Unit 3

WBI13
Core practicals

Dr. Nihal Gabr

 003
Core practicals guidance

or
1. Core practical 1 ( serial dilution, food test including Benedict’s, Buiret and iodine solution)
2. Core practical 2 ( vitamin C )
É 3. Core practical 3 ( investigating membrane properties )
s
4. Core practical 4 ( measuring reaction rate )
5. Core practical 5 ( using graticule)
6. Core practical 6 ( prepare and stain a root tip squash to observe mitosis )

§¥ 7. Core practical 7 ( observing plant cell under microscope ..drawings )

8. Core practical 8 ( tensile strength)
9. Core practical 9 ( aseptic tequnique)

Recommended additional practical

1. Structure of mammalian heart by dissection

2. Tissue water potential using plant tissue and graded conc of solute
3. Semiquantitative test to estimate protein concentration using Buiret and color standards
4. Factors affecting growth of pollen tubes
5. Investigating plant minerals deficiencies.

003
Before starting
Things you should know :

1. Error bars and SD 1 .detecting significant difference between data

To show Reliability by calculating mean value and SD and plot a graph

1. Draw error bars ..given SD value……..

on

E.
um Overlapping
mx
E
Given mean value is 13 , S.D is o.5cm
:
Touching 2. Comment on size of error bar ………longer error
o
bars ……more data are spread / variable from the
o
mean value ……….so less reliable results .
o

Cinema 3. Compare data / bars

100 m
Overlapping …..so no significant difference
300m
Least reliable No overlapping …there is a significant difference

SD / error bars overlap …no SD / error bars dont overlap …so there
SD / error bars overlap …no
significant difference is a significant difference
significant difference
2. Controlled variables

Constant variable as it affects the results

( you should mention how it will affect the results if not kept constant )
To have only one changeable variable
To have fair test and valid comparison

Explain why ….enzyme and substrate solution should be kept in a water bath at temperature 40C , before and
after mixing ?

To allow equilibration ..( to allow them to reach to experimental temperature )

As mixing the solution at different temperatures would make the temperature not equal to 40 C
40 C its the optimum temp for enzymes
If temp is cooled / warmed up the rate of collision os affects the rate of the reaction would change during the
experiment
To have only one changeable variable
Fair test and valid comparison .

DR. MODY IS CRAZY

I 1
Dependent Measurable Independent Changeable
3. Plan an investigation

I. Independent value mention values

1. Controlled variables ( biotic and abiotic) + methodology

:
Water bath, buffer, thermostatic seedling mat, equilibration, .........controlled variables the way
being controlled is considered as standardization , reset at zero before measuring ,
a blank – a cuvette containing distilled water and dilute iodine/KI solution – is used to set the
absorbance of the colorimeter to zero.
The blank – water and dilute iodine/KI solution – will be pale brown. If we set the absorbance to
0 this eliminates any absorbance due to just the iodine solution sowe are measuring absorbance
due to iodine/ starch complex. (1)
Serial dilution :
Prepare 5 different concentrations of .....using a stock solution of concentration ....%
Use a dilution factor of 0.1 ( 1:10) where we use 9 ml of distilled water 1 ml of solution for the
's
volume in each test tube 10 ml main "
no ¥:*
*

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Volume of sample 1mL
Dilution factor =
°

?÷=
Volume of sample+ volume of diluent +
sin .

Instruments
Technique 100
my
os

100×0-1
ogɀ yori

3 Time interval
'
100% =
10
MY 10×0-1 ogÉ°
1mg
=
101
U Dependent variable
.

1×0-1
.

5 , Safety precaution 1% =
01mg
6
Control o It

Repeat
 Who to write steps for serial dilution

Using a stock solution of 500 gmdm-1

And use dilution factor of 0.1 ( 1; 10)
Where we use 9 ml water and 1 ml of solution to have total of solution in each test tube 10 ml

Stock § r

1ml

oÉ
solution

9ml
E-
100mg
100x 0.1= 10 x 0.1=
In 10 ml 1 x 0.1=
10 mg 1mg 0.1 mg
100%

1ml
Volume of sample = 0.1
Dilution factor = 10 ml
Volume of the sample + volume of the diluent
Sum up plan an investigation.

Plan an investigation

Independent variable ........numbers

Methodology .........details (serial dilution / calibration of instrument ( reset at zero before
measurements are taken / instrument / technique / equilibration)
Controlled variables for fair test and valid comparison
Safety precautions
Time interval ‘
Dependent in details
Repeat using larger sample to calculate mean value and the standard deviation.
You are provided with a solution 500 g dm-3 of sucrose solution
Devise a procedure to investigate the effect of different concentration of sucrose on rate of growth of
pollen tubes ..using the sucrose solution .

Independent :
Carry out the experiment using 5 different concentrations of sucrose
( 50, 5, 0.5,0.05, 0.005 g dm-3)
3 times at each sucrose
Methodology : Repeat
concentration and calculate
Use serial dilution method
value and SD and plot a graph
By using the stock solution of 500 gm dm-3
And dilution factor of 0.1 ( 1: 10)
Where we use 9ml of water and 1 ml of solution to have total volume of each solution in each test tube 10 ml

Controlled variables
Same temperature using a thermostatic water bath …( abiotic)
Same type and age of pollen grains …( biotic)
Same volume of solutions of different sucrose concentrations ..( abiotic)

Time interval

Leave the pollen grains in each sucrose concentration for 2 hours

Dependent Use a microscope ( eyepiece graticule and stage micrometer ) to measure the length of
pollen tubes grown
 To evaluate the data

1. Comment number of readings taken/ sample size ………….reliability

2. The range of measurements taken ………….accuracy and validity
3. Check reading that are inconsistent with other reading .. anomalies
4. Controlled variables ( same ) ……..validity
5. To improve reliability …..repeat to calculate mean value
and plot SD / error bars
Spot anomalies

Validity Reliability Accuracy

1. Constant variables 1. Repeating and getting similar results 1. Way of measurements
2, repeat
 Core ractical 1
Core Practical 1
P

Dr. Nihal Gabr

 Qualitative test Semi quantitive test Quantitative

Rough estimation to the concentration

Indicates the presence Exact determination to
of a substance present comparing it
or absence of a the concentration of
against standard / known
chemical substance substance present .
concentration
Measure the intensity of
Source of error : subjective judgement to
color using a colorimeter
color change
and using calibration
curve ..

ROYGBIV
1% 3% 6% 10%

Colorimeter…measure
absorbance of unreacted
bendict;s ( blue)
1% 3% 6% 10% 0
024

Core practical 2
12/8/2021
-0--808
Vitamin C

Dr.Nihal Gabr
024
Core practical 2
1C.5025Testing for vitamin C
Objectives


1. To be able to calculate the vitamin C concentration of fruit juices using the
titration method
2. To solve problems set in practical contexts
3. To process and analyse data using appropriate mathematical skills (mean,
median mode, use arithmetic mean)

Safety:
1. Wear eye protection.
2. Avoid skin contact with the DCPIP and test tube solutions.
3. Do not taste the fruit juice.

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Procedure:

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Steps:
1. Extraction of fruit juice by crushing and adding distilled
water to extract vitamin c.
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2. Controlled variables: same conc. Of DCPIP, same same
mass of fruit, same volume of extract, same storage DCPIP
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conditions.
3. Use DCPIP
4. Titration by adding DCPIP to vitamin c sample drop by Endpoint
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Stop
drop where colour change from blue to colourless .
5. Count number of drops /measure the volume of DCPIP
decowuiuh
Vitamin C
Dr

added for the colorless to change to blue. sample

i-
•
Blue .

6. Standardization, repeat using 1% solution of vitamin C so

that the volume of DCPIP decolorised by fruit juice is
compared with volume decolorised by standard vitamin C Keep adding DCPIP
till blue colour of DCPIP
solution.
7. Repeat and take average. ! = remain.

As DCPIP will be

Remember : role of vitamin C on CVD ↳ decow

÷ reduced( gain electrons)

Vitamin C will be
oxidised (lose electrons)
Affect the endothelium lining of blood vessels End part
So low vitamin C. Damage endothelium lining of artery
causing atherosclerosis
As vitamin C act as an antioxidant that reduce plaque and
atheroma formation. Donate electrons 023
Dr.NIhal Gabr 025
006
IMP
'
026

Procedure:
µ ①µ -
Iomguitaminclloomli
grapes .
LEO ……GER

DCPIP.EE
1. Extraction of fruit juice ....crushing and add distilled water to extract vitamin C
2.independent ; different extracts of fruit juice with different vitamin C content
......or use a stock solution and carry serial dilution method

3. Controlled variables;
Fruits of same age . Same mass same volume of extract , same storage conditions , same
concentration of DCPIP
Once

4. Methodology
1.
reduced ..
colorless
Titration of the fruit juices by adding DCPIP to juices drop by drop and mix gently upon
adding each drop where the color change from blue to colourless

§¥§
reductionIDCPIP
withutaminc
5. Dependent ; measure the volume / number of drops of DCPIP added for the colourless
.

to change blue / for blue color to remain

Add till blue color of

6. Standardization : compare to standard solution of known vitamin c concentration
DCPIP persist
Repeat using 1% of vitamin C solution so compare the volume of DCPIP decolorised by
fruit juice with the volume decolorised by standard vitamin c solution
→ standout
10 mg st solution .......5ml DCPIP
Titrant DCPIP .

X mg unknown solution ..........10 ml DCPIP

026
 027

Independent : get the three fruits and extract the juice from them by crushing and
adding water to dissolve the vitamin c

Controlled variables: fruits all of same age, same mass and extracted with same
volume of distilled water.
Methodology: titrate using DCPIP
by adding drop by drop of DCPIP to fruit juice extract where colour
change from blue to colourless
Dependent : measure the volume of DCPIP added for the colorless to change to blue.
Standardisation: by repeating using 1% solution of vitamin C so that the volume of
DCPIP decolorised by fruit juice is compared with the volume decolorised by standard
vitamin C solution
Repeat 3 more times for each fruit extract and calculate average

027
 standard solution at C. → Long / 1mL
Read
-

028 1.1mL to decolorize DCPIP

Lo
→

11
.

←
→
-
-
unknown conceit of 5- 23.17mi to Declare
DCPIP
E- -23-17 Concentration of vit C in fruit
.

standard solution

1% vitamin C Vit C Volume of st solution

DCPIP
.

10mg /ml .

x concentration
Volume of fruit juice of standard
10mg
5MIDCPIP.sc#lom1DCPIP
→

solution
1.1
=
✗ 10=0.67 my 1mL .

23.17

0.47
my → 1mL

o 0¥ so Iooml
d-
→
.

g.
DCPIP
Vit
*C ¥
End point …..blue color persist End point …..blue turn
colorless ..persist colorlress
The more DCPIP used …the
higher the concentration of The more vitamin c used….the lower the
vitamin C concentration of vitamin C in sample
Role …compare against standard of I
known concentration of vitamin C …
cross multiplication. 028
Titrant is DCPIP ….end point blue color persist
Titrant is vitamin C …end color is colorless

Titrant vitamin C sample …. Volume of st solution long

-

Calculate the vitamin C conc of unknown = X concentration of standard

Volume of fruit juice
solution

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corepradical23wm-o.IN
029 calculating vitamin C conc .

Titrant is vitamin c Memorise role when

vitamin c is the titrant

÷±T"
→
Hate . -

=
-

✓ =

Tom/
'

DCPIP

C,v = volume and concentration of DCPIP

016
029
 1. What
030 were the independent and dependent variables in this
investigation?
Important
The independent variable was the type of fruit juice used. The dependent questions

variable

 Was the volume of juice added to decolorise the DCPIP.

2. Why was each titration completed three times to calculate a mean?

Calculating a mean value makes it easier to spot anomalies. Repeated readings also reduce the effect
of errors.

3. Suggest one reason why syringes were used in this investigation rather than burettesOne of:

• Using very small volumes of solution requires a more precise piece of apparatus than a burette.

• Syringes are easier to control, allowing smaller volumes to be added.

• Ease of use

• Relatively low cost

4. Which of the fruit juices tested contained the highest concentration of vitamin C?

Your own answer according to your results.

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5. The reagent DCPIP tests for the presence of ascorbic acid (vitamin C). It is
blue when oxidized and colorless when reduced. During the reaction between
Important
questions DCPIP and
ascorbic acid, DCPIP becomes reduced and ascorbic acid becomes oxidised.
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✓
(a) Describe the direction of movement of electrons during the reaction between
DCPIP and vitamin C. (2marks)
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DCPIP gains electrons (1) from the ascorbic acid. (1).

(b) Explain why vitamin C is described as an antioxidant(2 marks)

Vitamin C / ascorbic acid readily loses electrons / becomes oxidised (1) and this prevents other
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cellular components / chemicals from becoming oxidised. (1) .

✓
Dr

(c) . Suggest why, when adding ascorbic acid to DCPIP, the tube should be shake gently and not
vigorously with the addition of each drop of DCPIP.(3 marks)

DCPIP becomes colourless when reduced and that is the end point. (1) Shaking

too vigorously would introduce oxygen to the DCPIP / reoxidise it (1) and the Blue colour would return /
would make it difficult to find the true end point. (1)

✓
6. A student tested (assayed), by titration with DCPIP, a solution of blackcurrant juice. The juice
was first decolorised by adding a piece of activated charcoal. The data
obtained are shown in the tables. I
23.17×10
-

O
-

024 ①
Dr.NIhal Gabr 030
007
 031why the blackcurrant juice was first decolorised (2 marks)
(a) Explain
This juice was decolorised to make it easy to spot the endpoint, i.e. the point whenDCPIP just
becomes decolorised. (1) This point would be difficult to judge in
coloured juices. (1)

(b) Calculate the mean values missing from both tables. (2marks)
Note: mean values can be to 1 d.p. more than the raw data values.

(c) . Calculate the ascorbic acid concentration, in mg/100 ml, of the blackcurrant juice. Show your
-

working. (4 marks)
In 1I Egan .

ml 1% DCPIP there are 10 mg DCPIP per ml

•

In 1 ml 1% ascorbic acid solution there are 10 mg ascorbic acid per ml

1 ml (10 mg) DCPIP is decolorised by (1.1 × 10) mg ascorbic acid = 11.0 mg
ascorbic acid (1)
Therefore there are 11.0 mg ascorbic acid in 23.17 ml blackcurrant juice (1)
So in 1 ml blackcurrant juice there are 1/23.17 × 11.0 =-
0.47 mg ascorbic acid (1) So in 100 ml
←

blackcurrant juice there are 0.47 × 100 = 47.0 mg ascorbic acid /

o.u⑧

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=

vitamin C (1)
Correct answer with no working gets 4 marks

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7. Vitamin C is water soluble and cannot be stored within the body. Many mammals,
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including dogs, can make vitamin C in certain cells. Humans cannot synthesise vitamin C and
have to obtain it from their diet. However, if they consume more than needed,
the excess is filtered from the blood in the kidneys and passes out in the urine.
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Imagine that you are a scientist in a physiology research lab. Outline the design of an
investigation to determine whether taking vitamin C supplements benefits people.
(8 marks)
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Any eight from:

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Large sample of volunteers (1)

Dr

Matched age / gender / mass / lifestyle / diet (1)

Questionnaire about diet (1)

Collect a day’s urine and test for vitamin C to ascertain base levels – are they already

taking plenty of vitamin C or not enough? (1)

Divide into groups – one group given no vitamin C supplement, one group given small
supplement, one group given larger supplement (1)

Could make it blind / double blind and give first group a placebo (1)

Make sure they stick to normal diet / stay in lab so scientists can control their diet (1)

Collect urine again (one day’s worth) and test for vitamin C content – is the extra being passed
out? (1)

Other valid point (1)

025
Dr.NIhal Gabr 031
008
Core ractical 6
Core Practical 6

Dr. Nihal Gabr

Procedure Core practical 6

1. Removal of the last 5-10 mm of root tips ( meristem).

2. Place root tip in hydrochloric acid to separate the cells/macerate tissue/ soften tissue (making
it easier to stain).
3. Addition of appropriate stain such as toludine (blue) , acetic orcein, methylene blue to high light
chromosomes.
4. Place the root tip on microscope slide, teasing cells (using needle to spread cells),
Squashing the cells underneath a cover slip to separate cells (for easier view),
and heat the slide to intensify the stain.
5. Ensure the students view the preparation under the microscope on low power first to identify the
area of dividing cells and to focus. Higher power will be required to examine the dividing cells for the
stages of mitosis
6. Counting number of cells in mitosis, then counting total number of cells , then Calculate the
mitotic index .

Cells in mitosis
Mitotic index X 100
Total number of cells For safety : wear eye goggles
Avoid contact with HCL irritant /
Measuring how actively the cells in a tissue are dividing corrosive …wear gloves
Core practical 6
3B.2 Prepare and stain a root tip squash to observe the
stages
 of mitosis
Objectives
1. To know how to prepare a temporary slide of a root tip to observe mitosis
2. To recognise the stages of mitosis in dividing cells
3. To identify hazards, associated risks and control measures for the procedure

Safety:
1. Eye protection must be worn.
2. Acetic orcein stain is corrosive, causes burns, has an irritating vapour and will stain.
Wear eye protection and avoid contact with skin. If contact does occur, wash the
area thoroughly with water for 10 minutes and tellMmm
your teacher.
3. Avoid skin contact with the hydrochloric acid, by using gloves ( irritant/corrosive).

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Procedure:
1. Removal of the last 5-10 mm of root tips ( meristem).

2. Place root tip in hydrochloric acid to separate the cells/

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macerate tissue/ soften tissue (making it easier to stain)

3. Addition of appropriate stain such as toludine (blue) , acetic

orcein, methylene blue to high light chromosomes.

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4. Place the root tip on microscope slide, teasing cells (using
-

needle to spread cells), Squashing the cells underneath a

cover slip to separate cells (for easier view), and heat the slide
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to intensify the stain.

5. Ensure the students view the preparation under the

Dr

microscope on low power first to identify the area of dividing

- -
cap X
cells and to focus. Higher power will be required to examine
the dividing cells for the stages of mitosis
For high
6. Counting number of cells in mitosis, then counting total Magician.jp
number of cells , then Calculate the mitotic index .

How to calculate the mitotic index( % of

cells undergoing mitosis)

Calculating the mitotic index:

mitoticindex = cellsinmitosis ÷ totalnumberofcells X 100

Cells with visible chromosomes= 12
Total number of cells= 25
Mitotic index = 12/25= 0.48

042
Dr.Nihal Gabr
 1. Mitotic index : is a measure of how
actively the cells in a tissue are dividing


. It is the ratio between the number of
cells in a tissue sample that are in
mitosis and the total number of cells in
the sample.

Once the cell leaves interphase and enters mitosis, the

2. We can use the mitotic index to identify chromosomes can be seen. Yo can use this when counting cells
actively dividing tissues, including to work out the mitotic index.

cancerous tissue . As cancerous tissue is

dividing more rapidly than it should , so we
can use the mitotic index to measure the
effectiveness of treatment of cancer, as if
treatment is working then the mitotic index
of the tumor will fall.

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1. Explain why the root tip is heated with acid.
lG
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. .
(softening I macerate tissue
)
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2. State the effect of maceration and pressing the slide preparation on the dividing cells.
Maceration and 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.

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•
3. Describe what information the cell counts give about each stage of mitosis.

Dr

• 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. Describe the role of mitosis in the life of an organism.

Mitosis produces identical daughter cells for growth, replacement and repair.

- - - -

cell tissue .

043
Dr.Nihal Gabr
5. Observations of meristem cells of an onion root tip were made and the stage of the
cell cycle was recorded for each cell. The following results were obtained.



2¥ E ÷

80
.

(a) Calculate the percentage of cells that are in interphase. (1 mark)

(a) 200/250 × 100 = 80% (1) .

=
€ 18×60=1080 Mins .

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(b) Assume that the mean total duration of the cell cycle is①18 hours for these cells.

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Calculate the actual time, in minutes, spent in anaphase. Show your 18×60=080 Mins
working.
(2 marks)
1080 → Loo's
-

OR 0.36 × 60 = 22 (minutes) (1)

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18 × 60 = 1080 (minutes) OR 2/100× 18 = 0.36 (1) × 1080 = 22

I → 2
.

Allow 21.6.

180=21.6 .
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6.Describe how cells from the root of a plant could be prepared in order to observe
mitosis. (5 marks)
Reference to use of the root tip / area with meristem (1)

Add acid / named acid / acetic alcohol (1)

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Add appropriate named stain, e.g. acetic orcein or toluidine blue O (1)Warm / use a water
bath at 55 °C (to intensify staining) (1)

Dr

Break apart tip with a (mounted) needle / equivalent (1)

Mount on a slide and (gently) squash under a coverslip (1)

7. Some students are planning to take measurements of root tip cells and compare the
size of cells in different stages of mitosis. They would like to make sure that their
results are valid, accurate and reliable.

(a) Explain what is meant by the term ‘valid results’. (3marks)

• Valid results measure what they are supposed to (or equivalent wording).(1)

• The measurements that have been made are affected by a single independent variable / only by
the stage of mitosis. (1)

• All variables apart from the independent variable / stage of mitosis have been controlled. (1)

• There is no observer bias (1)

- validity → Controlled variables ( example) .

→ no observer bias .

Reliability
044
→
repeat and calculate mean .

Dr.Nihal Gabr Calculate SD .

EE(b) Explain how the students could ensure that their results are as accurate
(near to the true value) as possible. (3marks).

Use appropriate equipment for measurement, e.g. eyepiece graticule (1)

Correct calibration with a stage micrometer (1)

Accurate identification of the stage of mitosis (1)

Do a large number of measurements / repeats for each stage and calculate a more accurate
mean (1)
and calculate SD
Suggestion of a variable that should be controlled, e.g. distance from root tip /age of root / use
the same root (1)

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lG
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Dr

045
Dr.Nihal Gabr
 Core Practical
Core ractical 8 8
 Core practical 8 :
Tensile strength

Effect of different length on tensile strength

1. Get fibres from same plant , of same age and same diameter .
2. But of different lengths …….carry the experiment under same temperature , humidity , and thickness
( diameter) .
3.clamp one of the 20 cm string between two retort stands
4. Add Cushion underneath the string as as not to hurt your feet / to avoid hitting the bench when string
breaks
5. Gradually add increasing masses until the fibre breaks .
6. Record the mass of which the fibre breaks
7. Convert mass of which the fibre breaks
Tensile strength = breaking force / cross sectional area indef
8. Repeat same steps with other fibres of other lengths . →

Improve accuracy

Use smaller mass intervals

Improve reliability

Repeat 3 more times at each length

Calculate average value ( SD and plot a graph)
Core practical 8
4A.5 Determining tensile strength:


Objectives
1. To know how to determine the tensile strength of plant fibres
2. To be able to comment on experimental design and evaluate scientific methods.
Safety
1. Use a sharp knife, place the celery on the tile and cut down onto the tile.Take care
when using the knife to cut the celery, Cut away from your hands.
2. Wash hands with soap and water after the practical work is over.
3. Goggles to prevent fibres from getting into your eyes.
4. Add sponge material under mass (cushioning ) so that you protect your feet , and
also for not striking the bench causing loud noise.

Procedure:

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1. Select fibres of the different (length) and same diameter.

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2. Clamp one of the 10 cm strings between two retort stands, ensuring its held securely.
3. Add cushion underneath the string so as not to hurt your feet , and to avoid striking the bench when
the string breaks. lG
4. Gradually add increasing masses until the fibre breaks.
5. Record the mass of which the fibre breaks.
6. Convert mass to force :
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• Tensile strength= breaking force/ cross section area.
7. Repeat same steps with each other string/ fibre.
8. Ensure validity: all other variables should be kept constant example, temperature, humidity, and
same thickness.
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9. Improve accuracy:
- use sharp knife to be cut precisely to same size/ diameter(wet knife to reduce frequency.
Dr

- Smaller mass intervals to find more accurate results. → loads being added .

10. Improve reliability:

- repetition at each length and taking average.

Notice:
Another independent
variable can be introduced
to the investigation by
preparing the celery fibres in
-

advance and then allowing

- -

them to dry for varying

-
periods of time to determine
-

whether this has an effect on

the tensile strength.

051
Dr.Nihal Gabr 034
 


1. Use your graph to predict the tensile strength of a celery string that is 12.5 cm
long.
This answer will depend on your data. To find a value, you should have drawn a line from
12.5 cm on the x-axis up to the line of best fit, and then across to the y-axis.

2. Suggest one way in which this procedure could be modified to provide more accurate

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data.
Use smaller masses, so the mass at which the string breaks is closer to the true value.

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3. Identify one variable in this investigation that was not controlled and describe how this
may have affected the data obtained.

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One of:
• The strings may have come from different parts of the stalk. Some parts of the stalkmay have
denser fibres, so the data may have provided higher or lower values accordingly.

• Storage condition or age of the celery. The tissues may have been degraded or weaker, so they
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snapped at a lower value.

• The time between cutting the strings and using them may have allowed some strings to dry out.
This may have caused them to be more brittle and to snap at a lower value.

4. Suggest one further investigation that could be undertaken to determine the validity of the
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conclusions drawn from this investigation. Tcu -1

One of:
repeat
Test fibres from other types of plant to see if the pattern shown by the celery strings is replicated.

Dr

Use a greater range of lengths of celery string, to determine if the pattern continues.

052
Dr.Nihal Gabr 035
1. Write a plan to investigate the tensile strength of banana skin (8 marks).
Testable hypothesis / prediction stated (1)

Mark lengths of e.g. 0.25 cm, 0.5 cm, 0.75 cm, 1.0 cm, 1.25 cm, 1.5 cm along an intact


banana (1)

Cut the banana into sections, cutting at these points. Push the edible part out of the

banana and discard (1)

Hang a loop of banana skin on a clamp and secure it (1)

Hang slotted mass from the lower end of the loop until breaking point is reached (1)

Repeat three times and find mean (1)

Repeat with different lengths of banana skin loops (1)

Tabulate and graph data (1)

Other valid point (1)

2. A length of fibre from a pumpkin stem broke when a mass of 300 g was suspended
from it. The diameter of the fibre was 2 mm.

r
Calculate the tensile strength of this fibre in N m−2. (3 marks)

ab
Tensile strength = breaking force/cross sectional area

Breaking force = 300 g = 0.3 kg = 2.94 N

Diameter = 2 mm = 0.002 m, so radius = 0.001 m (1)

lG
Cross sectional area = 3.142 × (0.001)2 = 0.000 003 142 m2 (1)

Tensile strength = 2.94/0.000 003 142 = 935 709 N m−2 (1)

3. Plant fibres consist of the polymer cellulose. Name the monomers that make up
iha
cellulose.( 1 mark).

• Beta glucose.

4. Silk and wool are fibres obtained from animals. What is the nature of the polymers in
.N

these animal fibres?( 1 mark)

Protein

Dr

053
Dr.Nihal Gabr 036
Core practical 9
Aseptic technique

Dr. Nihal Gabr

Core practical 9

Aseptic technique :

1. Keep bunsen burner on yellow flame on bench

To creat convection current to carry away airborne bacteria

2. Sterile tools such as the inoculating loop and the neck of the flask containing the bacterial sample by
heating them in bunsen burner

3. Always when opening the lid of petri dish containing bacteria ....allow slight degree opening to avoid
contamination with another microorganisms causing disease .

4. Fastening to the lid using cello-tape, but dont tape all way round to avoid anaerobic conditions
 Continue with core practical 9 Risk : bacteria might mutation , and
become pathogenic + contamination
How plant can be detected for antimicrobial properties to the culture by microrganism and
then spread out.
1. Test for different parts of the plant/ different plants
2. Grinding plant materials to make extract
y inoculating loop
3. Prepare a lawn of bacteria .
.

4. Allow aseptic technique : using bunsen burner at yellow flame to creat a convection current to carry a
way air borne bacteria , sterile the inoculating loop and the neck of flask containing bacterial lawn .
5. On agar petridish . Distribute bacteria evenly using the streaking method
6. Creat 4 equal sized wells ( opening in the gel) .
7. Place the paper discs soaked with different plant extracts . ….and the fourth paper disc soaked with water
as a control experiment .

BAA
8. Seal the lid at both sides but not all way round with cello-tape, to prevent
anaerobic conditions that might kill the bacteria in culture and cause growth growth
of anaerobic bacteria
9. Incubate at temperature 20-35 ( 27 C) for 24 hours ( to 7 days) to prevent
the growth of pathogenic organisms.
10. Measure the diameter of zone of inhibition
11. Then disposal ..putting petri dish in a plastic bag , seal and sterile at 121 C for 15 mins under high pressure
and temperature then throw it away .
12. Repeat and calculate the mean value
 Core practical 9
4A.6 investigating the antimicrobial properties
of plants.



Objectives
1. To successfully compare the effect of plants on microbial growth
2. To understand the safety issues of microbiological work and how to apply
good aseptic techniques .

Safety:
1. Wear eye protection.
2. Wash your hands with soap and water before and after the activity.
3. Do not open your inoculated plate. Instead, use the alternative plate provided by your
teacher.
4. Do not decant ethanol near a naked flame and stopper the bottle after use.
5. Use aseptic techniques when transferring the bacteria to the Petri dishes.

r
6. Only open the Petri dish slightly when adding the discs with forceps (not fingers) and, once

ab
it is closed with some strips of tape, do not open it again.
7. Disinfect the bench before and after working. Leave the disinfectant on the bench for about
10 minutes.
lG
8. If the agar plate shows signs of contaminating growth, do not open the plate. A technician
will destroy it by autoclaving.

Procedure:
iha
1. Aseptic techniques:
- Inoculating loop
- Bunsen burner on yellow flame to be put on bench
.N

• to creat convection currents to carry away airborne bacteria from plates.

• Sterile tools / instruments ( including inoculating loop, neck of flask containing bacteria) used
↳ Blue
Flame
Dr

for inoculating the agar plate (adding bacteria to plate) . .

- Open lid on petri dish containing bacteria slightly to avoid contamination with another
-

pathogen/ microorganism causing disease.

- Fasten the lid using cello tape ( don’t tape it all way around). avoid anaerobic
conditions .

"
agar.

054
Dr.Nihal Gabr 037
Procedure:
Steps:
1. Allow aseptic technique, using bunsen burner yellow flame to sterile inoculating loop.


2. On the agar petri dish , distribute bacteria evenly using the streaking method( lawn
distribution).
3. Creat 4 equal sized wells/ openings into the gel.
4. Place paper discs of same size soaked with three different plant extracts , and fourth
paper disc soaked with water acting as a control.
5. Seal lid at both sides ( but not all way around) to prevent anaerobic conditions that might
kill bacteria in culture and cause growth of anaerobic pathogens.
6. Incubate at temperature 27 C to prevent growth / culturing of pathogenic organisms at
higher temperature for 24 hours.
7. Measure diameter of zone of inhibition / trace zone of inhibition using tracing paper and
calculating surface area.
8. Then disposal , by putting dish content in plastic bag , seal it and sterilisation at 121 C for
15 mins under high pressure then throw it away .

r
9. Repeat experiment and calculate mean ( taking several diameter measurements and

ab
calculate mean).
10. Risk factors: bacteria might mutate becoming pathogenic , contamination of culture
by microorganisms ( pathogen from environment)
lG
1. Suggest a reason for including the control discs in this investigation.
To allow comparison and to check that the paper discs themselves were not

responsible for the inhibition of growth. The control discs enable us to be sure that The
iha
only factor that was different was the independent variable.

2. Explain why the plates should be incubated at a temperature lower than 30 °C
.N

Temperatures above 30 °C are closer to human body temperature, so there is a risk of incubating
human pathogens which could infect you.

Dr

3. Sterile (aseptic) technique is important in microbiology. Explain why this is the case
even in this activity, where the bacterial species have been selected because they are
harmless to humans..

• Aseptic techniques are vital in microbiology to ensure there is no contamination of cultures by

microorganisms from the environment, and that people and the environment are not
contaminated by the microorganisms being handled. Even cultures thought to be low risk
should be treated with caution because:

• bacteria may mutate to form pathogenic strains

• our knowledge of the hazards may be incomplete

• the culture may have become contaminated.

4. Based on the class data, suggest which of the two types of plant material had the
stronger effect on the growth of bacteria.

Dependent on student results but must include reference to the size of the clear zone.

055
Dr.Nihal Gabr 038
5. Explain why measuring the area of the clear zone around each disc is more
accurate than measuring the diameter of the clear zone.
The shape

 of the clear zone may be irregular, so the diameter may not represent an
accurate measurement. Measuring the area of the clear zone enables a fair

comparison to be made between discs.

6. The table shows data obtained from an investigation into the effectiveness of tea tree
oil and garlic extract at inhibiting the growth of two species of bacteria, using the disc
diffusion method.
Replicates were carried out and the mean diameters of the zones of inhibition found.

r
(a) Comment on the way in which the data have been recorded in the table.(2 marks).

ab
All numbers should be recorded to the same degree of precision. They

should be recorded to the same number of decimal places so should be 22.50, 32.00, 25.00,
39.50. (1) The data from the replicates should havebeen recorded and then the mean values
lG
shown. (1)

(b) Calculate the area of the zone of inhibition for tea tree oil when used on S. albus.(3 marks)
Radius = 22.5/2 (1)

Area = 3.14 × (22.5/2)2 (1) = 397.66 mm2 (1)

Forms:{int
iha
•

punier
.

(c) Calculate the percentage increase in effectiveness of garlic extract at inhibiting The growth of E.
coli compared to inhibiting the growth of S. albus (2 marks).
.N

• 39.50 − 32.00 = 7.5 (1)

• 7.5/32 × 100 = 23.44% (23%) increase in effectiveness (1)

Dr

(d) Tea tree oil is poisonous if taken internally. It contains many compounds, including
terpenes, which produce the characteristic odour. These are unsaturated
hydrocarbons containing a cyclic molecule with the formula C10H16.
The antibacterial ingredient in garlic is allicin, produced when alliin in chopped garlicis acted upon
by the enzyme alliinase. Its formula is C6H10OS2.

(i) Discuss the problems with using the disc diffusion method to compare the

antibacterial properties of different compounds.

The active ingredients have different molecular sizes and so this affects their diffusion into the agar gel. (1)

- -

Tea tree oil may have many different ingredients. (1)

Some # of the chemicals may remain bound to the paper disc. (1)

(ii) Discuss the problems of using data from the disc diffusion method to promote these products
as effective antibacterials.

000 Because a substance works in a Petri dish does not mean it will work in the

same way on skin. (1)

⑧ Garlic is not poisonous but when ingested may be digested by enzymes and the active ingredient
u

may not remain intact / active. (1)

•
Tea tree oil is poisonous so cannot be used internally. (1)
056
Dr.Nihal Gabr 039
 Core practical 3
Investigating effect of temperature and alcohol on the cell
membrane
 Core practical 3

Investigating effect of temperature and alcohol on the cell membrane

Safety :
1. Ethanol is highly flammable ..keep it away from the flame and keep the stoppers on the bottles .
2. Wear eye googles

A) effect of temperature : 10 20 30 no 50°C

1. Carry out the experiment at 5 different temperatures ( 10 C to 50 C) using a thermostatic water bath
2. Get 5 test tubes of same size and add equal volume of water
3. Cut 5 pieces of beet roots ( containing Anthocyanin pigments ) using a cork borer all of same size and
from beetroots of same species and age
4. Rinse and dry to remove pigments from surface due to cutting
5. Allow equilibration of the 5 test tubes of water to reach to experimental temperature by leaving them in
the water bath at required temperature for 5 mins.
6. Add the beet root pieces to the water
7. Use the colorimeter to measure the degree of absorbance
Zero the colorimeter using a blank cuvette filled with distilled water .

É
is
§
jÉ me

¥ ¥

Temp Temp
 Absorbance

Sudden steep increase in rate of diffusion when protein molecules in the cell membrane
denatures so begining to lose their tertiary structure .

A at low temperature
Cell membrane / tonoplast still intact
Pigments are large molecules
So too large to pas through membrane

As temperature increase at B :
1. Increase in kinetic energy of molecules
Increase movement and diffusion of pigment molecules
2. Phospholipid membrane becomes more fluid ….by decreasing interaction between fatty acid tails ..xo become
more loosely packed ..
SO < INCREASE IN PERMEABILITY of the membrane so more pigment molecules can diffuse outside so % of
light absorbance increase , light transmission decrease .
At ver high temperature :
The protein molecules in the cell membrane becomes totally denatured by disrupting the bonds holding
the tertiary structure in place , also the phospholipid melt .
Gaps are formed i the membrane though which pigments flood out

At C ….the level of transmission / absorbance level off /out As the concentration of pigments inside
is equal to that outside the cell ( equilibrium ) .

B) effect of alcohol :

1.Carry out the experiment at 5 different alcohol concentrations .

2. Get 5 test tubes of same size and add equal volume of alcohol
3. Cut 5 pieces of beet roots using a cork borer all of same size and from beetroots of same species
and age
4. Rinse and dry to remove pigments from surface due to cutting
5. Leave them in the different alcohol concentrations for 15 mins.
6. Use the forceps to remove the cylinders from each tube …and discard the cylinders but keep the
supernatant liquid .
7. Use the colorimeter to measure the degree of absorbance
Zero the colorimeter using a blank cuvette filled with distilled water .
 •
Ñ

QQ
*
g.gg

Explain the results

QQ
9 a-

¥
1. Phospholipid are soluble in alcohol , where alcohol dissolve the fatty acids in cell membrane
Altering the hydrophobic orientation …increasing the fluidity of cell membrane
2. At very high alcohol concentration , protein molecules will get denatured, resulting in gaps or holes in
8,9 the membrane which allow more pigments to diffuse out of the cell …increasing rate of absorbance .
Decreasing light transmission .

Variables should be kept constant

1. Age of beet root and species
2. Size of beet root cylinders
3. Time of equilibration.
4. Time of soaking
5. Volume of water / alcohol
6. Volume of solution added to cuvette
Core practical 3
2A.1 investigating membrane properties:


Objectives:

To know how the effect of temperature and alcohol on membranes can be determined.
To be able to recognise quantitative variables that should be controlled in an investigation .

Safety:

1. Wear eye protection.

2. Water baths at temperatures above 50 °C may scald. Take care when removing lids to
allow steam to escape away from the face or body. If you are splashed by hot water, or
scalded by steam, cool under cold running water immediately.
3. Take care with sharp items such as the cork borer and knife. Always cut or push
downwards onto the tile.

r
4. Ethanol is highly flammable so keep away from naked flames and keep the stoppers on

ab
bottles.
5. Do not handle electric plugs, sockets or switches with wet hands.

Procedure:
lg
iha
.N

Uff
Dr

A. Effect of temperature:

Procedure:
1. Set six water bathes at a range of temperature (0°C, 10 °C, 20°C, 30°C, 40°C, 50°C).
2. Get 5 test tubes with equal volume of distilled water 10 cm3.
3. Allow equilibration (leaving in each water bath for 5 mins) to reach experimental
temperature. equilibration .

4. Get 5 beet root pieces all cut to same size, and all should be of same age.
5. Rinse then dry to remove pigments from surface due to cutting.
6. Add beetroot pieces to test tubes.
7. Then use the colorimeter to measure degree of light absorbance.

Dr.NIhal Gabr 105

A. Effect of temperature:

Results:
A. At low temperature:


Tonoplast / cell membrane of vacuole remain in tact.
Betalain /pigment molecules are too large to pass through membrane easily.
So high light transmission.

B. At higher temperature:
Kinetic energy of molecules increase
Increasing movement and diffusion of pigment molecules

Phospholipid of membrane becomes more fluid, bond/interaction between fatty acid tails
begin to separate .
Increasing permeability of the membrane .
So pigment molecules can pass more freely through the cell membrane to the liquid outside.

r
So less light can pass through liquid.

ab
C. At very high temperature:
The protein molecules in the membrane become completely denatured (losing their tertiary
structure by disrupting the bonds that hold their tertiary structure in place), (also
lg
phospholipid may melt) and gaps are formed in the membrane through which the pigment
can flood out .
The change in transmission levels out as the concentration of pigments is the same inside
iha
and outside the cells(equilibrium).

rate of diffusion percentage transmission of light

.N

C
18 16 A
Dr

13.5 12 B
B
9 8
C

4.5 A 4

0 0
10 20 30 40 50 60 70 10 20 30 40 50 60 70
Temperature /°C Temperature /°C

Dr.NIhal Gabr 106

 B. Effect of alcohol:

Procedure:
1. Take five test tubes and add 10 cm3 of ethanol to each one. Use a different
concentration

 of ethanol in each tube (distilled water can be used for a 0%
concentration). ↳
using serial dilution method .

2. Use a cork borer to cut five beetroot cylinders. Use a knife, ruler and white
tile to trim them all to the same length (1 cm is sufficient).
Wash the cylinders thoroughly with water until the water runs clear, then gently pat dry
with a paper towel, to remove pigments from surface due to cutting.
3. Add one beetroot cylinder to each of the five tubes and leave for 15 minutes.
4. Shake the tubes once. Then, working quickly and carefully, use forceps to remove the
cylinder from each tube.
Discard the cylinders but keep the supernatant liquid (the clear liquid above the solid). It

r
may be easier to decant this liquid into clean test tubes.

ab
5. Set the colorimeter to a blue/green filter and percentage transmission. Zero the
colorimeter using a blank cuvette filled with distilled water.
6. Transfer liquid from each test tube in turn into a colorimeter cuvette, place in
lg
the colorimeter and take the percentage transmission reading. Record your results in a
suitable table.
iha
A. Effect of alcohol:

Results:
.N

The change in alcohol concentration affects the integrity of the phospholipid bilayer.
Because
Dr

1. phospholipids are soluble in alcohol, where alcohol dissolves the fatty acids in cell
membrane,
2. so the membrane loses the ability to orientate towards and away from water
( altering the hydrophobic interaction)
3. The lose of hydrophobic and hydrophilic interaction disrupt the phospholipid bilayer,
4. As well as at very high concentration ,proteins in cell membrane denature.
5. resulting in holes forming in the membrane which allow the pigment out of the
cells.
6. The increase in pigment outside the cells reduces the ability of light to penetrate
the solution therefore decreasing transmission.

Dr.NIhal Gabr 107

1. List the variables that were controlled during the experiment and state
how

 they were controlled. This could be done using a table. Important
The variables controlled during the experiment are:
questions

- the volume of bathing water or ethanol in each tube (10 cm3)

- the surface area and volume of the beetroot cylinders (dependent on size of
cork borer; 1 or 2 cm in length)

- the equilibration time in the temperature experiment (5 minutes)

- the soaking time for the cylinders (15 minutes)

- the volume of coloured liquid in the cuvettes (e.g. 4 cm3)

- the colorimeter filter / wavelength used (blue / green)

- the part of the beetroot the core was taken from (e.g. the centre)

- the age, variety and storage time of the beetroot (the same beetroot or beetroots from the
same batch may have been used).

2. Suggest why the tubes were placed in the water baths for 5 minutes before the cylinders

r
were added.

ab
The temperature must be equilibrated to ensure the tubes contain water at the correct

temperature before the experiment begins. This allows confidence that the effect of The correct
temperature is being assessed.

experiment began?
lg
3. Why were the beetroot cylinders washed with distilled water and dried before the

• The cylinders are washed and dried to remove excess surface pigment from the cut

• cells at the edge. This excess pigment would distort the transmission readings, giving inaccurate
iha
results.

4. Use the trend line of one of your graphs to describe the effect of temperature or alcohol
concentration on the percentage transmission.
The percentage transmission decreases as the temperature rises. Initially there is little increase, but
.N

at around 40–60 °C the percentage transmission decreases sharply. You should use values from
your own graph. At higher temperatures, the rate of decrease

usually levels out.

Dr

The percentage transmission decreases as the alcohol concentration increases. At low

concentrations of alcohol (0%–10%) there may be little noticeable effect but as the

concentration is increased the decrease in transmission should be proportional until

Leveling off between 60%–80%.

5.Explain your results in detail in terms of what is happening to the beetroot membrane.

Answer using the effect of temperature and alcohol parts mentioned before☝ .

Dr.NIhal Gabr 108

6. Describe how you would investigate the effect of acetic acid on beetroot
cell membranes. (6)
1. Same equipment and protocol but use different concentration of acetic Important
questions
acid. Award



marks for hypothesis, equipment, procedure, IV and DV, control variables, how
to deal with the data. (6)

7. Explain how (a) high temperatures and (b) ethanol damage cell membranes. Make
reference to the fluid mosaic model in your answer. (4 marks)
Any four from:

1. Cell membranes (surface and around organelles, e.g. vacuole) consist of proteins

2. floating in a phospholipid bilayer. (1)

3. High temperatures can denature the proteins by disrupting the bonds that hold their
tertiary structure in place. (1)

4. High temperatures increase the fluidity of the phospholipid bilayer and may melt the
lipids, causing gaps in the bilayer. (1)

r
5. Ethanol at high enough concentrations may denature some proteins by altering

6. hydrophobic interactions. (1)

ab
7. Alcohol disrupts the phospholipid bilayer and this is more severe in plant cell

8. membranes as they lack cholesterol which helps stabilise the membrane. (1)

lg
8. The cellulose cell walls of plant cells are permeable whereas the cell membranes of plant
cells are partially permeable.
Explain the meaning of the terms
iha
(a) permeable and
(b) partially permeable.
(2 marks)
Permeable: allows any type of / size of molecule to pass through (1)

.N

Partially permeable: only allows some molecules (of certain size or type) to passthrough (1)

Dr

9. By what process does water pass across cell surface membranes?(1 mark)m

Osmosis. (1)

10. Complete the table to compare active transport, facilitated diffusion, exocytosis and
endocytosis.

Dr.NIhal Gabr 109

 Some key ideas about graphs

If the particles can move through the lipid bilayer by



A simple diffusion, then there is no limit to the number
that can fit through the membrane. The rate of
diffusion increases linearly as we add more particles
to one side of the membrane.

If the particles can only pass through protein channels

by facilitated diffusion, then the rate of diffusion is
determined by the number of channels as well as the
number of particles.
Once the channels operate at their maximal rate, a
further increase in particle numbers no longer
increases the apparent rate of diffusion. At this limited

r
rate we describe the protein channel as being

ab
saturated.

B
lg At Point A :steep increase in concentration of substance
X

Due to highest concentration gradient so highest rate of

C
iha
diffusion.

B
At point B : less steep increase in concentration of
substance X

Due to lower concentration gradient so lower rate of

.N

diffusion

A
At point C: no change in concentration of substance X

Dr

Equilibrium is reached this no diffusion occurred..

Dr.NIhal Gabr 110

Core practical 4
Core practical 5
Core practical 7

Core practical 4

Dr. Nihal Gabr

 Factors affecting enzyme activity
1. Temperature
2. pH
3. Concentration of enzyme
4. Concentration of substrate
Rate of
reaction At higher substrate concentration
Enzyme concentration becomes a limiting factor
All active sites are occupied
Maximum number of ESCs
Substrate is no longer a limiting factor

At low substrate concentration: Substrate

Substrate concentration is a limiting factor. concentration

Few collision between enzyme an substrate
More Free active sites
Fewer ESCs .
Yet, increasing substrate concentration ….more active sites will
Rate of reaction
be saturated ..increasing number of ESCs.

Time
1. To be able to measure the initial rate of enzyme activity
2. To understand why measuring the initial rate is important
3. To understand the variables that can affect the rate of an enzyme-
catalysed reaction and the result
of changing each variable
4. To be able to calculate the rate of a reaction using the gradient of a
line.

Safety:
1. Wear eye protection.
2. Avoid skin contact with the trypsin solution. If you get it on your skin, wash it
off with cold running
water.
3. Hot water from the water baths may be a hazard. If a water bath is above 50
°C, take care not to scald yourself.

Procedure:

2cm's
trypsin
+2cm milk
? .

A ; effect of different temperatures on rate of enzyme catalysed reaction

1. Carry out the experiment under 5 different temperatures ( 10, 20, 30, 40, 50 60 C) and
control temperature using a thermostatic water bath
2. Get 5 test tubes and add equal volume of ( 2cm3) of 1 % trypsin
3. Get another 5 test tubes add equal volume 2cm3 of milk to each one
4. Allow equilibration ( leaving test tubes for 5 mins so they reach to the required temp)
5. Place 2 cm3 of trypsin and 2 cm3 of distilled water in a cuvette and set the colorimeter to
zero absorbance .
6. Mix the trypsin solution and the milk by shaking gently , then place the solution in the
colorimeter and start reading the absorbance of light .
7. Measure immediately at at 15 seconds intervals for 5 mins
Repeat with the other 4 test tubes ( make sure to wash the cuvette with distilled water)
Rememebr to use the reference cuvette to zero the colorimeter before each new set of readings.
 B
Effect of different pH on rate enzyme catalysed reaction:
1.Select the buffer solutions for the pH values being investigated ( 2,4,6,8,10). Place 1 
Y
cm3 of trypsin solution, 1 cm3 of buffer solution and 2 cm3 of distilled water into a cuvette.
Use this as a reference cuvette to set the colorimeter absorbance to zero.
2. Add 1 cm3 of trypsin solution and 1 cm3 of your first buffer solution to a cuvette.
3. Measure 2 cm3 of milk suspension into a second cuvette.
4. Add the mixture made in step 2 to the milk in the cuvette. Working quickly, mix the solution and
the milk by shaking gently, then place the solution into the colorimeter and start the data logger.
5. Measure absorbance immediately and then at 15-second intervals (more frequently if recording
electronically) for 5 minutes, or until there is little change in absorbance.
6. Rinse the cuvette with distilled water and repeat steps 2–5 for each of the other four pH values.
Remember to use the reference cuvette to zero the colorimeter before each new set of
readings. Record the data collected in a results table.
i

C Effect of different enzyme concentration on rate enzyme catalysed

r
ab
reaction:
1. Prepare five different concentrations of 4% trypsin solution using serial dilution ( 4%,
2%,1%,0.5% and 0.25%)
lg
2. Place 2 cm3 of trypsin solution and 2 cm3 of distilled water in a cuvette. Use this as a reference
cuvette to set the colorimeter absorbance to zero.
3. Measure 2cm3 of milk suspension into a second cuvette.
iha
4. Add 2 cm3 of your first trypsin solution to the milk in the cuvette. Working quickly, mix the
trypsin solution and the milk, then place the cuvette into the colorimeter and start the data
logger.
5. Measure absorbance immediately and then at 15-second intervals (more frequently if recording
.N

electronically) for 5 minutes, or until there is little change in absorbance.

6. Rinse the cuvette with distilled water and repeat steps 3–5 for each of the other four
concentrations. Remember to use the reference cuvette to zero the colorimeter before each
Dr

new set of readings. Record the data collected in a results table. Trypsin + water

D Effect of different substrate concentration on rate enzyme catalysed

reaction:
1. Prepare five different concentrations of 4% milk solution using serial dilution ( 4%,
2%,1%,0.5% and 0.25%)
Same procedure as above .
2. Get 5 test tubes and add in each equal volume of 4% trypsin
3. Add to same test tubes same volume of different substrate concentrations …mix then together by gentle shaking
4. Control temperature using a thermostatic water bath
5. Leave for 5 mins ..then take equal volume from each test tube into a cuvette to measure the degree of light
absorbance.
6. Don’t forget to to zero the colorimeter using a cuvette with trypsin and water each time before measuring
absorbance at each substrate concentration
Dr.NIhal Gabr 132
1. What were the independent and dependent variables in this investigation?
Independent: depends on the investigation but appropriate answer from: temperature,
pH, trypsin concentration, substrate concentration

Dependent: rate of reaction in absorbance units s−1

2. Ideally, you would repeat the procedure for each factor several times. Explain why it is
important to measure the initial rate of the reaction rather than an average rate over a
longer time period. ?
Because the reaction is rapid and the milk (substrate) concentration quickly declines.

The rate slows as the substrate is used up. Therefore, it is only possible to make valid
comparisons at the start of the reaction, when controlled variables such as substrate

concentration are the same for all levels of the independent variable.

3. If the surface of the cuvette is scratched, this can result in a greater absorbance of
light. If the cuvette used for the reaction was scratched (but the reference cuvette was
not), would this give a random or a systematic error? Explain your answer.

r
• A systematic error, because it would cause absorbance readings to be higher than the true

ab
value for every measurement.

4. Suggest one variable that would normally be controlled in an enzyme-catalysed

reaction but which has not specifically been controlled in your investigation. Explain
lg
why this variable would usually be controlled carefully and suggest how this could Be
done.

For the pH investigation: The rate of reaction of enzymes varies with pH, due to changes in the
shape of the active site. An enzyme has the highest rate of reaction at its optimum pH. A buffer
iha
might be used to maintain pH at a suitable level.

For the temperature, substrate concentration and enzyme concentration investigations.

.N

Temperature, because the rate of reaction of enzymes varies with temperature. As

temperature increases, particles gain more energy and more collisions take place

between enzyme and substrate particles.

Dr

Enzymes have an optimum temperature at which the rate of reaction is at its peak.

Above that temperature, enzymes will begin to denature, changing the shape of the active site
and preventing further catalysis. A water bath and thermometer could be used to maintain a
suitable temperature.

Dr.NIhal Gabr 133

5. When the reactivity of an enzyme is unknown, a pilot study using serial dilutions
may be carried out.
(a) Explain the term ‘serial dilution’ (2marks).
A stepwise dilution of a solution (1)

The dilution factor at each step is constant / the concentration decreases by the same
factor with each step. (1)

(b) Explain the advantages of using serial dilutions. (3 marks)

The dilutions cover a wide range (and are therefore good for pilot studies). (1)Dilutions are
easy to carry out as the steps are similar in each case. (1)

Dilutions cover the range evenly.. (1)

(c) . Describe how you would carry out a serial dilution of the 1% trypsin stock solution to
make concentrations of 0.1%, 0.01% and 0.001%. You may use a labelled

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diagram to help with your explanation. (5 marks)

Mix 1 cm3 of the 1% stock with 9 cm3 of distilled water. (1)

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This produces a 10-fold dilution / a 0.1% solution. (1)

Then mix 1 cm3 of the 0.1% solution with 9 cm3 of distilled water to make the 0.01% solution. (1)

Then mix 1 cm3 of the 0.01% solution with 9 cm3 of distilled water to make the 0.001% solution.
(1)

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Mention of suitable equipment for measuring volumes, e.g. pipette. (1) Allow other suitable
volumes that would give dilutions by a factor of 10. Marks can be awarded for a diagram.

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(d) What type of scale would you use when plotting these concentrations on a
graph?
A logarithmic scale (1)
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PH change =change in hydrogen ion concentration

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Dr.NIhal Gabr 134

Core practical 5
Microscope
Cell measurement
Core practical 5
3A.1 using a graticule:
Objectives:
1. To be competent in the use of a microscope at high

 and low power, including the use of a graticule
(eyepiece micrometer) to make measurements
2. To know how to record observations using appropriate biological drawings
3. To understand the importance of staining specimens in microscopy.
Safety:
1. Methylene blue may be harmful if swallowed but is not otherwise classified as hazardous. However.
2. Avoid skin contact with iodine and methylene blue stains. Wear gloves and eye protection when
handling the stain. Clean up any spills immediately.
3. Make sure all biohazardous material is placed in disinfectant solution as soon as possible. Cotton
buds should only be used once, by one individual.
4. If you are using a microscope with daylight illumination (a mirror) do not place it where sunlight
might strike the mirror as this will damage your retina and may cause blindness.

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Procedure:

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First allow calibration
1. Place the micrometer slide on the stage of microscope and focus on it using low power objective
lens (10χ).
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2. Move the micrometer slide and rotate eye piece to align the scale of the eye piece graticule and
stage micrometer in the field view.
3. Count eye piece unit (epu) on eye piece graticule which is equivalent to (100µm ) divisions on
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stage micrometer.
4. Measure cell using eye piece scale and convert into length unit (µm)
- For example,if 100 µm is equivalent to 2 epu,
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- then 1 epu=100 / 2= 50 µm at this magnification.

5. Repeat steps 1 -3 using medium and high power objective lenses.
Dr

• so stage micrometer. Calibrate eyepiece

• Eye piece graticule. Draw cells with correct proportions
• Together allow accurate measurements to be taken.

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028
Dr.Nihal Gabr
 Second plant /animal tissue preparation:
1. Wash your hands with water an soap
2. Put sample on glass slide. 

Precaution: cut away from fingers to avoid injury , use wet scalpel to minimise friction and
reduce damage to tissue.
3. Sample from

From Cheek IT Plant tissue

1. On a chopping board , use a scalpel to

1. Using cotton buds
cut a transverse section of tissue .

2. Gently rub it on inside of cheek .

2. Keep sample as thin as possible to
3. Rub the cotton bud in the small circle allow light to pass through it .

center of glass slide.

3. Use water droplet to hold your sample.

mouwintn water

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4. Put cotton bud in disinfectant.
4. Add tissue on slide .

5. Add few drops of methylene blue then

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5. Use forceps to gently lower the cover

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cover with cover slip. slip.

6. Remove excess water using absorbent.

Third examine a specimen under microscope:
1.
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Place specimen slide on stage under the lens (low power).
- No questions
2. Switch on light below specimen, and examine the stained slide. Yet .

3. While looking though eyepiece , use the coarse focus knob to focus.
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4. Then use the fine focus until a clear view of cell is established.
5. Sketch few cells (use the eyepiece graticule to measure diameter of cell).
6. Add a title and include magnification
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7. Change to medium power objective lens to locate finer details .

8. Finally use higher power objective lens to focus and spot fine details.
Dr

9. Draw and label details on cells accurately .

Tips for drawing:

1. Use sharp HB pencil.
2. Never shade or colour.
3. Lines drawn clear and continuous ( not feathery or sketched).
4. Start with outline . Keep it large ( covering more than 50% of space provided).
5. Make sure proportions are proper.
6. Label line drawn in pencil , ruler with no arrow heads, just touching item to be labelled, and
shouldn’t intersect.

029
Dr.Nihal Gabr
Overall uses of microscopy:
1. Cell and tissue identification.
2. Check for foreign organisms/ bodies.
3. Size , measurements of structures.



Errors:
1. In slide preparation: sample contamination , artefacts, wrong staining , dirty slides/lens.
2. In structure identification: leading to misdiagnosis.
3. Calculation errors or inability to use graticule.

Limitations:
1. Bad preparing , staining or specimen presentation.
2. Some features are not stainable.
3. Poor resolution or magnification of microscope.
4. Improper focus leading to blurred image.

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5. Only a sample of tissue is used, not all tissue leading to misdiagnosis.

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Improvement:
1. Careful preparation.
2. Recheck calculation.
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3. Calculate mean , measure more cells to estimate more reliable mean.
4. Measure volume of cells would be a better description of size than linear diameter.
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Reasons for staining:
1. Add contrast to image.
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2. To locate / see the position of particular type of chemical component.

3. To differentiate between different types chemicals/ components/ tissues ( make it easier to
identify type of cells or parts of cells under microscope).
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4. To identify presence of chemical components of interest.

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Dr.Nihal Gabr
Rey
1. Use the scale bar on your diagram to calculate the magnification of
the image you have drawn. Important
questions


This will depend on the size of the drawing and the objective used. You should
divide the image size (length of scale bar measured with a ruler, converted to
µm) by the

actual size (the length that the scale bar represents in µm).

2. Explain briefly how cells can be measured using a microscope.

A suitable summary might include the following points.

- Use a stage micrometer and eyepiece graticule (eyepiece micrometer).

- Calibrate the eyepiece graticule with the objective lens that will be used. Do this by measuringk
the eyepiece scale against the scale of the stage micrometer.

- Measure the cell using the eyepiece scale and convert into length units (µm).

3. Suggest improvements to the method that was used to describe the size of cells in a
tissue.

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• For example:

• Calculate a mean. Measure more cells to estimate a more reliable mean.

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• The volume of the cells would be a better descriptor of size than the linear dimensions.

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Dr.Nihal Gabr
pay
4. Under a high-power objective lens, 300 µm on a stage micrometer was Found to be
equivalent to 87 eyepiece units.
(a) What is the size of each eyepiece unit in µm? (1mark)
(a) 3.4 µm (1)

Allow 3.45 / 3.5 µm.

(b) Using the same objective, the diameter of a red blood cell was measured as2.5 eyepiece
units. What is the actual diameter of the red blood cell?
(2 marks)
(b) 2.5 × 3.4 = 8.5 (allow in range 8–9) (1)

µm (1)

Allow error carried forward from part (a).

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5. A cell from epithelial tissue in the trachea was found to be approximately cylindrical in shape.
The cell had a length of 20 µm and a diameter of 4 µm.
The volume (v) of a cylinder may be calculated from v = πr2h.
Calculate the volume of the cell. Show your working. (3marks)
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r2 = 2 × 2 = 4 (1)

v = 3.14 × 4 × 20 = 251.36 (allow 251 / 251.4) (1)

µm3 (1)

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Award 3 marks for a correct calculation with units but no working.

6. State the purpose of staining specimens in light microscopy.(2marks)

Award up to 2 marks for any of the following:

.N

To add contrast to the image (1)

To identify the presence of chemical components of interest (1)

To locate / see the position of particular types of chemical component (1)

Dr

To differentiate between different types of chemicals / components / tissues (1)

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Dr.Nihal Gabr
Core practical 7
Drawings
Core practical 7
4A.1 observing plant cells under microscope:
Objectives


1. To be able to use a microscope competently to observe biological specimens
2. To learn how to draw and label plan diagrams accurately
3. To be able to identify sclerenchyma fibre, phloem, sieve tubes and xylem vessels
Safety:
1. Wear eye protection.
2. Avoid all skin contact with the toluidine blue stain; disposable gloves may be worn.
3. Take care when using razor blades and mounted needles and ensure you hand them back to
your teacher when you have finished with them.
4. If you are using a microscope with daylight illumination – a mirror – you must not put the
microscope in a position where direct sunlight might strike the mirror and be reflected into the
eyes through the microscope. This could cause permanent retinal damage or blindness.

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Stain:

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1. Toluidine blue O is a dye. It is a metachromatic stain and interacts with different chemical
components of cells to produce a variety of colours.
• It can help us distinguish between phloem and xylem tissue as it stains the pectins in primary
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cell walls of phloem cells pinkish purple and stains the lignin and tannins of secondary cell
walls, found in xylem vessels, blue or blue-green.
• This is an example of differential staining – different types of cells are stained differently. This
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stain also shows up nuclei, plastids such as chloroplasts, and vacuoles.blindness.

2. Acetic orcine, hematoxylin, acetocarmine, methyl blue.

• These stains absorb more light making it easier to see the structures.
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• And allow making the transparent structures visible.

Dr

105
Dr.Nihal Gabr
÷
Procedure :
1. Collect a piece of plant stem. Add a few drops of water to the centre of the white tile and wet
the razor to reduce friction.


2. Cut several transverse sections (across the stem ) keeping them as thin as possible.
3. Use a brush to transfer the sections to water in a watch glass.
4. Place a thin section on a slide, and add a drop of water.
5. Add two drops of toluidine blue O stain and leave for 2-4 mins.
6. Then add cover slip and gently remove excess stain with a paper towel.
7. Turn the objective lens to low power . Examine the slide under the microscope.
8. Then while looking through the eyepiece , focus using the coarse focusing knob.
9. Use the fine focus until a clear view of the section is established.
10. Draw a plan diagram without including any cell details .

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Dr.Nihal Gabr
106
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Cell organelles:

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Dr.Nihal Gabr
teo
llenchyma .

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Palisade mesopnyll

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cwemmma .
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1. Suggest why a stain was used on the stem and root cells.?
The thin sections of stem and root do not absorb much light, so they are difficult to see
using a light microscope. The stain absorbs more light, making it easier to see the



structures. Not all of the cells are stained so differences can be seen.

2. Explain why it was important not to use too much stain when preparing the slide.
If too much stain is used, it will be difficult to see anything using the microscope. Any
differences between the cells will be hidden as the stain will absorb all the light.

3. Describe a method for comparing the size of the xylem tissue in the root and stem
samples. :

• By using a micrometer slide and an eyepiece graticule. The graticule is calibrated using the
micrometer slide. The size of the xylem tissue can then be calculated and compared.

4.
(a) Complete the table to compare and contrast phloem tissue with xylem tissue(5
marks)

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←

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+ hydrostatic pressure

(b) The movement of substances in the phloem is called translocation. gradient .

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I)Describe how the companion cells in phloem contribute to translocation.( 2 marks)

Translocation in the phloem happens through tubes connected in chains called the sieve-
tube element. The sieve-tube element is next to the companion cell.

Dr

The companion cells are connected to the sieve-tube element with

plasmodesmata, which allows materials to flow between the cells. (1)

The sieve-tube elements are alive, yet they lack organelles such as nuclei or

ribosomes. The organelles of the companion cells carry out all the metabolic

functions of the sieve-tube element. Without the companion cells, translocation would not
be possible because the sieve-tube element would die. (1)

ii) . Explain how the companion cells are adapted to their function..(2 marks)
RER
•

The companion cell is able to carry out translocation because it usually has a

larger number of ribosomes and mitochondria than a typical plant cell. This

Mitochondria
makes the companion cell more metabolically active than a 'typical' plant cell
Plasmodesmata
-

and it is therefore able to carry out translocation. (1)

Nucleus
The cytoplasm of a companion cell is connected to the sieve-tube element by
i.
.

plasmodesmata. This allows materials to flow between the companion cell and the sieve tube
element. (1)

Folded cell . mcnh .

109
Dr.Nihal Gabr
 Read
Sieve tube elements are connected with the companions cells by the plasmodesmata.....which
is a cytoplasmic bridge between them allowing the diffusion of sucrose ( assimilates) by
symplast pathway from the companion cells to sieve tube elements .
Sieve tube elements have peripheral cytoplasm with no nuclues and fewer cell organelles
offering little resistance to mass flow
Companion cells have mitochondria for aerobic respiration to produce ATP for the active
loading of sucrose ...RER ...cell membrane of companion cells have many foldings to
increase surface area to transport more sucrose ....

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companion ""
Sieoetube
element

sucrose .

{
secondary
active transport