A.

S Biology
Unit 3
Booklet 2023

Prepared by
Dr.Sahar Haggag
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Unit 3 Basic Principles

❖ Outline of Any Experiment:
1- Independent variable: the factor you change ex. temperature:
2- Dependent variable: the factor you measure
ex. rate of the reaction /mass of product. ( always include SI unit)
3- Controlled variables kept constant.(for / ensures* Validity)
(not increase* validity) . They are factors that can affect your results.
-You need to give ( whenever possible)
a--Examples of controlled variables. (+/- suggested values).
b--How to control (apparatus).
c--Effect on the results if not controlled ( obligatory in unit 6 ).
-Mention controlled variables in:
a- organism: (same age , species ,mass,gender)
b- experiment: ex. same pH, volume and concentration of (enzyme).
c- surrounding conditions: (temperature, light intensity)
d- others : same type and mass of food provided to an organism.
4-Time scale : suitable to show the expected results.
5- Replications: Minimum 3 times at EACH….
(For reliability, NOT to increase xxx reliability)!
-(to spot anomalies , minimise the effect of individual variations,)
-to calculate the mean , show variability, SD, and allow statistical analysis).
6- Control: , Definition: Identical experiment, with identical apparatus and
conditions, without the factor you are testing (ex. without enzyme).
- Purpose of the control: Allows comparison –( Ensures* VALIDITY) to
be sure that your results are really due to this factor & no other factor.

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e.g.. Experiment result = 50 au ,, control result = zero au

Actual result of experiment is: 50 – zero = 50 au.
e.g. Experiment result = 50 au. Control result = 4 au.
Actual result of experiment is: 50 – 4 = 46 au.
❖ Validity : How TRUE/correct your results are?? This is achieved by:
1- Keep all controlled variables constant.
2- Use a control to compare.
3- Choose the correct accurate instrument to measure your parameter.
4- Non overlapping error bars→ SIGNIFICANT difference /correlation.
❖ Reliability: ( Can you trust the results) ?
1- Large sample size ( individuals / repetitions) to be representative.
Also anomalies can be identified and excluded.
2- Low Variability: Consistent /similar/ close results of repetitions,
/clustered around the mean → Small SD / narrow range / error bars.
3- Non overlapping error bars→ SIGNIFICANT difference or correlation.
4- Repeatable by other scientists, giving the same/similar results.
NB: (repeating does NOT make your measurements more reliable, it just
helps you to spot and remove anomalies, asses and judge the degree of
variability, hence reliability!)
• Random error: Unpredictable , occasional error in one* or more of the
measurements, usually caused by:
1- problem in the controlled variables , or
2 -inaccurate instrument.
-Ex. when weighing yourself, and standing differently on the scale,
or looking at the level of a liquid each time from a different angle, or
difficulty in standardising the pressure or force applied.
-Corrected by : Performing many repetitions, exclude the anomalies.

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• Systematic error: persistent error, always obtained, with the same , due
to poorly calibrated apparatus
- Corrected by: proper calibration of your apparatus, during the planning
stage, or preliminary work.
[ If a non-calibrated balance gives an extra 10 grams mass each time you
use it , ( reading is always 100 grams, while the real is 90 grams)→
(systematic error). This means your results are not accurate!!,
❖ Accurate: your measured results are close to the real/ actual/ standard
values. -How to improve accuracy :
1-Use a more sensitive instrument, and repetitions to minimize random
errors.
2-Calibrate your apparatus ( to remove systematic error).

-Qualitative result: describing what is present / not present. ex judging the

colour by the eye ex biuret test, -ve = blue, +ve purple
-Semi-Quantitative result:
ex. comparing the colour obtained to a colour chart.
Disadvantage of both of the above : subjective , inaccurate,
-Quantitative result: numerical/digital, accurate,
( result indicates exactly how much is present)
ex by using a colorimeter..
Advantage : objective, more accurate , can be used to draw a graph
and do statistical analysis.

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Conducting an Investigation

Preliminary work( Planning ):

1-Try the proposed method to see if it will work

2-To decide upon the best factors that would give meaningful results:
SELECT: Most suitable/best:

1- Organism: Type/species, source, age, ….

2- Independent variable: The most suitable range and intervals.
3- Dependent variable: (What will you measure?) Best parameter
e.g. mass, length for growth.
4- Apparatus: Choose the correct, most accurate, calibrated apparatus.
5- Method: Try different methods to see which works best.
6- Set most important controlled variables to ensure validity.
7- Consider the possible risks and safety precautions, ethical concerns.

• Ethical concerns: Explain clearly:

1- Consider the welfare of animals used, and the concept of animal rights
in vertebrates and invertebrates.
2- Ethical concerns of harming or disturbing the surrounding environment.
Subjects should give consent to participate.
3- Otherwise say: { There is no significant ethical concerns }

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A) Risks and Safety precautions (just examples)

Risk How to minimise it

Injury when cutting with a scalpel Cut away from one’s self , wear gloves
or knife and from broken
glassware.
Infection from bacterial cultures Use sterile equipment,,, see the experiment

Allergies, irritation, corrosion from Wear protective gloves, masks, goggles

chemicals e.g. enzymes, stains ….
Risk of fire from flammable Handle flammable substances away from
substances e.g. alcohol used near any fire or Bunsen burner, wear heat
Bunsen burner flame. resistant gloves. Heat indirectly, use electric
plate, water bath.

Splashes from heating liquids or Wear goggles

specs of broken fibres hitting the
eyes.
Insect bites or falling branches in Wear protective clothes
field ecological sampling studies
Harm from handling animals Wear protective gloves ,eye protection

(Go back to each Individual experiment for more safety precautions)

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B) Method: (Clear and detailed)

1- Identify ( mention) the independent variable : clearly.

2- State 5 specific values,(should be in the range and intervals to give

meaningful results) / or
-the presence and absence of the IV variable.

-Put your units clearly and apparatus used to set the values
(ex. incubator for temperature)

3- Identify the dependent variable: ( must be a measurable parameter,)

(ex dry mass/Length to measure growth )

State the apparatus used to measure it. -Use SI units.

5- Use a control to compare ( in most experiments ).

6- Describe your method briefly

-Mention and justify the tool or instrument used to measure the

dependent variable .

-Indicate any calibration if needed, ex that of colorimeter, or spirometer.

7 - Time scale: Suitable to show the results

8- Controlled variables ( in the organism, conditions,apparatus)

- Mention at least two ( in the organism, + chemicals used ).

- Put suitable values if possible.

- Mention how each is achieved ( at least one)

9- Replications: several times at EACH…. and used to get average.

reasonable number to ensure reliability ( too few →non-representative,
too many → not practical + time consuming)

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C) Recording, presenting and analysing the data:

➢ Recording : in clear tables with SI units.

➢ Presenting: GRAPH
➢ Analysing : Name + justify (reason) suitable statistical test.
-t- test: for difference between 2 or more groups ( independent variable
is discontinuous, ex. in males versus females , or in different countries)

- Spearman rank correlation: when the independent variable is

continuous, ex, increasing light intensity.

- Chi – square test for the difference between the observed and
expected (ex in genetic crosses).
-Interpretation: In all of them: If the calculated value is greater than the
critical value at 0.05 significance level, -→ then there
IS A SIGNIFICANT DIFFERENCE / CORRELATION!
-The closer the correlation coefficient to 1, the stronger the correlation.

D) Limitations (Mostly unavoidable!): .--> affects the validity!

❖ Difficulty in controlling all the variables: ex.
- Light intensity a field (a cloud passing).
- Genetic variation in organisms.
- Antibiotic molecules/ chemicals may have different sizes, so different
rates of diffusion through the agar.
❖ Difficulty in determining the end point or measuring the dependent
variable e.g. colour change in DCPIP.
❖ Cells overlapped in root tip squash.
❖ Behaviour of organisms in the lab may be different from real life.
❖ Difficulty in identifying the species when similar ones exist.

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GRAPHS

Axes: Correct orientation (choice):

• Independent variable on X axis (that you change).
• Dependent variable on Y axis (that you measure).
Labelling: Very clearly with SI units!!! ( IN BRACKETS )
Type of graph: - Bar , it the IV is discontinuous.
- Line , if the IV is continuous.
Scale: Take care!!
1- Use more than 50% (1/2) x axis , and also more* than 50% (1/2) y axis.
2- If all your readings are within a narrow range of large numbers e.g. 201,
203, 207, 209, you can interrupt ( split / Truncate) the axis from the zero,
or start from 200.( However, You Cannot do that in BAR graphs!!!)
3- SD or RANGE ..VIMP!!!, to be considered when deciding your scale,
(maximum value , see sample old syllabus!)
Plotting the data: Rules:
1- Use clear dots / crosses ± circles. If bars are used = equal width !.
2- If 2 or more sets of data = use a Key and different symbols / shades.
3- Never go out of the graph paper (white area).
4- Never extrapolate forwards or backwards !! (you lose one mark if you do)
[Never assume that at zero X, the value of Y was zero !]
5-The points joined clearly by broken line ( ruler !) (not free hand)!
6- Don't use line of best fit unless you are asked to do so.
( Fairly equal points on either side of the line).
• Error bars (variability) using S.D. or Range ,
Only plotted if the examiners asks for it ! (don't volunteer) (too much time).

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Describing graphs:
❖ Graph 1
M
Risk of CVD (au)

Smoking level (au)

➢ Trend, Steepness, Manipulation

1] Describe the relation between smoking level & CVD risk in males and females
1-In Both males and females, as smoking level increases ,the risk of CVD increases.
(+ve correlation).
2- Linear increase in both.
3-Risk in males is always higher than females .
4-Steeper increase in males than in females.
5-Manipulation: Subtract, ratio , % (choose the easiest). not just copy figures !
ex. At smoking level 2 au, the risk in males is higher than females by 4 au. ( never
forget the unit!!!) fixed the 2 au on the X axis ,and subtracted 8 - 4 on the Y axis
-wrong: At smoking level 2 au, the risk in males is 8 au while that in females is 4 au
-wrong: the risk in males by - - - - XX (because you didn’t mention females) !!
2] Give evidence from the graph that there are other factors/ genetic factors
linked to increased risk of CVD -Even non smokers (smoking level zero!) have some
risk of CVD in both males & females.
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❖ Graph 2
In this graph, points of trend and manipulation are
Risk
the same as graph 1, however, since both lines are
parallel, they have same steepness !! (point 4 )

Smoking level

Error Bars (variability)

Show the degree of spread of the results of the repetitions around the mean.
• Error bars can be obtained by:
a) Range: simply difference between highest & lowest results of the repetitions.
(However, one anomaly would give a wide error bar based on the “Range”.,,,so
better use SD→
b) Standard deviation: S.D. shows the average degree of spread of the data around
the mean, considering the sample size ( The number of repetitions.)
It is calculated by a certain equation ( later). This is more objective, as not greatly
affected by only one or two anomalies within large number of repetitions.

S.D. of the same

Range experiment &
same results

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A) Overlap of error bars ( significance)

- When assessing whether there is /is not a “ SIGNIFICANT
difference/correlation” between groups ex. blood pressure with and without
using a certain drug.
overlap

No
overlap

   

- No overlap of error bars - Overlap of error bars

- No common values bet. gp 1 & gp 2 - Common values bet. gp 1 & gp 2
- The highest values of gp are
- similar to the lowest values of gp2
- There is a significant difference - Non* significant difference
between the results of the 2 groups.
- You can make a valid conclusion→ - May be due to chance.
This drug may be effective. -

Confirm by statistical tests in A2 !!

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B) Size of error bars (variabilty)

-Narrow error bars -Wide error bars
-Low variability, -High variability,
→ - High reliability → - Low reliability

mean 10 mean 10

(A) (B)

The mean of both groups can be the same, but in (A), the
results of repetitions are more similar/close/clustered to the
mean, → lower variablity so more reliable!!

Tabulation:
1- Independent variable → first column.
2- Dependent variable in the second.
3-Units in headings only* , between brackets ,use SI units.
4-Include columns for the repetitions if required, and the mean.
5- Use horizontal lines to separate the data.
6- Use consistent number of decimal places. Ex if 2.87 in the data, the
zero has to be 0.00.
7-
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❖ Diluting a stock solution:

Making serial dilutions: With a dilution factor 2:

1- Add a certain volume from the original stock solution (ex 5 cm3)
2- Add EQUAL volume of distilled water ( also 5 cm3).
3-Total volume would be 10 cm3.
4- Do this repeatedly,,,Ex. obtained concentrations would be :
100%, 50%, 25%, 12.5%, 6,25%.....

𝒕𝒐𝒕𝒂𝒍 𝒗𝒐𝒍𝒖𝒎𝒆
• Dilution factor =
𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒔𝒕𝒐𝒄𝒌 𝒕𝒓𝒂𝒏𝒔𝒇𝒆𝒓𝒆𝒅
10
=2
5

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What would be the dilution factor be in the examples below?

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Q) What is meant by molar solution of glucose?

- It is the molecular weight of glucose in grams dissolved in distilled water and
made up to [ 1 dm³ ].
Q) How can you prepare serial dilutions from a stock of glucose solution of
concentration 1 mol /dm³?

Apply the formula C₁ V₁ = C₂ V₂

C₁ is the concentration of stock solution
V₁ is the volume you are going to take from the Stock to be diluted.
C₂ is the concentration of the solution you are going to prepare.
V₂ is the volume of the solution you are going to prepare.
Suppose you want to prepare 10 cm³ with the following dilutions respectively

0.5, 0.25, and 0.1 mol dm̄ ³ from the stock (1 mol dm-3)

A)for first dilution (0.5 mol dm-3)

C₁ V₁ = C₂ V₂
1 X V1 = 0.5 X 10
0.5 𝑥 10
V₁ = = 5 cm-3 from the stock, then make them up to 10 cm³ by distilled
1
water.
B)For second dilution [0.25 mol dm̄ -3),
C₁ V₁ = C₂ V₂
1 x V₁ = 0.25 x 10
V₁ = 2.5 cm-3 from the stock, then make them up to 10 cm³ by distilled water.
C)For third dilution [0.1 mol dm-3]
C₁V₁ = C₂ V₂
1 x V₁ = 0.1 x 10
V₁ = 1 cm³ from the stock, then make them up to 10 cm³ by distilled water

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Questions:
1. Describe how you would make 50 cm³ of a glucose solution of concentration
0.05 mol dm̄ ³ from a glucose solution of 0.1 mol dm-3 ?
C₁ V₁ = C₂ V₂
0.1 x V₁ = 0.05 X 50
0.05 𝑥 50
V₁ = = 25 cm³
0.1
-Measure out 25 cm³ from the stock [0.1 mol dm-3], make up to 50 cm³ by adding
25 cm³ distilled water.
2. Starting with stock solutions of 10% glucose and 2% sucrose how would you
make 100 cm³ of a mixture of final concentration of 1% sucrose and 1% glucose?
Stock glucose solution: C₁ V₁ = C₂ V₂
10 x ? = 1 x 100
𝟏𝟎𝟎𝒙𝟏
V₁ = = 10 cm³ from stock glucose solution
𝟏𝟎
Stock sucrose solution: C₁ V₁ = C₂ V₂
2 x V₁= 1 x 100
𝟏𝟎𝟎𝒙𝟏
V₁ = = 50 cm³ stock sucrose solution.
𝟐

Add 10 cm³ of 10% glucose to 50 cm³ of 2% sucrose and make up to 100

cm³ with distilled water.

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Experiments
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Experiments Unit 1
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Core practical 1: Semi quantitative estimation of reducing*

sugar in foods/drinks.
Obtaining the juice : crush, the food sample with distilled water in a
blender, then filter using a narrow sieve to obtain the juice.

- Benedict’s test: Add excess* Benedict’s reagent* to the solution*

(juice) you are testing and boil* in a water bath* for a few minutes.
-- If a reducing sugar is present, the solution will change colour , from
blue to:→ - Low concentration → green yellow precipitate
- Medium concentration→ orange precipitate
- High concentration → Red-brown(brick red)ppt
- Negative ----→ remains blue!!
The intensity of the red color is directly related to the concentration of
the reducing sugar.

➢ HOW MEASURED:
(estimate!!!)
1- Colour chart Comparing the
colour obtained to a colour
chart done using reducing
sugar solutions of known
concentration.
2- Measure the time taken for
the colour to change (stop
watch) The less the time→the higher the concentration.
 -3-

• More accurately :
3- Use a colorimeter
measure light absorbance/transmission→numerical results.
4- Mass of the precipitate: Filter the ppt and dry , and use a digital
sensitive balance.
➢ Repeat the same experiment several times with each* type of food
and calculate the average
➢ Controlled variables kept constant : food =same→
variety, mass, storage conditions, concentration of the solution).

➢ Safety: 1-Use heat resistant gloves to avoid skin burns.

2-Use goggles to avoid splashes.
• Test for non-reducing sugar: if the result of the Benedict test is -ve,
there may sugar present , but a non reducing one (ex. Sucrose)
-Acid hydrolysis first: Use a fresh sample. Boil first with dilute HCL,
( this breaks the glycosidic bonds in the disaccharide forming two
monosaccharides). Neutralise the acid with some Na HCO3, then repeat
-Benedict test, it would give a +ve result→ brick red ppt.
How to prepare different concentrations : ,,each volume 10 cm3
Given 100% glucose solution

Concentration Volume of Volume of Total volume of

of glucose glucose stock distilled water ( the
solution (%) solution(cm3) cm3) solution(cm3)
0 0 10 10
20 2 8 10
40 4 6 10
60 6 4 10
80 8 2 10
100 10 0 10
 -4-

• Test for Starch: Use iodine solution.

+ve = blue black, -ve = yellow brown.
• Test for protein (Biuret test): add a few drops of NaOH , then a few
drops of Cu2 SO4 , +ve = purple , -ve = blue.
• Test for fat : Mix with water and shake, if +ve = milky emulsion.

Core practical 2: Estimating vitamin C content of foods

Obtaining the juice : crush, the food

sample with distilled water in a blender,
then filter using a narrow sieve to obtain
the juice.

A. Qualitative method:
1. Use 1 cm3 of 1% concentration of blue DCPIP in a test tube.
2. Titrate* juice* of fruit or vegetable over it drop by drop and shake
gently between the drops till the color turns from
blue to colourless!!.
3. Determine the volume* of juice needed to decolorise this fixed
volume and concentration of DCPIP.
a-Comparative estimate: The higher the volume of juice needed
to decolourise DCPIP → the less the vitamin content!! Or→
b-Compare it with “Vit C calibration curve”-> numerical value.
4. Repeat the experiment several times at each*(…) and get the
average ( for reliability),plot a graph of the results.
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5. All controlled variables kept constant (for validity).

- DCPIP: Same volume and concentration .

- Fruit /vegetable: Same species, age, storage conditions, mass.

-Limitations: difficulty in determining the exact end point of colour

change ( human error)

B. Quantitative method: Using ‘standard vitamin C solution’

e.x. -Given: 1 cmᵌ of 1% DCPIP needs 6 mg vitamin C to be decolorized.

- In the experiment: (Measured):

-1 cmᵌ of 1% DCPIP needs 2 cmᵌ of fresh orange juice to be decolorized.

-So 2 cmᵌ orange juice must also contain 6 g vitamin C!!!
- So , Fresh orange juice content of vitamin C per cmᵌ is:

2 cmᵌ---> 6 mg vitamin C.
1 cmᵌ---> X mg vitamin C
𝟔𝒙𝟏
X= = 3 mg vitamin C per cmᵌ
𝟐

This method is better as you can use it to draw a graph , or do

statistical analysis to test significance

➢ Other independent variables:

- Another type of food juice.
- Different method and temperature of storage.
- Different method of cooking. Fresh-/cooked food juice.
- Compare 2 fruits from different sources/ countries….ect.
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➢ What decreases Vit C content:

- Heat : breaks down Vit C.
- Prolonged storage decrease Vit C content.
- Cooking in large volumes of water: Vit C is water soluble,,
- Cutting into small pieces→ larger total surface area → faster loss.

Juice tested Volume required Vit C content ( mg cm3-1 )

(cm3)
Grapefruit juice 1.61 3.8
Orange juice 2.12 2.8
Fresh lemon juice 1.73 3.5
Bottled lemon 24.00 0.25
juice

Comment on the data in the table

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Core practical 3a : Effect of temperature on membrane

permeability :
The sap vacuole of beet root contains a large* molecule pigment that
cannot* diffuse out unless the membranes are damaged*.

1- Cut equal sized discs from a single* fresh beet root (using a
cork borer and slicer /or a knife and ruler).
2- Rinse well with running distilled water (and blot dry to
remove any pigment released due to cutting). (validity)
3- Put one disc in 10 cmᵌ distilled water and put the tube in an
electric water bath at: I.V. = 10◦C ,20,30,40,50,60◦C .
4- Leave for 20 minutes in the water bath.
5- Remove the beetroot disc, shake, and put a sample of the
water in a clean colorimeter CUVETTE.
6- Measure the colour intensity using a calibrated
Colorimeter: (Calibrated= adjusted to read zero
absorbance using clear distilled water)
7- Light absorption ( D.V.): The greater the colour
intensity→the greater the light absorption , meaning the
higher the membrane permeability, and greater damage.
 -8-

8- Reliability : Replicate several times at EACH temperature and

calculate the average , plot a graph.
9- Controlled Variables ( ensure validity):
- Same beet root source/variety/ age/ storage conditions.
- Same, surface area, size of the discs.
- Same duration of time. - Same volume of water.
- Same light filter in colorimeter (e.g. Green filter in this case).
10-Control: same
experiment with identical
conditions but with no
beet root disc (to
compare).
11- Safety: wear lab coat
and gloves, to avoid
staining , and heat
resistant gloves with high
water temperature.

-Qualitative: The colour can be compared to a colour chart, but this is

subjective, inaccurate.
-Quantitative: The Colorimeter ( light absorbance) : gives numerical
accurate data, allowing drawing of graphs and statistical analysis.
 -9-

○Theoretical: increasing temperature→damage cell surface membrane

and membrane of the sap vacuole.
1- Membrane proteins are denatured, leaving wide gaps, pigment
leaks/diffuses out. …
2- High temperature increases kinetic energy of the phospholipids,
they move more, so more gaps and so the pigment can diffuse and leak
out of the vacuole .
3b: Effect of different alcohol concentrations on the
permeability of the cell membrane:

Same steps as before!.

3-Put one disc in each of 20, 30, 40, 50 ,60, 70 % alcohol.
4-Replicate 3 times at each concentration of alcohol and get
the average.
Controlled Variables: Same as before, in addition to:
Same temperature , same type* of alcohol.
○Theoretical: Phospholipids disperse/dissolve in alcohol
(organic solvent), leaving gaps , so the pigment can diffuse out.

-The higher the concentration of alcohol→ the wider* the gaps

between the phospholipids→ so MORE pigment diffuses out .

NB The tonoplast (sap vacuole membrane) is the same structure of

the cell membrane.
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Core practical 4: Enzymes: Independent variables: Changing:

1-Temperature→ electrostatic water bath.
2-pH→ Buffer solution.
3-Substrate concentration: Use different concentrations of hydrogen
peroxide : 20,30,40,50,60,70,%. Or of skimmed milk.
4-Enzyme concentrations. make serial dilutions.
❖ Effect of different enzyme concentrations on the rate of the
reaction:
1. Put skimmed milk suspension in test tube A.
2. Put trypsin enzyme solution (protease) in test tube B.
3. Allow them to reach 30◦C, separately for 5 minutes in water bath
(acclimatise). Mix together, and immediately start the stopwatch.
4. Dependent variable: Measure the time needed for the milk to
become clear, using a colorimeter !!
( calibrated by Trypsin + distilled water)
5. Independent variable : Use different concentrations of enzyme
trypsin :1%, 0.8%, 0.6%, 0.4%, 0.2% .
6. Reliability: Replicate experiment several times at each*
concentration and calculate average. Plot graph of results
7. Validity: Keeping all other controlled variables constant.
- Same pH , using a buffer solution;
- Same temperature 30◦C using an electrostatic water bath.
- Same volume and concentration of milk suspension.
- Same volume of enzyme solution.
8. Use a control: to compare ( validity) : same experiment with same
conditions , without enzyme.
 - 11 -

9. Risks: enzyme may cause skin allergy / irritation.

alkaline/acidic buffer solutions can be corrosive.
Safety: - Use gloves and minimize skin and eye contact with
enzymes. -If heating is involved take care of burning hands, wear
heat resistant gloves (only in effect of temperature on enzyme
activity experiment )

How to dilute: 1 % trypsin stock solution in order to obtain different

concentrations of trypsin: 0.8%, 0.6%, 0.4%, 0.2% , each to be 10 cm3

Test solution Volume from Volume from Total volume of

concentration the stock distilled water the obtained
needed (%) solution (10%) in (cm3) solution (cm3)
in (cm3)
1.0 10 0 10
0.8 8 2 10
0.6 6 4 10
0.4 4 6 10
0.2 2 8 10
0.0 (Control) 0 10 10
 - 12 -

➢ Alternative method : Catalase enzyme : from potatoes.

-Hydrogen peroxide as a substrate
1- Cut large number* of equal sized* discs from a single potato.
2- Change enzyme concentration by putting different number of discs
over the H₂O₂ .ex. 1,2,3,4,5 discs in each tube.
3-Control: Use one with same volume and concentration of H2O2,
same temperature and pH , but no potato disc .
4-Validity: Keep all controlled variables constant (as before).
Also… same potato / variety/ source /age / storage conditions.
5- Use buffer solution pH 7, and water bath 30◦C.
6-Initial Rate of the reaction: Record the volume of oxygen evolved
/second in the first 30 seconds using a gas syringe* and stop watch*.
7-Reliability : Replicate several times at EACH concentration of
catalase and get the average . Plot a graph of the results.
• -Risks and safety: Same, but take care of injuries while cutting the
discs, cut away from yourself.

• Enzyme Immobilization: Keeping the enzyme in ‘beads’ made of an

inert material ( usually in industrial processes)
-Advantage: The enzyme is more resistant to pH extremes and high
temperature , so can be reused, → saves cost.
Disadvantage: Slower rate of the reaction because less free to move
and collide.
 - 13 -

Rate at 20°C was 2.5 au, rate at 30 °C was 5 au

Q10 = 5/2.5 = 2 . Up to 40°C , the Q 10 is around 2 ( rate is doubled).

Above 40°C , the rate decreases so Q 10 becomes less than 1.

❖ The initial rate : (In the first 20-30 seconds):Measured by:

-Increase in Volume/mass of product , or per unit time!!!.
-Decrease in substrate vol/mass per unit time!!!.
is best a there is plenty of substrate so the substrate is not the
limiting factor, only on the enzyme concentration is the limiting
factor.!! As the reaction goes on, the substrate is used up and
substrate becomes a limiting factor.
𝒄𝒉𝒂𝒏𝒈𝒆∗ 𝒊𝒏 𝒚
Rate=
𝒄𝒉𝒂𝒏𝒈𝒆∗ 𝒊𝒏 𝒙
- 14 -
 - 15 -

Unit 2 Experiments

How to set up a light

microscope
1- Turn on the light source, clean
the lenses, open the diaphragm.
2- Put the slide on the stage, fix it
with the clip.
3- Begin by the low power. Choose
a low power eye piece (up) +
objective lens (down).
4- View the field with low power
using the coarse adjustment.
5- Turn the objective lens to
stronger one in order to view
with the high power. Use the
6- fine adjustment.
 - 16 -

Slide viewed by the low power With High power showing

→ Shows many cells, smaller in size Less number of cells but larger cells
 - 17 -

Core practical 5 ) Eye piece Graticule and Stage micrometre

Q. What is a graticule?- It is a glass or plastic disc with a scale on it,

divided into 100 divisions. Its scale is super imposed on the specimen.
Q. What is a stage micrometre? It is a special microscope slide with a
scale on it ( given), example, each unit is 10µm.
It is used to calibrate the graticule.
A)Calibration of the graticule: First calibrate your eye piece graticule,
(as its scale is variable according to the magnification you are using
1- Remove the eye piece lens, and insert the graticule in its place.
2- Place the stage micrometer on the stage, move it till its scale is in
line with (parallel to) that of graticule.
3- You will be given a known scale for the micrometer
e.g. each micrometer unit = 10 µm.
4- Now observe how many graticule units (epu) are in line with how
many micrometer units. In the diagram above:
5- 100 graticule units = 30 micrometer units
1 micrometer unit = 10 µm
So, ,,,100 graticule units = 30X10 µm= 300µm
i.e. 1 graticule unit = 300/100= 3µm
B) Now:
- Remove your stage micrometre
and put your tissue slide.
- Keep the graticule in its place (now you know its scale!)
- (your required cell) measures how many graticule units.
If e.g. it measures 10.5 graticule units (and you know from your
calibration that each graticule unit measures 3 µm) so diameter of your
cell is 10.5 X 3 = 35µm
 - 18 -

example:, If 20 graticule units are aligned with 10 stage micrometer

units. Given that each micrometer unit is 10 µm , calibrate this eye
piece graticule.
Answer :
20 epu = 10 micrometer units
so 20 epu = 10 units x 10 µm
𝟏𝟎𝟎
20 epu = 100 µm ,,,, So 1 epu = = 50 µm
𝟐𝟎
Alternative method
 - 19 -

Core practical 6 ) Plan Diagrams :

Should show only outlines , indicating the relative positions , no details

Tips for a good drawing:

1- Draw only what you can see, don’t make up additional structures.
2- Use a sharp HB pencil. Use clear continuous lines.
3- No shading or colour.
4- If enlarging, keep the proportions, ie enlarge all the parts equally.
5- Label with a pencil and ruler, no arrow heads , no lines intersecting.
 - 20 -

Vascular bundle in the stem

epidermis
sclerenchyma
Phloem
Xylem

Plant Root T.S.

 - 21 -

Core practical 8: Root tip squash To Observe Mitosis

1- Cut the final 5 mm from the tip an
onion root/garlic clove.
(this is a MERISTEM, the cells are
dividing by mitosis)
2- Put in a watch glass and add :

a) Dilute HCL acid .→( softens the cell

wall*, by breaking the pectin in the
middle lamella →
-helps entry of the stain into the cells ,
- separates the cells to one thin layer .)
b) Acetic orcein stain, /Schiff’s reagent / Toluidine blue stain. -->

-→view/ dye chromosomes! ( not view cells!!)

3-Indirect heating in a water bath/hot plate at 55ºC for 5 minutes.

4-Transfer to a glass slide . Macerate* ( break open/separate) the
tissues with 2 needles. ( additional drops of stain may be needed)
5- Put over it a cover slide, wrap it with a paper towel and SQUASH
GENTLY* with your thumb, to obtain a thin layer of cells (so as to see
them clearer, with no cells overlapping).
 - 22 -

6- View with a light microscope:

a- Low power first: to count total number of cells, and those in
mitosis.
b- High power: to view the chromosomes in detail, and count the
number of cells in each stage :
- Interphase.
-Mitosis ( prophase , metaphase , anaphase , telophase) , and
-Cytokinesis.
- and get the % from the total stage of mitosis.
- The number of cells in each stage is directly proportional to the
duration* of this stage. ex The least number of cells would be in
anaphase , as it is the shortest stage ( least duration).
-Most cells would be in interphase (longest duration).

𝐧𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐜𝐞𝐥𝐥𝐬 𝐢𝐧 𝐦𝐢𝐭𝐨𝐬𝐢𝐬

Mitotic index = x 100
𝐓𝐨𝐭𝐚𝐥 𝐧𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐜𝐞𝐥𝐥𝐬

➢ Risks and Safety:

1-The stain is irritant and acid is corrosive >>> Avoid skin contact, wear
protective gloves and lab coat.
2-Harmful fumes during heating >>>wear mask and goggles.
3-Acetic orcein stain contains alcohol, so flammable >>> keep away
from direct heat ( use hot plate not Bunsen flame.)
5-Injury from scalpel >>> Cut away from oneself.
➢ Try at different distances from the root tip, As the distance
increases, the mitotic index (% of cells in mitosis ) decreases .
--Many cells in mitosis > rapidly dividing cells
- 23 -
 - 24 -

Core practical 8 : Measuring the tensile strength of plant

fibres.
Tensile strength:
The minimum force/tension needed
to break / cut a fibre. The breaking
force in NEWTONS divided by the
cross-sectional area in m² (unit is N / m²)

1- Suspend the fibre using a force meter or a pulley .

( alternatively ,you can clamp it horizontally between 2 retort stands,
and apply the masses in the middle.
2- Attach to its lower end a small known mass (10 grams) .
3- Keep adding small masses* gradually*e.g.10 grams at a time till the
fibre breaks. Determine the minimum mass needed to break it.
4- Controlled Variables: -
Fibre: -Same diameter, length, source
Environment-Same temperature (AC), Humidity (Humidifier), air
currents in the room.
5- Replicate several times at each (IV ?)…..using the same controlled
variables and get the average.

.. The higher the mass needed to break the fibre…. the higher the
tensile strength (stronger fibre)

6- Risks and Safety :

1- Specks of broken fibres can get into the eyes >>.wear goggles.
2- The masses may fall and injure the feet >> use a mat.
 - 25 -

Core practical 9: The antibacterial effect of plant

extracts:
1. Use already prepared sterile nutrient* agar plates seeded
(inoculated) with a fixed mass of HARMLESS bacterial culture.
2. Preparing plant extract: ex garlic clove / mint.
Crush the plant in alcohol ( solvent) , and then filter it.
(R = alcohol kills the bacteria that may be present on the plant itself.)
(validity).
3. Soak equal sized sterile filter paper discs with the extract and place
on the agar using a sterile forceps.
- (OR pipette 0.1 cmᵌ of extract in holes (wells) in the agar).
- The discs or holes should be equidistant.
- Control : one disc / well with no extract ( put solvent only,,
alcohol.. or sterile water) For Comparison!!
4. Tape the lid vertically: allows some air to enter →prevents growth
of anaerobic bacteria harmful to humans.
5. Incubate for 24 hours at 25◦C (R = higher temperature(37◦C).. human
body temperature, would encourage harmful bacteria to grow.)
6. Observe without opening the lid (R = prevent spread of bacteria)
7. The clear area around the extract is the inhibition zone/Clear zone .
Measure its diameter using a ruler ,,and area ( TTr²)

The larger the diameter / area → the more powerful is the

antibacterial effect of the extract).

• Compare with the control disc / well !!-->Subtract..

7- Replicate the experiment several times at each*…. and get the
average ( for reliability).Plot a graph of the results.
 - 26 -

Q)How to measure the area?

A) If circle: measure diameter using a ruler or vernier caliper then, if
irregular, get 2 diameters , at different planes, get their average and
use it to calculate the area

B) or use ( graph paper tracing). (Exclude the area of the well)

• Bacterial colonies appear on the agar except in the areas around

the discs ,,Clear zones,,,. This is because the extract DIFFUSES
through the agar! , killing the bacteria around the discs )

Experiment can be used to:( independent variables ):

- Compare different types of extracts on one type of bacteria.
- Different concentrations of one type of extract.
- Same extract on different types of bacteria (use different plates
with different bacteria, in each add one extract)
- Compare different methods of extraction, storage time , different
solvents ,, raw versus boiled extract……etc.

Preparing sterile agar plates : Dissolve agar powder in boiling water in

a flask. Leave to cool, but not solidified (around 50 ◦C). Add a fixed mass
of bacterial culture to the warm molten agar .This ensures that the
bacteria are evenly distributed in the agar. Now pour the agar with the
inoculated bacteria into sterile petri dishes)
 - 27 -

Safety:

1. All equipment should be sterile in a Bunsen flame or autoclave. Work

near a safety Bunsen flame (or work in a laminar flow chamber if
available ).
2. Wear sterile gloves, masks, hair cover and lab coats.
3. Alcohol is flammable, avoid using it near flame.
4. Use harmless bacterial strain e.g. Lactobacillus
5. Don’t open lid totally during pouring agar, or adding the extract. This
minimizes contamination from bacteria in the air.
6. Don’t open lid after incubation.
7. Avoid eye or skin contact.
8. Autoclave the plates before disposal.
9. Wash hands with an antibacterial soap before and after working.
10. Clean the bench with disinfectant before and after working.
11. Care from burning while pouring hot molten agar.
(use heat resistant gloves) .
- 28 -
 - 29 -

Recommended Practical: Effect of mineral ion deficiency on

plant growth
1- Use wheat seedlings from same* plant (genetically similar).

2 -Independent variable- Put one seedling in a nutrient culture

solution , but the nutrient solution without one of the mineral ions ex.
Without Nitrates.

( alternatively: 5 stated different concentrations of nitrates)

- Control: Place one similar seedling in a tube containing solution with

all mineral ions in optimum* concentrations (to compare).

3-Controlled variables: SAME !!!

• Plant : Same type, age ( same source ) Better use CLONES ,, and
same starting mass (sensitive balance)!!
• Conditions :
-Same temperature (incubator, or AC in a closed room) ex. 20 ◦C,
-Same light intensity and direction, use a (light bank.)/ (same light
source at a fixed distance)
- Same CO₂ and O₂ concentration in the air.
• Solution Same concentration of other mineral ions (same mass
dissolved in the same volume of distilled water)

4-Method-Cover the tubes with foil or black cover ( to prevent entry of

light, to prevent growth of algae, can consume minerals so affect
growth of the plant (-->so nonvalid results.)
 - 30 -

-Close the mouth of the tube with cotton wool.(not a plug) (This allows
some air in for respiration of roots)

-Time scale: -Leave for 1 or 2 weeks , then observe.

5-Depedent variable: How to measure “growth???”!!!

>Compare to the control: (calculate or measure)

• Fresh or dry mass.. % change (better than length.)

• length of shoot or root.. % change .
• surface area of leaves→graph paper tracing

5-Replicate: use several seedlings with each* mineral ion defecincy….

and calculate the mean and SD. Plot a graph with error bars.
( Not just repeat the experiment!)

6-Safety (culture solution is a good medium for bacterial growth→risk of

contamination and infection)
-. Avoid eye or skin contact. - Autoclave the plates before disposal.
- Wash hands with an antibacterial soap and clean the bench with
disinfectant at the end.

>Why mass better than length:

- Length measures growth in only one plane.

- The plant may have increased in diameter, with same length.

-plant may be curved or non uniform→difficult to measure length

 - 31 -

>Why dry mass better than fresh mass:

The dry mass shows the mass of the organic matter, excluding water ,
which may vary , so dry mass is more valid.

>Why % increase is more valid:

The initial mass of the plants may not be the same.

> Why use genetically identical plants {from cloning}:

-So that any difference observed is due to the environmental factor you
are testing,,,,,not genetic causes. (Validity )

Other Independent variables:

-Similar experiments can compare the effect of a certain mineral ion
deficiency on different types of plants,
- or different concentrations of a certain mineral ion
( mention 5 values).
> How to obtain the dry mass: Place the plant in an oven at
50→70 ◦C. Weigh every hour till a CONSTANT mass is reached