hydrochloric
acid for 5 minutes

4. Rinse the tips well in cold water using a pipette and blot dry with a paper towel

5. Cut approximately 2mm off the tip and place onto a microscope slide
 6. Add a drop of a suitable stain (eg. warm, acetic orcein, which stains chromosomes a deep
purple)

7. The stained root tip is gently squashed on a glass slide using a blunt instrument (eg. the
handle of a mounting needle or a cover slip using your fist to gently apply pressure in a
rolling motion)

8. View the slide under the microscope

9. Cells undergoing mitosis (similar to those in the images below) can be seen and drawn

10. Annotations can then be added to these drawings to show the different stages of
mitosis

Results

 You should be able to observe cells in different stages of mitosis from the stained
meristem

 Video tutorial:
https://www.youtube.com/watch?v=K2_FZRdjQH4&ab_channel=BiologyPracticalsandRev
isionBiologyTutor



 Video tutorial: https://www.youtube.com/watch?v=5-

ur7bWqlDQ&t=20s&ab_channel=ThomasTKtungnung

 Answers to the questions:

 1.The root tip is heated with acid to break up the tissues into individual cells. The
cellulose walls of plant cells are held together by pectins such as calcium pectate.
Treatment with hydrochloric acid breaks this down.
 2. Pressing the preparation will separate the cells in the meristem tissue into individual
cells in a single layer. This makes it easier to see the chromosomes and to identify the
stages of division.
 3. 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. 4.
Mitosis produces identical daughter cells for growth, replacement and repair.
CORE PRACTICAL 7: USE A LIGHT MICROSCOPE 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

 In order to identify tissue types within stems, a permanent pre-prepared slide could
be used

 Alternatively, a section of a plant stem could be cut and stained before preparing
a temporary slide

Apparatus

 Plant stem

 Scalpel

 Suitable stain

 Microscope slide
  Cover slip

 Forceps

 Dissecting needle

 Light microscope

 Gloves

Method

1. Cut a very thin cross-section of the stem using a scalpel

2. Carefully transfer each section into a dish containing a suitable stain and leave for one
minute

o A stain such as toluidine blue O (TBO) will make xylem and sclerenchyma fibres
appear blue-green, while phloem will appear pinkish purple

3. Rinse off each section in water and mount onto a microscope slide, before adding a
cover slip (take care to lower the coverslip slowly over the sample from one side to the
other to avoid trapping air bubbles, which can then be mistaken for plant
tissues/structures)

4. View under a microscope and adjust the focus to form a clear image
 5. Make a labelled drawing of the positions of the xylem vessels, phloem sieve tubes and
sclerenchyma fibres

Plan diagrams

 When drawing tissue plan diagrams you need to:

o Read the instructions carefully

 o Draw a large diagram

o Use a sharp pencil and do not shade (including the nucleus)

o Use clear, continuous lines (no sketching)

o When using an eyepiece graticule, use it to ensure you have correct proportions or
if you are not using a microscope then endeavour to keep the proportions between
tissues to scale

 If drawing from a low-power image:

o Do not draw individual cells

o Read the question carefully as you may only have to draw a portion of the image

o Include the magnification on the drawing

 If drawing from a high-power image:

o Draw only a few of the required cells

o Draw the cell wall of the plant cells

o Include the magnification on the drawing

 When labelling, remember:

o Use a ruler for label lines (and scale lines if appropriate)

 o Label lines should stop exactly at the structure (do not use arrows)

o Don't cross label lines over each other

o Label all tissues and relevant structures that are requested


Video:practice drawing
https://www.youtube.com/watch?v=CduEel3vgDM&ab_channel=GenieusHub
CORE PRACTICAL 8: DETERMINE THE TENSILE STRENGTH OF PLANT FIBRES
 The tensile strength of a fibre refers to the maximum load it can carry before breaking

o For example, this information would be important when determining the strength of a rope made of plant
fibres
Apparatus

 Plant fibres

 Retort stand (clamp stand)

 Clamp

 Weights

Method

1. The fibre should be attached to a clamp stand

2. Attach a weight on the other end of the plant fibre

3. Carefully continue to add one weight at a time until the fibre breaks

4. Record the mass at which the fibre broke

5. This represents the tensile strength

6. To increase the accuracy of your results, this process should be repeated with more samples of the same plant fibre

o These values can be used to calculate the mean tensile strength for the fibre

o It is important to ensure that the fibres are all of the same length and that all other variables are kept
constant
Video watch:
https://www.youtube.com/watch?v=bCc2WAsYSZ4&list=PLHa3EHIQpBFk3XX9CORxdGpCj0oYIWFy1&index=7&ab_channel
=AyaMansour
CORE PRACTICAL 9: INVESTIGATE THE ANTIMICROBIAL PROPERTIES OF PLANTS,
INCLUDING ASEPTIC AND TECHNIQUES FOR THE SAFE HANDLING OF BACTERIA

Part 1: Anti microbial properties of plants.

  Certain plant species have the ability to kill or prevent the growth of micro-organisms

 These antimicrobial properties can be incorporated into the development of new drugs

Apparatus

 Broth containing bacterial culture and nutrients

 Agar plate

 Pipette

 Plastic spreader

 Plant tissue

 Pestle and mortar

 Ethanol

 Funnel

 Glass beaker

 Filter paper

 Forceps

 Stopwatch

 Incubator

Method
1. Prior to this practical, bacteria would have been grown in a mixture of distilled water and nutrients, along with a
specific bacterial culture

o This mixture is called a broth

2. Transfer some of the bacteria from the broth onto an agar plate (which is a petri dish filled with agar jelly that will
serve as a growth medium for the bacteria) using a sterile pipette

3. Make sure the bacteria is evenly spread out by using a sterile plastic spreader

o Open the lid of the of the agar plate as little as possible when doing this to avoid contaminating the plate with
other fungi or bacteria present in the surrounding air

o Place the lid back on top of the agar plate immediately afterward to prevent contamination

4. To prepare the plant extracts, plant tissue must be dried and ground finely

5. This should be soaked in ethanol to extract the antimicrobial substances, after which it should be filtered

6. Equal sized discs cut from sterile absorbent paper should be dipped in the plant extract using sterile forceps

7. Leave the discs in the extract for the same amount of time to ensure that they absorb a similar amount of the plant
extract

o The disc that will serve as the control will only be dipped in ethanol

8. Space the discs out evenly on the agar plate, before taping the lid on, inverting the plate and incubating it at 25°C

o This temperature will ensure good bacterial growth without stimulating the growth of human pathogens

9. Incubate for 24 to 48 hours

Analysis

 The area around each disc where bacteria cannot grow is known as the clear zone

 The larger the clear zone, the more effective the antimicrobial properties of that plant extract was

 The size of the clear zone can be determined by measuring the diameter or by calculating the area (area = πr2)

 Repeat the experiment at least three times and calculate the mean of the results

Video watch:
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sicsandmathstutor.com%2F&source_ve_path=Mjg2NjY&feature=emb_logo
Part 2: Aseptic technique.
Aseptic technique is used to avoid contamination of the sample from outside substances such as microorganisms. This is
important to get reliable and repeatable data
 These techniques are important to use in order to prevent the bacterial cultures on the agar plate from being
contaminated by other micro-organisms or human pathogens from outside

 Contamination will have a negative impact on the growth of the bacteria under investigation

 When doing the investigation above, use the following aseptic techniques

 Wipe down surfaces with antibacterial cleaner both before and after experiment.

 Use a Bunsen burner in the work space so that convection currents draw microbes away from the
culture.

 Flame the wire hoop before using to transfer bacteria.

 Flame the neck of any bottles before use to prevent any bacteria entering the vessel (air moves
out so unwanted organisms don’t move in).

 Keep all vessels containing bacteria open for the minimum amount of time.

 Close windows and doors to limit air currents.