UNIT 4: ENERGY, ENVIRONMENT, MICROBIOLOGY AND IMMUNITY

TOPIC 5 – ENERGY FLOW, ECOSYSTEMS AND THE ENVIRONMENT

PHOTOSYNTHESIS
- It is the process by which green plants capture sunlight energy using chlorophyll and use it to
convert co2 water into simple sugars
Light energy
Carbon dioxide + Water Glucose + Oxygen
Chlorophyll

Light energy
6 CO2 + 6 H2O C6 H12 O6 + 6O2
Chlorophyll

-It occurs in the chloroplasts

CHLOROPLASTS

• They are large organelles surrounded by a double membrane (chloroplast membrane /

envelop).
• The outer membrane is smooth and fully permeable to molecules like CO2, magnesium ions
and H2O.

It has many open protein channels (fixed protein channels) that allow movement of molecules.
• The inner membrane is smooth and partially permeable i.e. regulates passage of substances
in and out of chloroplasts e.g. sugars and proteins.
It contains many transporter protein molecules and carrier proteins. These membrane
proteins regulate the passage of substances such as sugars and proteins synthesized within
the chloroplast, in and out of the chloroplast.
• In addition, chloroplasts have a system of membranes called thylakoid membranes. These
contains photosynthetic pigments. The photosynthetic pigments are attached to proteins. The
protein and the pigment is called photosystem.
• The thylakoid membranes are highly folded and arranged as stacks at regular intervals to
form the grana (sing. granum). This increases the surface area on which reactions take place.
The thylakoid membrane also have electron carriers these are molecules that are capable of
accepting one or two electrons from one molecule and transferring them to another in the
process of electron transport.
As the electrons are transferred from one electron carrier to another, their energy level
decreases, and energy is released.

1
 • Proteins, photosynthetic pigments and electron carriers are embedded in the thylakoid
membranes.
• The thylakoid membrane contain a protein called proton pump that actively pumps hydrogen
ions from the stroma into the thylakoid lumen to create a concentration gradient
(electrochemical gradient) for synthesis of ATP.
• The thylakoid membranes also contain an ATP synthase involved in ATP synthesis. ATP
synthase is a membrane protein that allows hydrogen ions (protons) to pass through from the
thylakoid lumen into the stroma. The ATP synthase can also combine an ADP molecule and
inorganic phosphate to form ATP.
• The thylakoid membrane enclose a fluid filled cavity called thylakoid lumen / space. The
thylakoid lumen / space contains enzymes involved in photolysis of water / splitting of water
molecules.
Thus;
- The thylakoid membrane are - the actual site for light dependent reactions
- the site for photophosphorylation
- The thylakoid lumen is the actual site for photolysis of water.
• The thylakoid membranes are surrounded by stroma which contains the enzymes involved in
light – independent reactions of photosynthesis e.g RUBISCO.
• Thus; the stroma is the site for light – independent reactions i.e. Calvin cycle
• The fluid filled interior is called stroma and contains:
- the enzymes necessary for photosynthesis -starch grain - loop of DNA
- lipid droplets -70s ribosomes

Note: - Chlorophyll pigment is arranged in the thylakoid membranes.

2
3
In summary;
Structure Function (s) Features
1. outer Fully permeable to molecules e.g. CO2 and Many open protein
membrane H20 needed for photosynthesis channels
(fixed protein channels)
2. inner Partially permeable i.e. regulates passage Transporter proteins
membrane of substances in and out of chloroplasts (gated channel proteins
e.g. sugars and proteins and carrier proteins)

3. Stroma Site for light – independent reactions i.e. Contains enzymes for
Calvin cycle Calvin cycle e.g. RUBISCO

4. Granum Site for light dependent reactions Large surface area

Site for photophosphorylation

4
 5. Thylakoid Actual site for light dependent reactions Contains photosystems
membranes Site for photophosphorylation (clusters of pigments) and
electron carriers

6. Thylakoid Photolysis of water Contains photolysis

space/lumen enzymes

7. Starch grains Carbohydrate energy store Compact, insoluble,

inert & large

8. Loop of DNA Controls protein synthesis Contains genes that code

for proteins in the
chloroplast.

QUESTION
Copy and complete the table of chloroplast functions below by suggesting, for each structure within
the chloroplast, the features that are adapted for these functions.

PHOTOSYNTHETIC PIGMENTS
- These are molecules that can absorb light energy and use it to provide energy for photosynthesis.
- Photosynthetic pigments are fat soluble.
- All types of photosynthetic pigments in plants are located in the chloroplast.
- They are a group of 5 closely related pigments namely
1] Chlorophyll a - blue green
2] Chlorophyll b - yellow green
3] Yellow xanthophyll
4] Orange carotene
5] Phaeophytin
- The chlorophyll molecules are primary pigments
-Their role is to absorb light energy and excite/emit electrons and they form a reaction centre
(RC)
- The xanthophylls, carotenes and phaeophytin are accessory pigments

5
 Their role is to absorb light energy and pass it to specialised chlorophyll molecules to increase the
rate of photosynthesis and they form a light harvesting centre (LHC)

- Chlorophyll a, is found in all photosynthesising plants and is the most abundant

- Each pigment absorb light at a different wavelength hence more light energy is available for the
plant than if only one pigment was involved. - This maximises the light absorbed by the plant
- In the thylakoid membranes photosynthetic pigments occurs in two complexes (clusters / reaction
centres) called photosystems namely;
1. Photosystem I / PS1/ P 700
2. Photosystem II /PSII/ P680
- The photosystems (photosynthetic reaction centre) have many pigment molecules that can be
designated as either reaction centre or antennae pigments.
- Each photosystem contains a different combinations of pigments which absorbs light at different
wavelengths,
- Photosystem I absorbs light at a wavelength of 700nm while, Photosystem II absorbs light at a
wavelength of 680nm

- Therefore not all light from the sun is absorbed by the plant.

- The two photosystems are linked by an electron transport chain

6
ABSORPTION SPECTRUM OF PHOTOSYNTHETIC PIGMENTS
Absorption Spectrum is a graph showing how different wavelengths of light are absorbed by the
different pigments in a leaf during photosynthesis.

7
(i) Absorption spectrum of chlorophyll a
Absorption peaks are in violet, lower part of blue and in red part of spectrum
The absorption peak in violet-blue is higher than in red
Very little absorption in the mid part of spectrum.
It reflects upper part of the blue and green hence it is blue-green in colour

(ii) Absorption spectrum of chlorophyll b

Absorption peaks are in violet-blue and red with the peak being highest in violet–blue
Very little absorption in mid part of the spectrum
It reflects green and partly yellow hence it is yellow green in colour.

(iii) Absorption spectrum of carotene

Absorbs the entire violet-blue and green and reflects all the other colours
They are accessory pigments. So, they absorb light energy in the green part of spectrum where
chlorophylls cannot and pass it (in form of electrons) to chlorophylls to increase the rate
photosynthesis.

8
ACTION SPECTRUM OF PHOTOSYNTHETIC PIGMENTS
This is a graph showing the rate of photosynthesis at different wavelengths of light

This graph shows that violet-blue and red give the highest rate of photosynthesis. However, violet
blue gives a higher rate than red.

Correlation between absorption spectrum and action spectrum

➢ From absorption spectrum, violet-blue and red are highly absorbed by chlorophyll a and b.
➢ From the action spectrum, it is the same wavelengths that give the highest rate of
photosynthesis

• Photosynthetic pigments absorb light only in the visible region of the spectrum (390nm –
760nm).
• The action spectrum peak of chlorophyll is almost same as that of absorption spectrum
indicating that is the primary pigment in photosynthesis.

9
Absorption Spectrum vs Action Spectrum
Absorption Spectrum
Absorption Spectrum is the graphic representation
of the different wavelengths of light absorbed by the
different pigments in a leaf during photosynthesis
Plot showing intensity of light absorbed relative to
its wavelength
Action Spectrum
Action Spectrum is the graphic
representation of the effectiveness of
different wavelengths of light on rate of
photosynthesis
Plot showing relative efficiency of
photosynthesis produced by light of different
wavelengths

Explains the relationship between quality of light Explains the relationship between quality of
and absorbing capacity of pigments light and absorbing capacity of pigments

Chlorophyll absorb blue and red light Carotenoids The maximum photosynthesis occurs in blue
absorb violet and blue light and red light

Absorption of different wavelengths of light by In action spectrum, the rate of

pigments can be measured using photosynthesis is measured as amount of
spectrophotometer. carbon dioxide fixation, oxygen production,
NADP+ reduction etc.

EXTRACTION OF PHOTOSYNTHETIC PIGMENTS

➢ Using mortar and pestle, grind fresh green leaves with organic solvent such as ethanol or
propanone to obtain green leaf extract that contains mixture of pigments
➢ Chloroplast pigments are organic in nature hence can be dissolved by ethanol or propanone
that are also organic.

10
SEPARATION OF PHOTOSYNTHETIC PIGMENTS
➢ It is done using paper chromatography technique
➢ Cut 2cm by 15cm chromatography paper
➢ Draw a pencil line 2cm away from the margin. This is called origin
➢ Using a dropper, add the green extract on the origin repeatedly allowing drying between
spots, until you get a concentrated pigment spot.
➢ Add 2cm3 of petroleum ether (solvent) in a beaker/boiling tube
➢ Vertically suspend the chromatography paper in the beaker/boiling tube ensuring that the
green spot does not touch the ether.
➢ Cover the beaker with a watch glass

➢ Leave the set up for 5 mins to 24 hours

➢ Solvent rises up paper
➢ Each pigment travels at different speed hence distance moved by each pigment is unique
➢ Pigments separated as they ascend

Diagram showing the plate after the solvent has moved about half way up it. The solvent is allowed to
rise until it almost reaches the top of the plate. That will give the maximum separation of the
components in this particular solvent.

11
➢ When the solvent is nearest the other end, remove the chromatography paper before it
evaporates.
➢ Immediately draw a pencil line to indicate solvent front (where the solvent reached)
➢ Let it dry and it is now called a chromatogram

12
IDENTIFICATION OF PHOTOSYNTHETIC PIGMENTS USING RF (RETENTION/RETARDATION
FACTOR) VALUE
Rf value is the distance moved by the pigment (from the origin to the centre of the pigment) divided by
the distance moved by the solvent.
This value is compared with the given Rf values in the table and corresponding pigments to identify
the pigment.

Measuring Rf values

Measurements are often taken from the plate in order to help identify the compounds present. These
measurements are the distance traveled by the solvent, and the distance traveled by individual spots.
Example 1 ;

If the red component traveled 1.7 cm from the base line while the solvent had traveled 5.0 cm, then
the Rf value for the red dye is:
Rf = Distance travelled by individual spot
Distance travelled by solvent
= 1.7cm / 5cm = 0.34

13
Factors affecting Rf values
• Temperature
• Type and purity of the solvent used
• Quality of the chromatography paper used
• Distance travelled by the solvent and solute

NOTE:
If you could repeat this experiment under exactly the same conditions, then the Rf values for each dye
would always be the same. For example, the Rf value for the red dye would always be 0.34.
However, if anything changes (the temperature, the exact composition of the solvent, and so on), the
value changes.
If the substances are colourless, they can be visualised by;
i) Using fluorescence dye
A substance that fluoresce is added to the chromatography paper, which will fluoresce when
exposed to UV light. That means that if you shine UV light on it, it will glow. That glow is
observed at the position where the spots are on the final chromatogram The spots show up as
darker patches.
While the UV is still shining on the plate, you obviously have to mark the positions of the spots by
drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear
again.
ii) Using chemicals dyes
In some cases, it may be possible to make the spots visible by reacting them with something which
produces a coloured product. A good example of this is in chromatograms produced from amino acid
mixtures. The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin.
Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple.

14
STAGES OF PHOTOSYNTHESIS
Photosynthesis is a two stage process;
Light dependent stage
Light independent stage
Reactions for light dependent stage requires light and produce materials which then used in light
independent stage.

1. LIGHT DEPENDENT STAGE

-Takes place in thylakoid membrane because that is where chlorophyll is found
- When a photon of light hits a chlorophyll molecule the light energy is taken up by electrons in the
chlorophyll molecule and the electron become excited.
- The excited electrons are raised to a higher energy level and leave the chlorophyll.
- The excited electrons leave the chlorophyll molecule and are picked by an electron acceptor and
moved along a series of electron acceptors
- The electrons release energy as they move which is used in the synthesis of ATP by a process
known as photophosphorylation

- PSII absorbs higher energy than PSI, some of which is used to split water molecules to produce H+
(protons) and OH- in a process called photolysis
- Thus, photolysis occurs in the thylakoid lumen.

❖ This stage involves;

ATP formation,
Reduction of NADP+ to NADPH
Production of oxygen

PHOTOPHOSPHORYLATION
Phosphorylation is the process of transfer of a phosphate group into ADP to synthesis ATP using light
energy.
Photophosphorylation can be:
i) Cyclic photophosphorylation
ii) Non cyclic photophosphorylation

a) Cyclic photophosphorylation

• Type of photophosphorylation whereby ATP synthesised using light energy released from
excited electrons that move through the electron transport chain and fall back to the same
molecule.
• involves only PS1
• When light hits a chlorophyll molecule in PSI, a light excited electron leaves the chlorophyll
molecule, and it’s taken up by the first electron acceptor

15
 • The electron moves along an electron transport chain through the second, the third and then
the fourth electron acceptor and finally back to PS1
• These reactions are a series of downward step each involving a different carrier molecule and
as the electrons move they lose energy which is used to drive the synthesis of ATP from ADP
and Pi.
• The ATP is synthesized on ATP synthase complexes located on the thylakoid membranes.
• It occurs when the conditions do not favour the non- cyclic photophosphorylation.
• The electrons return to the same chlorophyll molecule and become excited again
• The product of the cyclic photophosphorylation is ATP

b) Non- cyclic photophosphorylation

-It involves both PSI and PSII
-Light energy hits both PSI and PSII exciting electrons. The electrons in PSII are excited to a higher
energy level.
- The electron excited from PSII enter the first electron chain
-They are picked by the first electron acceptor designated Z and are transferred along a series of
acceptors (2nd electron acceptor, 3rd electron acceptor then to the 4th electron acceptor).
-At the end of the first electron transport chain, the electrons are transferred to PS1 (P700)
-They replace the ones that were excited and entered the second electron transport chain
- When light hits chlorophyll molecules in PSI the excited electron enters the second electron
transport chain move along carriers and are finally transferred to NADP+ (oxidized NADP), the final
electron acceptor which takes up the electron and H+ and become reduced to form NADPH

16
Note; The NADP+ is the last electron acceptor which takes up the electron and the H+ from splitting of
water molecule and it is reduced to NADPH
- Since the electrons from PSII enter PSI, PSII is left unstable and the electrons excited from PSII are
replaced by electrons from the reactions of the OH- from splitting of water molecules

❖ So, when photolysis occurs, in the thylakoids H+ and OH- are formed.
❖ The hydrogen ions (H+) are taken up by NADP which become reduced to form NADPH
❖ The OH_ react together to form oxygen and water releasing free electrons
OH- + OH- H2O + O + 2e-
OH- + OH- H2O + O + 2e-
Overall equation; 4OH- 2H2O + O2 + 4e-

In summary;

The electrons serve two functions:

• They reduce NADP+ to NADPH for use in the Calvin Cycle.
• They set up an electrochemical charge that provides the energy for pumping protons from the
stroma of the chloroplast into the interior of the thylakoid [View].

The protons also serve two functions:

• They participate in the reduction of NADP+ to NADPH.
• As they flow back out from the interior of the thylakoid (by facilitated diffusion), passing down
their concentration gradient), the energy they give up is harnessed to the conversion of ADP
to ATP.
• Because it is drive by light, this process is called photophosphorylation.
- Products of non-cyclic photophosphorylation are NADPH, ATP, oxygen and water

17
18
Differences between cyclic and non – cyclic photophosphorylation.

Cyclic Non - Cyclic

Needs light Needs light

Involves formation of ATP Involves formation of ATP

Involves flow of electrons Involves flow of electrons

Involves PSI Involves PSI

Involves PSI only Involves both PSI and PSII

There is photolysis of water

No photolysis of water

Products are NADPH, ATP,O2 and H2O

Product is ATP only

Involves one electron transport chain Involves two electron transport chains

Excited electrons come back to the same Exited electrons do not come back to the same
molecule molecule.

Last e- acceptor is P700 Last e- acceptor is NADP+

Overall; Products of light dependent stage are NADPH, ATP, oxygen and water.
Water and oxygen are by products while NADPH and ATP are used in the light independent
stage of photosynthesis where the NADPH releases the H+ to form NADP+ and ATP is
hydrolysed to ADP and pi releasing energy
- Some of the ATP is used in the light dependent reactions and some provides an immediate supply
of energy for biological processes

ATP synthesis by Chemiosmosis

• Chemiosmosis is the Movement of ions from down their concentration gradient.
• As the electrons move along the electron transport chain they release energy
• The energy released is used to pump protons through the proton pump, against a
concentration gradient from the stroma into the thylakoid lumen i.e the energy released
activates the proton pump.
• This leads to a higher concentration of protons in the thylakoid lumen more than in the stroma
• This forms an electrochemical gradient across the thylakoid membrane

19
 • The protons then move along the electro-chemical gradient into the stroma through the
enzyme ATP synthase
• The energy from this movement is used to combine ADP and inorganic phosphate by ATP
synthase to form ATP

2. LIGHT INDEPENDENT STAGE (CALVIN CYCLE)

• The reactions occurs in the stroma and uses the products of light – dependent stage (ATP
and NADPH).
• CO2 from air combines with a 5 carbon compound called Ribulose -1, 5- Bisphosphate
(RuBP)
• This reaction is catalysed by enzyme RUBISCO (Ribulose -1, 5 – Bisphosphate Carboxyale /
Oxygenase).- This is called carbon fixation
• The CO2 is said to be fixed [it cannot be reversed]
• This reaction results into a 6 - carbon compound which is highly unstable and immediately
splits into 2 molecules of 3- carbon compounds called GP (Glycerate -3-Phosphate)
• GP is then reduced by addition of hydrogen to form GALP [Glyceraldehyde 3-Phosphate ]
• The hydrogen for this reduction comes from NADPH and the energy from ATP, both products
from light dependent stage
• Some of the GALP is used to synthesise glucose ( a 6 carbon sugar)
• Some of the GALP is used to regenerate RUBP through a series of steps, to replace the
RuBP needed for the first step of the Calvin cycle. During regeneration of RUBP ATP formed
during the light dependent stage is used.
Note;
• RUBISCO is the rate limiting enzyme in photosynthesis. It’s a very slow enzyme and
therefore this reaction is the rate limiting step in photosynthesis.
• It catalyses a rate limiting step i.e. the slowest step in a metabolic which determines the
overall rate of the reactions in the pathway.
• The glucose synthesised is used for respiration or synthesis of new biological molecules such
as disaccharides like sucrose, polysaccharides like starch and cellulose, amino acids, lipids
and nucleic acids.

20
-Therefore Calvin cycle involves carbon fixation reduction reactions and regeneration of RUBP

21
Point to Note;
During photosynthesis, any excited electrons which are not picked up by electron acceptors releases
the energy in form of fluorescence.

Question
Describe and explain how reduction in RUBISCO or RuBP or CO2 in stroma causes
fluorescence and reduction in carbohydrate production.
➢ The short supply of any of these, reduce carbon fixation, reducing the number of unstable 6c
compounds that then causes the formation of less GP.
➢ Less GP means that only few NADPH will reduce GP so that most of NADP remain reduced.
This causes the electron carriers to remain reduced as they cannot pass the electrons to
reduced NADP. So, electron emitted by Chlorophyll (P700) will emit energy and fall back to
P700 by the reduced electron carriers. When these electrons flow back, they do so in form of
light. This is called fluorescence.
➢ Less GP means that only few GP will be reduced to GALP/TP by NADPH in presence of ATP.
Less GALP/TP means that only few glucose molecules will be synthesized reducing the
amount of carbohydrate formed. In addition, there is less regeneration of RuBP from
GALP/TP.

Photorespiration
RUBISCO can catalyse a reaction between RuBP and CO2 or RuBP and O2.
When CO2 levels are low and that of O2 is relatively high, RUBISCO can act as an oxygenase
combining RuBP and oxygen (i.e. RuBP becomes oxygenated rather than carboxylated).
Oxygenation of RuBP yields a 3 – carbon compound, GP and a 2 carbon compound.
One GP is produced per every reaction hence, less GP is produced and the 2 – carbon compound
formed cannot be used in the Calvin cycle. This is called photorespiration and it reduces the efficiency
of the carbon cycle.
Factors increasing the rate of photorespiration:
• Temperatures; Solubility of CO2 decreases than the solubility of O2 less CO2 dissolves.
The higher the oxygen concentration, the higher the rate of photorespiration.
• When water is scarce, stomata (partly) close, CO2 concentration does not diffuse in and
photorespiration increases

22
THE GLUCOSE PRODUCED DURING PHOTOSYNTHESIS IS USED FOR
A] Respiration
-It is broken down to release energy

B] Synthesise of new biological molecules like;

DNA, RNA, fatty acids, amino acids, NADP+, ATP
For example
During synthesis of DNA;
-The glucose molecule is converted to deoxyribose sugar
- It is also used to synthesise DNA bases in presence of nitrates
-The deoxyribose sugar and nitrogenous base combine with phosphates to form monucleotides
-The mononucleotides link by phosphodiester bonds through condensation to form a DNA strand
- Some of the glucose is broken down to release energy needed for the process.

Diagram showing some of the fates of the glucose made in photosynthesis

HOW OF GALP IS USED IN THE SYNTHESIS OF;

i) DNA
- GALP is converted to glucose
- Glucose is converted to deoxyribose
- GALP It is also used to synthesise nitrogenous bases in presence of nitrates from the soil
- Deoxyribose, nitrogenous base and phosphate absorbed from the soil by the plant roots join by
covalent bonds to form mononucleotides
- Nucleotides join by phosphodiester bonds to form polynucleotides / DNA strands
- Two antiparallel DNA strands join by hydrogen bonds between the base pairs to form a double helix
structure of DNA

23
ii) RNA
- GALP is converted to glucose
- Glucose is converted to ribose
- GALP It is also used to synthesise nitrogenous bases in presence of nitrates from the soil
- Ribose, nitrogenous base and phosphate absorbed from the soil by the plant roots join by covalent
bonds to form mononucleotides
- Nucleotides join by phosphodiester bonds to form polynucleotides / RNA strands
iii)Amylopectin
- GALP is converted to alpha glucose
- Alpha glucose molecules join together through condensation reaction forming alpha 1,4-glycosidic
bonds to form straight chains and alpha 1,6-glycosidic bonds forming branches every 20 to 30
monomers

iii) Amylose
- GALP is converted to alpha glucose molecules
- Alpha glucose molecules join through condensation reaction forming alpha 1,4-glycosidic bonds to
form straight chains.
- Hydrogen bonds forms making amylose helical.
iv) Cellulose
- GALP is converted to beta glucose
- Beta glucose molecules join through condensation reactions forming beta 1,4-glycosidic bonds to
form cellulose.
v) ATP
- GALP is converted to glucose
- Glucose is converted to ribose
- GALP It is also used to synthesise adenine, a nitrogenous base in presence of nitrates absorbed
the soil by the plant roots
- Ribose, the nitrogenous base and three phosphate groups absorbed from the soil by the plant roots
join by covalent bonds to form ATP
➢ ATP is made up of ribose and adenine, collectively called adenosine and three phosphate
groups that are joined by phosphate bonds.

24
Factors increasing the rate of photorespiration:
• Temperatures; Solubility of CO2 decreases than the solubility of O2 less CO2 dissolves.
The higher the oxygen concentration, the higher the rate of photorespiration.
• When water is scarce, stomata (partly) close, CO2 concentration does not diffuse in and
photorespiration increases

Factor Affecting Photosynthesis

• Light Intensity: When CO2 and temperature are not limiting, the rate of photosynthesis
increases with an increase in light intensity. At a point saturation may be reached, and the
rrate of photosynthesis remains constant.
• At high intensities, the temperature of the plant increases which leads to increased
transpiration in the plant. This leads to the closing of the stomata which leads to a reduced
CO2 intake. Thus, leading to a reduction and finally stoppage of photosynthesis. Therefore,
excessive light inhibits photosynthesis.
• Light Quality (Wavelenth): chlorophyll most effectively absorbs red and blue wavelengths
from the entire spectrum of light. Thus, maximum photosynthesis occurs when the plant is
exposed to the light of these wavelengths.
• Light Duration: The longer the plant is exposed to light, the longer the process of
photosynthesis will continue. As long as the temperature of the plant remains optimum,
photosynthesis will occur.

25
Carbon Dioxide Concentration
The atmosphere contains 0.03% of carbon dioxide. Plants take in carbon dioxide from the air. But,
since the amount of CO2 in the air is very less, it acts as a limiting factor for photosynthesis.
Experiments have been performed to study the rate of photosynthesis on increasing the concentration
of CO2 in the atmosphere.
It is seen that, when light and temperature are not the limiting factors, increasing CO 2 concentration
leads to an increase in the rate of photosynthesis. But, beyond a certain limit, CO 2 starts accumulating
in the plant and this leads to slowing down of the process. So, excessive CO 2 inhibits photosynthesis
especially when it starts to accumulate.

Temperature
When CO2 and light are not limiting factors, the rate of photosynthesis increases with increase in
temperatures till the optimum level for that plant. Below optimum levels the enzymes are deactivated
and above optimum, rate of photosynthesis declines. The decline may be due;:
(i) Accumulation of the end products of photosynthesis.
(ii) Inhibitory effect of high temperature on the activity of enzymes.
(iii)Failure of carbon dioxide to diffuse rapidly.
The enzymes are denatured and photosynthesis stops.

Water
The rate of photosynthesis increases with increase in amount of water till the optimum level for that
plant as more water molecules are split to release H+ ions.
Also, when there is a reduced water intake or availability, the stomata begin to close to avoid loss of
any water during transpiration. With the stomata closing down the CO2 intake also stops which affects
photosynthesis.

26
Investigate Photolysis of water and production of reducing power NADPH
- Photolysis of water and production of reducing power during the light-dependent reactions of
photosynthesis can be demonstrated using DCPIP as a substitute for NADP+. The DCPIP take up
the excited electrons and become reduced.
- When the chloroplasts are illuminated with the corrected wavelength and all other factors kept
constant DCPIP become reduced
- It accepts the excited electrons released from the chlorophyll and the H + from photolysis of waters
and the colour changes from dark blue to colourless.

Note:
- The production of oxygen and starch are used as evidence for photosynthesis.
- The light-dependent reactions produce a reducing agent. This normally reduces NADP, but in this
experiment the electrons are accepted by the blue dye DCPIP. Reduced DCPIP is colourless. The
loss of colour in the DCPIP is due to reducing agent produced by light-dependent reactions in the
extracted chloroplasts.

ENERGY TRANSFER THROUGH ECOSYSTEMS

• The main route by which energy enters an ecosystem is photosynthesis by producers.

• Not all the energy from sunlight is used by plants;
e.g.
- Some is the wrong wavelength that cannot be trapped by chlorophyll or the
accessory pigments
- Some is reflected by the leaf
- Some passes straight through the leaves,
- Some hits parts of the plant that can’t photosynthesise, e.g. the bark of a tree.
- Some is used for evaporating water from the leaves
- Some wasted through biochemical inefficiency of photosynthesis
- Photosynthetic pigments may be saturated with energy hence cannot absorb more and the
electrons emit the energy in form of fluorescence

27
PRODUCTIVITY
Gross Primary Productivity (GPP)
It’s the rate at which energy is incorporated into biological molecules in plants during photosynthesis.
It shows how much energy is available from the producer.

• After some organic molecules are produced, plants use some of the organic molecules in
respiration.

Net Primary Productivity (NPP)

Refers to the rate at which energy is transferred into plant biomass that can be eaten by herbivores or
decomposers.
NPP is the potential energy for primary consumers
The more the NPP the more the consumers can be supported.

Plant respiration (R)

Refers to the rate at which some of the GPP is used by plants for respiration and will eventually be
lost as heat energy.
Thus; NPP = GPP - R
All these variables (i.e. GPP, NPP and R) are measured in kilojoules of energy per square meter per
year (kJm-2 yr-).
Tropical rainforests have more NPP and GPP than deserts
• Tropical rainforests have high temperature, high light intensity, high rainfall, large and many
ever-green leaves and a high minerals content hence high photosynthesis and high GPP and
NPP.
• In deserts, there is little water, sparsely distributed vegetation with small leaves where some
are reduced to spines or thorns, high transpiration due to high temperatures hence lower GPP
and NPP.
Efficiency of energy transfer
Refers to amount of energy transferred between trophic levels divided by the amount potentially
available to that trophic level multiplied by 100 (NPP).
Its expressed as a percentage.

energy transfered into new trophic level

𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 𝑜𝑓 𝑒𝑛𝑒𝑟𝑔𝑦 𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟 = 𝑥 100
Energy available in previous trophic level

Although NPP energy is available for organisms in the next trophic/feeding level, not all NPP is
transferred to the next trophic level.
• Only about 2-10% of the energy in the producers is transferred to the primary consumers
(herbivore) because;

28
 (i) Not all parts of the plant are edible yet they have a lot of energy e.g. tree trunks, roots, thorns
and parts around these thorns.
(ii) Some energy is lost in faeces (undigested food materials)
(iii) Some energy is lost in excretory products such as urine
(iv) Some energy is lost in respiration in form of heat

• Likewise, only about 2-10% of the energy in the primary consumers (herbivores / prey) is
transferred to the secondary consumers (predators) because;
i) Not all parts of the prey are edible yet they have a lot of energy e.g. teeth, bones, horns and
hooves.
ii) Some energy is lost in faeces (undigested food materials)
iii) Some energy is lost in excretory products such as urine
iv) Some energy is lost in respiration in form of heat

Therefore, transfer of energy from one trophic level to another is never 100%

Worked out examples

1. Use the information given in the diagram below to answer the questions that follow.
Light absorbed by plants (4.6 X 106 kJ m-2 year-1)

Energy trapped in glucose (44090 kJm-2 year-1)

R = 2920 kJ
Energy transferred to primary consumers (1500 kJ m-2 year-1)
R = 700 kJ
Energy transferred to secondary consumers (500 kJm-2 year-1)

a) Calculate the efficiency of energy transfer from producers to primary consumers.

NPP = GPP – R
44090 kJ – 2920 kJ = 41170 KJ/ m /yr (NPP of Producer)

In this case we are to calculate efficiency of energy transfer from producers to primary consumers.
So, from the equation;
Efficiency of energy transfer =
(Energy transferred to a trophic level ) X 100
(NPP) Net productivity of previous level

i.e. 1500 kJ X 100 = 3.64%

41170 kJ

29
 b) The diagram below shows the energy content of two trophic levels.

Calculate the percentage of energy transferred (not efficiency) from trophic level 1 to trophic level 2.
(2)
NPP = GPP – R
= 2800 – 1750
= 1050 (NPP of L2) - – This is the actual energy that they incorporated to the body mass
as the rest was respired

NPP of L1 = 5300 – Because its given as biomass and NPP is the amount incorporated as
biomass
So; 1050 X 100 = 19.8%
5300
2. If the energy in the producer (grass) 4500kJ, and the rabbit as the primary consumer received
500kJ during energy transfer, calculate the efficiency of energy transfer from the grass to the rabbit.

500kJ X 100 = 11.11%

4500kJ,

STUDY OF ECOSYSTEMS
Definition of terms
Ecosystem

• This is a natural unit area in which organisms interact with each other and their environment.
• The interaction is through energy flow that involves photosynthesis, feeding and
decomposition; and recycling of nutrient.
• An ecosystem is self-sustaining and self-regulating hence stable
• The largest part of the Earth and its atmosphere that is inhabited by living things is called
biosphere.
Community
A group of organisms of different species living in the same habitat at the same time e.g. in a habitat
like a rock pool- the community consists of different - seaweeds, sea anemones, shrimps, small fish,
grabs.
Population.
A group of organisms of the same species living and breeding together in a habitat at the same time
e.g. zebra in Nairobi National park constitute a population.
- Population size

30
 The number of organisms of the same species living in the same habitat at the same time
e.g. 450 zebras in Nairobi National Park
- Population density
The number of organisms of the same species living in the same habitat at the same time in
a unit area e.g. 50 zebras / km2.
Habitat
A place where an organism lives e.g. a stream, a tropical rain forest, sand dune.
Microhabitat.
A small part of a habitat. e.g. a single leave on a tree.
Species
A group of very closely related organisms that freely interbreed and give rise to fertile off-spring.
Trophic level
This is the position in food chain or food web where an organism obtains energy.

Abiotic factors
These are non-living components of an ecosystem that control the distribution and abundance
of organisms. They include;
a) Water d)Temperature g) Earhquakes
b) Oxygen availability e) Topography h) Fires
c) Minerals content f) Flooding i) Volcanic eruptions
Usually, abiotic factors are density independent factors whose effect is not as a result of population
density.
Biotic factors
These are living components of an ecosystem that involve organisms in an ecosystem and control
distribution and abundance of organisms.

These factors include;

- Parasitism - Diseases - Food - Mates
- Predation - Competition. – Space - Territory
Usually, biotic factors are density dependent factors because they are factors whose effect is as a
result of population density (number of organisms of a species per unit area).

- Distribution of organisms
Refers to spreading / location of organisms in an area.
- Abundance of organisms
Refers to the amount of a species in an ecosystem. When carrying out ecological studies,
abundance of organisms it is estimated through;

31
a) Counting the total number of individuals of a given species in a unit area. It is used when the
individuals of a species are countable, e.g. 315 oak trees.
b) Percentage cover: The area on the ground covered by the individuals of a given species. It is
applied when the species is growing in a spreading manner and its not countable, e.g. grass.

Both biotic and abiotic factors control the distribution and abundance of organisms in an
ecosystem.

Explain how the biotic and abiotic factors in an ecosystem affect the distribution and
abundance of organisms
Predation
Increased predator population leads to decrease in prey population. However when population prey
decreases the predator population decreases as well because their food supply (prey) lowers.
Decrease in predator population allows prey population to increase as they reproduce.
Therefore predator and prey population fluctuates in a repeating cycle.

However predation affect even plant population as the changes that occur in the in turn affect plant
population e.g. increase in prey (herbivores) leads to decrease in plant population.
Mates
Chances of finding a mate or achieving pollination affect distribution of organisms in a habitat. e.g. -
A single individual of an animal species cannot result to an abundance of animals of that
particular species unless there are males and females to mate.
- Dispersal of a single seed to an area may not lead to abundance of that particular species of
plant even if the seed germinates and grows unless there are other plants of the same
species for pollination to occur or the plant can reproduce asexually.
Territory
This is an area defended by organisms against other organisms of the same or different species.
The type and size of territory defended determines which the species live in a community.
Animals use territories to ensure that the males have females for breeding, purposes, breeding
animals have enough resources to raise the young ones in terms of space and food.
This is a density dependent factor. The more the individuals in a territory, the more the higher the
success of breeding and survival of the species.

32
Parasitism and disease
Parasites cause disease and diseased animals do not reproduce successfully, diseased predators are
not able to obtain food well and diseased prey are easily hunted and killed.
Diseased plants are not able to photosynthesis and reproduce successfully.
If the population of parasite increases, they kill their
hosts, so their population decreases. This means there are fewer hosts for the parasite, so their
population
decreases. This allows the host population to recover, so the parasite population also recovers:
Therefore presence of diseases leads to decrease in population of organisms or can wipe out the
whole population.

Effects of disease are higher in areas of low biodiversity than areas of high biodiversity.
Spread of disease is higher in areas of high population density than in sparsely populated areas. This
is also a density dependent factor.
Competition
Occurs when organisms compete for the same resource which is in limited supply e.g. sunlight,
minerals food, mates, space, territories.
It leads to selection where the most suitable individuals survive while the weaker ones may be
eliminated or forced out.
It may also lead to evolution. E.g. competition for mates leads to sexual selection and to evolution
where the males and females of the same species look different.
Competition can be;

• Intraspecific
• Interspecific

Intraspecific competition
Refers to competition for limited resources between members of the same species. It leads to
decrease in in the abundance of the species because some weaker individuals may not survive to
reproduce. In contrast when there are sufficient resources for the members of the same species, with
little or no competition, the organisms increase.
it is density dependent. If the population gets too big, intraspecific population increases, so the
population falls again. If
the population gets too small, intraspecific population decreases, so the population increases again.

33
Interspecific competition
Refers to competition for limited resources between members of different species. It leads to
decrease in in the abundance of the competing species or may lead to extinction of the weaker or the
slow breeding one. i.e. the fast – breeding species reproduces faster and increase in numbers and
competes more effectively. e.g. Two different species of Paramecium grow well in lab flasks when
grown separately, but when grown together P.aurelia out - competes P.caudatum for food, so the
population of P.caudatum falls due to interspecific competition:

ABIOTIC FACTORS AND HOW THEY CONTROL THE DISTRIBUTION AND ABUNDANCE OF
ORGANISMS

Oxygen Availability
Cold and fast flowing hence aquatic contains sufficient amount of dissolved oxygen hence support
higher no. of organisms. As temperature rises and as the water becomes still and stagnant, oxygen
concentration lowers and this affect survival of organisms in the soil.
The air spaces in water logged soil are filled with water and the plant roots are deprived of oxygen.
The plants may die and others like mangroves develop adaptations to survive.
• Its role in primary productivity:
- Needed for aerobic respiration to produce ATP needed for energy hence increases growth.
Rainfall
High amounts of rainfall favours high distribution of organisms while ares of low rainfall organisms are
sparsely distributed.

34
 • Its role in primary productivity;
(i) It is split by light into protons and oxygen releasing free elctrons. Electrons and protons
synthesize reduced NADP and ATP in light dependent reactions to be used in light
independent reaction to synthesize carbohydrates and non-carbohydrates.
(ii) Provides turgidity to plant cells to give support.
(iii) It’s as a medium for chemical reactions.
(iv) It’s a medium for transport.
(v) Water is a solvent

Edaphic factors
These are factors that affect the soil.
Soil structure
Sand soil has loose structure that is easily warmed but easily drained. Water passes through them
rapidly carrying minerals deep depriving the top soil of minerals. This is called leaching. Leaching
reduces the no. of plants growing in a habitat.
However, sandy soils, supports few plants e.g. those that have massive root network or form
rhizomes.
These plants have an added advantage in that they bind sand particles together and this makes the
soil suitable for colonization by other species. Some of these plants e.g. murram adapt to survive in
sand soil by having leaves that curl round on themselves with the stomata inside to reduce water loss.
Clay soil is made of tiny particles that are not easily leached but are heavier, difficult for water to drain
through, take longer to warm up and are easily water logged.
They support few organisms.
Loam soils have particles of average size, are heavier, less prone to leaching and are easier to warm
up.
They support a wide range of plants and animals.
Soil mineral content
Soils with more mineral content support more organisms
• Role in primary productivity;
1) Phosphates and nitrates are used to synthesise nucleic acids (DNA and RNA).
2) Nitrates and sulphur are used to synthesise amino acids.
3) Magnesium is needed for are used to synthesise chlorophyll for trapping light energy for
photosynthesis.
4) Calcium is needed in to synthesise the middle lamella in plant cell wall.
Soil pH
Most organisms are found in areas with alkaline soils.
• Role in primary productivity –
- Alkaline soil has more calcium ions which encourages availability of other important minerals
which are needed for plant growth. Acidic soil has less calcium ions hence less of important

35
 minerals and this leads to few plant supported.
However, some plants are adapted to survive in acidic soil while most of them are adapted to
survive in alkaline soil.
Soil Water
Refers to amount of moisture in the soil. Areas with more soil water support more plants hence, more
organisms in an area
• Role in primary productivity;
(i) It’s absorbed by the root hair cells. It’s then split by light energy into protons and oxygen
releasing free electrons. Electrons and protons are needed in light dependent reactions of
photosynthesis to generate NADPH and ATP.
(ii) Provides turgidity to plant cells to give support.
(iii) It’s as a medium for chemical reactions.
(iv) It’s a medium for transport.
(v) Water is a solvent.
Measurement of soil water / moisture;
- Soil sample is weighed, dried slowly in an oven until constant mass is obtained and
then re-weighed and the difference is the mass of water.
- To calculate the percentage mass of soil water, the following equation is used;

original mass−final mass

% mass of soil water = × 100
original mass

Soil organic matter

When organic matter decomposes, it releases nutrients such as carbon, nitrates and phosphates
hence, more organisms in an area
• Role in primary productivity;
These nutrients are needed to synthesize biological molecules such as ATP, nucleic acids
and amino acids needed for growth.
Light intensity
Plants that receive maximum light intensity have high number of plants and animals.
• Its role in primary productivity;

(i) Needed in light dependent reaction of photosynthesis where it splits water into
protons and oxygen. H+ and electrons synthesize NADPH and ATP in light
dependent reactions to be used in light independent reaction to synthesize
carbohydrates and other biological molecules.
(ii) It causes excitation of electrons from photosystems in light dependent reactions to
synthesize NADPH and ATP in light dependent reactions to be used in light
independent reaction to synthesize carbohydrates and other biological molecules.

36
 (iii) Controls the opening and closing of stomata for gas exchange needed for
photosynthesis.

Temperature
Optimum temperature leads to more plants hence, more organisms in an area.
• Its role in primary productivity
➢ It controls enzyme-controlled reactions e.g. it controls the activity of enzymes in
light dependent, light independent reactions of photosynthesis and in respiration. Low
temperature inactivates enzymes, high temperature denatures enzymes and
therefore the best temperature is optimum.

Topography (shape of the land)

This refers to aspect - the direction the land is facing, either north or south.
It also refers inclination - the steepness of the land i.e. sloppy, gentle sloping or flat.
• Roles of topography in primary productivity;
a) Aspect
➢ Land facing direction of the sun is warmer hence productivity is higher due to more
active enzymes as a result of relatively high temperature.
b) Inclination
(i) Very sloppy land encourages loss of top fertile soil and less retention of water (high
drainage) hence less productivity.
(ii) Gentle slope – due to moderate drainage there is enough water and the top fertile soil
is retained hence has high productivity.
(iii) Flat area has poor drainage hence flooding that causes water-logged soil that
decreases productivity due to lack of oxygen needed for respiration.

ECOLOGICAL NICHE
An ecological niche is the place / position an organism occupies and the role it plays in its habitat.
This includes; how it meets its needs for food e.g. it’s a producer or predator, how it meets its needs
for shelter, how it survives, and how it reproduces, its effects on other organisms.
A species' niche includes all of its interactions with the biotic and abiotic factors of its environment.

Niche can be classified as;

- Structural niche
Where the organism lives e.g. nesting niches, the habitat niche of a lion is the dry grass niche while
that of a cheetah is a tree dwelling niche
- Functional niche
What the organism does or its role in the habitat e.g. feeding niche whereby a flowering plants role is
that it’s a producer that manufactures food for the herbivores, the food niche of a fox is a predator
niche, while that of a rabbit is a herbivore.

37
 • When two different species occupy and share the same niche, one will out-compete the other
and eliminates it.
• To avoid this and to allow for co-existence, the two species occupy different niches in the
same habitat.
• For example, lions hunt in the day and they area predators while hyenas hunt at night and
they are majorly scavengers. Therefore both may hunt at the same are at different times.

Effects of niche on distribution and abundance of organisms

✓ A species is adapted to its niche meaning it has characteristics that increases chances of
survival and reproduction leading to increase in population.
✓ A suitable niche provides suitable environment for organisms and results to increase in
abundance of organisms.
✓ If conditions change in the habitat, adaptations of a species may no longer be useful leading
to change or evolution of a species whereby the individuals that will adapt to the changes will
survive while those that cannot will be eliminated or forced out.

HOW ECOSYSTEMS EVOLVE.

They evolve through succession

SUCCESSION (ECOLOGICAL SUCCESSION)

• Ecological succession is a gradual sequence of changes in a community of organisms

where more adapted species of organisms succeeds other pre-existing species.
• The species with best adaptive features becomes more successful and colonize that site.
• It occurs through stages called seres.
• Every community in a sere is called a seral community.
• When ecological succession occurs in an area, the area attains ecological stability.
• Succession is always associated with increase in biomass and biodiversity.

TYPES OF SUCCESSION

1. Primary succession
2. Secondary succession

1. Primary Succession

• Primary succession is the gradual change in communities in an area from the start of an
empty inorganic surface e.g. bare rock, newly formed sand dune, a newly exposed land
surface such as volcanic eruption or land slide, that has never been colonised before.

38
 • The surface may occur e.g. after an eruption of a volcano or a land slide. When this occurs,
opportunists or pioneer species e.g. algae, mosses and lichens colonise the area.
• They colonise and grow on the new surface, penetrate the rock surface and in the growth
process, they break down the rock surface into particles and trapping organic matter that form
humus. This results in soil formation.
• Other species like ferns and grass grow on the soil, trap more organic matter and as they die
and decay, they add to the soil.
• Gradually other species grow and as succession continues, biodiversity increases, leading to
increase in plant and animal biodiversity.
• As the soil becomes deeper and richer in terms of nutrients and water holding capacity, it
supports more vegetation that in turn supports more animals. Eventually a climax community
is reached.

Example of a primary succession on a bare rock

The pioneer species are very simple organisms like lichens and mosses. They are able to grow in
little or no soil.
Lichens have rhizoids that attach onto the rock surface to absorb the little water and mineral ions
present. They produce acid that chemically breakdown the rock to form little soil which attracts the
mosses.
Mosses grow in large numbers.
Some lichens and mosses die forming organic matter adding humus into the soil that form.
Humus which produces acid that further breakdown the rock to form more soil.
As more soil forms and more organisms grow, there is improved water holding capacity in that area
Death of lichens and mosses attract decomposers and invertebrates. The invertebrates aerate the
soil.
The soil becomes better in terms of organic matter and water-holding capacity and attracts ferns.
Ferns have true roots and underground stems called rhizomes
The roots widen the cracks so that more water and more acid can penetrate, further breaking the
rock, forming more soil.
As ferns colonise the area, the soil conditions improves but become unsuitable for lichens and
mosses since they don’t grow in deep soils. The lichens and mosses die off.
This makes the soil rich and deep attracting other plants like grass, herbs, shrubs, trees, woodlands
and finally the woodland become dense. Plants lose leaves that die and decay adding organic
matter/humus into the soil. The soil is now able to hold more minerals and water and this increases
biodiversity and biomass in an ecosystem.
As the plants species succeeds each other, they attract different types of animal species like
herbivores that feed on the plants, birds that build nests on the trees and feed on plant seeds,
caterpillars and insects that feed on plant leaves and eventually a climax community is reached.

39
2. Secondary Succession
Secondary succession is the gradual, change in communities of organisms in an area that was
previously colonized by other communities.
It involves evolution of an ecosystem from existing soil that is cleared of vegetation.
They result; e.g. -When a river changes direction and leaves behind soil deposits.
- After fires or floods.
- Human influence such as clearing of a forest
Because the soil is already formed and it contains seeds, roots and soil organisms, the number of
organisms present at the beginning of the succession is higher than in the primary succession.
Secondary succession is therefore usually much quicker than primary succession.
The seeds, tubers, rhizomes present in the area germinate or regenerate faster.
The soil is developed in terms of organic matter, nutrients, minerals content and water holding
capacity.
The pioneer species are more complex and include species like grass and herbs.
Example of secondary succession is one involving an abandoned farmland.

Pioneer community
• The pioneer community is the first seral community and the climax community is the last
seral community.
• Pioneer species are also called opportunistic species because they colonise an area where
there is no other community than themselves.
• They are the first to establish themselves in a previously uncolonised or previously colonised
land that was later abandoned.
• They are good colonisers but poor competitors.
• A pioneer community in primary succession should have the following features to make it
successful;
i) Fast growth rate
ii) Short reproduction cycle through asexual reproduction
iii) Tolerance to harsh environmental conditions e.g. very little water and low mineral
content

40
 • Pioneer species may disappear because of the following reasons;
i) Outcompeted by more competitive species
ii) Shaded by larger plants
Climax community
• A climax community is one that remains generally constant over time, self-sustaining
and the most productive group that environment can support.
• A plagioclimax is a constant and self-sustaining and most productive group that is as a
result of human intervention. It is not stable because if the influence stops e.g planting more
trees, natural ecological succession will continue to get climatic climax community.
Note;

• The basis of succession is competition.

• The existing community improves the conditions for the incoming community.

CLIMATE CHANGE (GLOBAL WARMING)

• This is increase in mean temperature of earth’s surface due to greenhouse effect.
• Generally greenhouse gases reduce heat loss from the earth’s surface warming the earth’s
surface.
• It is believed that global warming will lead to a permanent change in the Earth’s climate.
• The evidence for climate change includes:

EVIDENCE OF CLIMATE CHANGE

1. Temperature records

➢ Long term data sets of temperature records are used. These allow changes in temperature to
be analysed.
➢ A general trend of increasing temperature is evidence that global warming is taking place.
➢ The data gives an indication of how temperatures have progressively changed and this
enables scientists to make conclusions

2. Dendrochronology (Tree rings analysis)

➢ This is the study of size of tree rings.

➢ Most trees produce one ring within their trunks every year
➢ The thickness of the ring depends on the climatic condition when the ring was formed.
➢ If the climate is warmer and wetter, the rings are wider and vice versa. A warmer and wetter
climate leads to increased metabolic processes like photosynthesis hence faster growth.
➢ Therefore the width of tree rings tell about the climate of a place.
➢ A trend of increasing wider rings suggests that the climate has been becoming warmer and
wetter and vice versa.
➢ Dendrochronology can give information about climate up to 3, 000 years into the past.

41
3. Pollen data (Peat bogs)

➢ Pollen grains are preserved in peat bogs.

➢ A bog is a wetland that accumulates peat
➢ Peat is an accumulation of partially decayed plant material and animal material.
➢ A peat bog is very acidic, cool and anaerobic. These conditions prevent decomposers like
bacterial from decomposing organic material hence the material are preserved in bogs.
➢ The acidic and cool conditions slows enzyme activity of the enzymes involved in
decomposition, the anaerobic condition affect survival of aerobic microorganisms.

➢ Two major components of the peat bog that are used as evidence of global warming are;
i) Pollen grains
ii) Exoskeletons of organisms like insects
➢ By sampling cores of peats, the pollen grains and the exoskeletons can give an indication of
the plants and animals that lived at that time when the material were preserved.
➢ Peats form in layers, the deeper the layer the older the peat layer. Carbon 14 dating allows
the age of a particular peat layer to be established.
➢ The age of the peat can also be estimated by counting the number of peat layers i.e the
deeper the layer, the older it is.
➢ Studies show that different levels of peat bogs represent different ages. Analysis of pollen in
peat bogs shows which plants were growing and how was the climate when the peat bogs
were formed.
➢ Pollen from peat is important to estimate past climatic conditions because;
i) Plants produce pollen in large numbers hence they are available in most places
ii) Pollen grains have a tough outer layer that is resistant to decay hence can be preserved
over a long time.
iii) Each plant species have a unique type of pollen grain. This enables identification of the
plant species from which the pollen grain came from.
➢ Pollen grains in the peat give information about climate up to 20,000 years into the past
NB: - Data from pollen grains might be unreliable as it relies on good preservation of pollen grain and
vegetation change may lag behind climate change.
- Pollen are only produced by mature trees. For trees to mature and produce pollen they must
grow for a long period of time

4. Ice cores (Frozen isotopes)

➢ As water freezes to form ice, air bubbles are trapped.

➢ Analysis of air trapped in ice cores can give information about temperatures and carbon
dioxide levels in the past.

42
 ➢ Records of O2 or carbon isotopes in different layers in the ice reflect the temperature at the
time when the layer was formed e.g the ratio of oxygen 16 : oxygen 18 will be higher in the ice
core if the temperature was low and vice versa.
➢ Ice cores give information about climate up to 20,000 years into the past

5. Records of carbon dioxide levels

➢ Increasing levels of carbon dioxide in the atmosphere are believed to contribute towards
climate change as carbon dioxide is a greenhouse gas and is involved in the greenhouse
effect
➢ Annual fluctuations in CO2 levels can be recorded and this can be used to show seasonal
differences in the fixation of CO2 by plants.
➢ An increase in CO2 levels indicates that less CO2 is being fixed probably due to deforestation
or excessive production of CO2
Note:
✓ Data from the above evidences gives an indication of the climate but not an exact condition.
✓ Therefore it involves looking for correlations and / or causations e.g. there is a correlation
between the increase in temperature and carbon dioxide levels.
Studies using different computer models suggest that increase in atmospheric CO2 increases
surface temperature. So, it is the increase in CO2 that causes global warming.
✓ The data from the above evidences is then analysed using different statistical methods.
✓ The reliability of data from the peat bogs and dendrochronology can be increased through a
process known as wiggle matching whereby the age of the wood or peat bog is dated from
radiocarbon measurement and the results are compared.

CAUSES OF ANTHROPOGENIC CLIMATE CHANGE (CAUSES OF GLOBAL WARMING)

• Anthropogenic climate change is climate change caused by human activity - such as burning
fossil fuels leading to the release of greenhouse gases, destruction of forests for land to be
used for things like building and agriculture.
• It occurs due production of These processes emit greenhouse gases emitted by human
activity.
• By examining evidences like polar ice cores, scientists are convinced that human activity has
increased the proportion of greenhouse gases in the atmosphere, which has highly increased
over the past few hundred years.
• Multiple lines of evidence confirms that the post-industrial rise in greenhouse gases does not
stem from natural mechanisms. In other words this is anthropogenic climate change, and the
significant increases in the atmosphere of these potent greenhouse gases are a result of
human activity.

43
 • it is estimated that about two thirds of anthropogenic climate change CO2 emissions have
come from fossil fuel burning (coal and petroleum) and about one third from land use change
(mainly deforestation and agricultural).

Greenhouse gas % estimated contribution to climate change

Water vapour 36 – 37
Carbon dioxide 9 – 26
Methane 4–9
Ozone 3-7
Generally greenhouse gases reduce heat loss from the earth’s surface.
When sun’s radiation reaches the earth, some of it is absorbed the earth’s surface. The radiation
which is mainly the infrared that reaches the earth’s surface is of fairly short wavelength. This is
absorbed by the earth’s surface and then and the radiated from the earth’s surface at a longer
wavelength.
Greenhouse gases in the atmosphere absorb some of the radiation from the earth’s surface and
reradiate it back to the earth’s surface. This maintains the temperature at the earth’s surface at a
higher level.

• Incoming short wavelength infrared penetrates atmosphere to reach the earth’s surface, the
earth radiates longer wavelength infrared.
• Water vapour and other greenhouse gases in the atmosphere absorb this infrared and re-
radiate it warming the earth’ surface.

Role of methane in global warming

It is produced in lower quantities than CO2 mainly from decay of organic material by some bacteria
particularly in wet conditions like rice paddies and from digestion in ruminant herbivores.
Increase in cattle farming and rice production leads to global warming because these activities leads
to increased methane production. However, older cattle produce less methane.
When released high in the atmosphere, methane naturally breaks down in a series of reactions and
eventually form CO2 and H2O.

44
Question.
Explain how increase in human population leads to increased methane production.

Role of CO2 in global warming

Increased release of CO2 into the atmosphere forms a layer that reduces heat loss from the earth’s
surface. It allows radiation from the sun to reach the earth’s surface but it traps some of the radiation
that is radiated back into the atmosphere from the earth’s surface. This is because they are radiated
back at a longer wavelength.
Due to human activities, extra CO2 is being added to the atmosphere altering weather patterns and
influencing oceanic chemistry.

THE CARBON CYCLE

• The carbon cycle is essentially nature's way of reusing carbon atoms in different ways and in
varying places.
• It is the process in which carbon travels from the atmosphere into organisms and the Earth
and then back into the atmosphere.
• It explains carbon circulates in nature.

• In the process of photosynthesis, atmospheric carbon is absorbed by plants.

• This carbon is transferred from plants to the animals feeding on them, and further moves up the
food chain.
• Respiration, digestion, and metabolism of plants and animals result in some transfer of carbon
back to the atmosphere.
• Some carbon also moves to the lithosphere (buried underground) when these living organisms
die or when wood and leaves decay or when animals excrete. Some of these living beings buried
millions of years ago have been converted to fossil fuels.
• Mining and burning of fossil fuels cause this carbon to move from the lithosphere to the
atmosphere.
• Some of this atmospheric carbon gets dissolved in the ocean.

45
The amount of CO2 is maintained by a balance between the process which withdraw CO 2 from the
atmosphere (mainly photosynthesis) and those which release CO2 to it e.g. respiration, combustion
and decomposition).
Carbon reservoirs
Carbon-storing natural feature (such as a forest or the land mass) that exchanges carbon with other
reservoirs. A carbon reservoir has accumulated carbon. It is usually considered that there are five
major reservoirs of carbon on the planet, which are interlinked by flow of exchanges.

The major carbon reservoirs are;

• Atmosphere
• Terestrial plants (forests / biosphere)
• The ocean ( e.g. in marine biota)
• The sediments (e.g. fossil fuels like coal, oil and natural gas)
• The earth’s interior -This interacts with the other components through geological processes.

Carbon sinks (stores)

A carbon sink is any natural reservoir that absorbs more carbon than it releases, and thereby lowers
the concentration of CO2 from the atmosphere. Globally, the two most important carbon sinks are
vegetation and the ocean.
They are reservoirs where carbon is removed from the atmosphere and get locked up in organic or
inorganic compounds.
A carbon sink accumulates carbon.
A carbon sink is an ongoing process which is increasing the amount of carbon stored in it.
Examples of carbon sinks are;

• Bodies of living organisms

• Soil -contains carbon rich material in form of humus
• Rocks –e.g. limestone and chalk
• Fossil fuels e.g.coal, oil and natural gas
• The oceans

Note; The difference is that a carbon sink accumulates carbon, whereas a carbon reservoir has
accumulated carbon.
i.e. A carbon sink is an ongoing process which is increasing the amount of carbon stored in it.

Reduction of atmospheric levels of CO2

• Use of biofuels
• Reforestation

46
Use of Biofuels

• Increased use of biofuels as opposed to fossil fuels can help reduce atmospheric level of
CO2.
• Their use is carbon neutral, because the CO2 released in burning them has recently been
fixed in photosynthesis.
• Biofuels are produced directly from plants unlike fossil fuels that have first to undergo the
fossilisation process.
• The frequent planting of crops and trees for biofuels increases removal of CO 2 from the
atmosphere due to increased photosynthesis.

Advantages of using bio fuels

1. They are sustainable.

They are plant based and the plants can be replanted after harvesting ensuring continuous
production.
2. They are carbon neutral
When they are burnt there is no net release of CO2 as they release the carbon that has recently
been absorbed by the plant for photosynthesis and not the CO 2 trapped long time ago like in fossil
fuels.
3. They provide an alternative to utilization of less productive land such as swamps.
4. They reduce overdependence on fossil fuels.
5. Their production leads to a source of income to those involved.

Disadvantages
1. It leads to a threat to food production especially when edible food such as corn is used to produce
corn oil as bio fuel.
2. They may lead to loss f agricultural land shortage in food supply.
3. They may lead to increase in CO2 levels in the atmosphere as plants ate cut down to give way to
their production.
4. Their production requires machinery use e.g. tractors for land preparation, combine harvesters for
harvesting and this leads to use of fossil fuels to run the machinery increasing CO 2 production.
Therefore they may not be carbon neutral.
5. Their pruction is time consuming and expensive compared to fossil fuels.

Reforestation

• This involves planting of trees.

• Forests acts as carbon sinks storing carbon in their organic compounds.
• Young growing trees are net absorbers of CO2 as when they grow rapidly, they turn CO2 into
wood. So photosynthesis is greater than respiration. This increase removal of CO 2 from the

47
 atmosphere compared to mature forests are carbon neutral i.e. give out as much CO 2 as they
take in.

DATA EXTRAPOLATION
Extrapolation is an estimation of a value based on extending a known sequence of values of facts
beyond the area that is certainly known.
It’s the process of estimating beyond the original observation range i.e. infer something that is not
explicitly stated from existing information.
Data can be extrapolated to make predictions and these predictions are used in models of future
global warming. However, these models have limitations.

Scientist extrapolate data on greenhouse gases and use them in;

• Models to make predictions about what will happen to global temperatures in the future
(i.e. will they increase or decrease?)
• Models to predict long term effects of increased temperatures on the environment.
When extrapolating, 2 assumptions are made;
(i) Present trends will continue.
(ii) We have enough data to establish the trend accurately.
Computer models are not expected to precisely predict the future but to make the best
prediction based on the evidence available.

These models are used in planning on responses to problems such as risk of flooding and rising CO2
levels.

Limitations of Computer Models

i) Some factors are very hard to predict CO2 emissions as this may decease if people reduce use of
fossil fuels or may increase in future.
ii) In adequate computing man power
iii) Lack of sufficient data
iv) Lack of knowledge of how the climate functions
v) It’s impossible to tell the exact impact of CO2 on global warming.
vi) It’s impossible to predict the impact of global warming on particular aspects on the world
climate.
vii) Extrapolation from past data cannot take into account unknown factors in the future because
trends in use of resources and technology may change.

48
EFFECTS OF CLIMATE CHANGE

1. Rising temperature.
- CO2 in the atmosphere allows radiation to reach the earth’s surface from the sun. When the
earth’s surface reradiate the heat at a longer wavelength, some of it is trapped by CO2 in the
atmosphere leading to increase in temperature.

2. Changing rainfall patterns.

- Rising temperature affect weather and rainfall patterns. It may lead to low rainfall in some
areas hence drought. It may also lead to high and heavy rainfall leading to flooding. This
causes damage and carries away top soil affecting development of plants.
- Warmer air can hold more water, so rainfall is increasing on average across the world. In
some places, rainfall is becoming more intense as well. However, some areas receive less
rain because of changes in wind patterns.

3. Changing seasonal cycles.

- It results to seasons that are different in length and intensity. e.g. warmer winter or a very hot
summer that is prolonged.

❖ The effects of climate change such as temperature rise, changing rainfall patterns and
changes in seasonal cycles which in turn would lead to:

a) Changes in distribution of organisms

- Due to temperature rise, species may move to new places.
- Species would move tocooler areas. This results to competition between the new and the
native species and either of the species might outcompete the other rendering one of them
extinct in the area. This could potentially lead to extinction of some species..
- Animals can move and therefore are able to survive the changes more easily moving to new
areas or colonise a bigger area. If plants have to survive they have gto adapt to the changes.
- If the changes positively affect organisms involved in spread of diseases, it leads to increase
in vector borne diseases resulting to population decrease.

b) Changes to development of organisms

- Studies show that in many reptiles temperature affect the sex of the young ones that hatch
because the embryos are sensitive to temperature as they develop. Therefore increasing
temperature could cause a change in sex ratio of these species or even extinction. e.g. male
crocodiles develop only if the eggs are incubated at 32oc – 33oc, if the temperatures are
cooler or warmer, females develop.
- If there is an increase of one gender, there will be completion for mates affecting reproduction
- If one gender is totally.

49
 - Oceans also absorb around 25% of the carbon dioxide that humans release into the air. The
oceans then become less alkaline, a process called 'ocean acidification'. Ocean acidification
is bad because it can have negative effects on marine organisms, like coral and plankton,
which are an important part of the food chain. Their enzyme activities are affected.
-

c) Changes on life cycles

- Increase in temperature may disrupt life cycles of organisms where temperature is an
important trigger for development for them to complete the life cycles. e.g. insects may go
through their life cycles faster and be ready to feed before the plants they feed on mature.
- Warmer temperatures mean that plants grow and flower earlier, insects have to become
active earlier and feed on the plants leading to increased population. This causes the birds
that feed on the larvae of these insects also to adapt to these changes and breed earlier so
that their young ones hatch when the insect larvae are available to feed on them.
- A faster breeding cycle leads to an increase in population as organisms reproduce more
times in a year.

EFFECTS OF CLIMATE CHANGE ON RATE OF ENZYME ACTIVITY IN ORGANISMS

Temperature affects enzyme activity and therefore the whole organisms and also microorganisms.
Increase in temperature leads to increase in in rate of enzyme controlled reactions but to an optimum
temperature beyond which the rate falls.
A rise in temperature in a few degrees may lead to a faster reproduction, growth and development of
organisms but, if the rise is too high, the enzymes will denature, reaction rate falls leading to death
and decrease in population.

- For every 100 c rise in temperature, the rate of enzyme activity doubles i.e. for every 10 0 c rise
in temperature there is a temperature coefficient (Q10) of 2.
(Q10) = Rate of reaction at (x + 10)o c
Rate of reaction at x o c
- Q 10 is a temperature coefficient which is a measure of the rate of change of a biological
system that occurs when the temperature changes by 10°C.
- The Q10 temperature coefficient is a measure of the rate of change of a biological or chemical
system as a consequence of increasing the temperature by 10 °C.
- The coefficient can be calculated and then used to predict the effects of climate change on
different biological systems, such as whole organisms and muscle systems.
- The temperature coefficient (Q10) represents the factor by which the rate (R) of a reaction
increases for every 10-degree rise in the temperature (T).
- Q10 is a way of describing the effect that temperature has on enzyme activity.

50
 - Theoretically for every temperature rise of 10 degrees, the rate of enzyme activity should
double, up to its optimum rate. Over the optimum, the increase in temperature will cause the
enzyme to denature.

SCIENTIFIC CONCLUSIONS ON CONTROVERSIAL ISSUES ON CLIMATE CHANGE.

• A controversial issue is the one with alternative points of view.

• Global warming is a controversial issue.
• Scientific conclusions about controversial issues, such as what actions should be taken to
reduce climate change, or the degree to which humans are affecting climate change, can
sometimes depend on who is reaching the conclusion
• While the vast majority of scientists agree on the human impact on climate change, it is the
governments and politicians that have the power to change things, often leading to less action
being taken than scientists themselves would.
• Governing bodies are also sometimes skeptical about claims about climate change and will
often use any disagreements within the scientific community to delay taking action.

Some of the controversial issues in regard to global warming include;

• What causes global warming?

• What actions should be taken to reduce global warming?
• To what degree are human beings affecting global warming?
• Who decides / concludes on issues relating to global warming i.e. scientists or politicians
among others.

Decisions on various issues are made by different groups like politicians, environmental activists and
scientists and they are influenced by pressure groups who are biased by their own interests e.g.
industrialists.
Therefore conclusions reached from scientific evidence depend heavily on who is reaching those
conclusions, who funded the original research, the prevailing financial and political conditions.

Actions
Some of the actions that can be taken to reduce global warming are;

• Controlling the use of fossil fuels.

• Making industrial processes and car engines cleaner and less polluting.

Role of the Scientific Community in issues regarding climate change

The scientific community plays a role in;
a) Validating new evidence

51
 When useful results and conclusions on effects and control of climate change are produced, they
are submitted to a scientific journal, goes through a process of peer review by experts to find out if
it is reliable and if reliable they are published.
NB: A paper should provide enough information for other scientists to carry out similar investigations.
b) Organising scientific conferences
For scientists working in the same field to get together to discuss ideas.
This helps to promote development of new techniques in research, provide opportunities to
challenge validity of results being presented at the conference.
c) Carrying out Molecular analysis to develop DNA and protein sequences that help identifying
individuals of particular group and those that are closely related. e.g. molecular analysis of
pollen grains
d) Analysis of evidence of scientific theory of evolution
They use evidence from DNA analysis of different species to develop a model of evolution from
a common ancestor. This shows relationships between organisms. This explains the impact of
climate change on existence of organisms.

PRACTISE QUESTIONS
1 The reactions involved in photosynthesis are affected

by environmental factors. The graph below shows the

effect of temperature on the rate of photosynthesis in

wheat.

a) Calculate the Q10 for photosynthesis between 20° C

and 30° C. (1 mark)

(Q10) = Rate of reaction at (x + 10)o c

Rate of reaction at x o c use (X – initial temperature)
Q10 = 8 ÷ 4 = 2

52
b) Explain the effect of temperature on the rate of photosynthesis in wheat. (3)

• Between 0oC and 30oC an increasing in temperature increases kinetic energy of molecules
• This hence increasing movement of both enzyme and substrate molecules,
• Therefore molecules collide more often, with more force causing the rate to increase
• At 30 -optimum temp. for the enzyme
• A temperature of above 30oC, results in {enzyme denaturation / change in bonding in the
enzyme, which causes active site shape to change shape causing the rate to decrease.

2. Many scientists think there is a link between global warming and increased levels of carbon dioxide
and methane in the upper atmosphere. Most organisms are found in regions where the
temperature range is between 0 °C and 40 °C at the Earth’s surface.
a) (i) Suggest why temperatures below 0 °C or above 40 °C would be unsuitable for most
organisms. (2)
o
• Below 0 C metabolic activities such as photosynthesis and respiration stops or become
extremely slow.
o
• At temperatures below 0 C, enzymes would be inactivated and having low kinetic
energy. Therefore few collisions would occur between enzymes and their substrates.
o
• At 0 C water freezes cell functions are disrupted as water is a medium for chemical
reactions.
o
• At temperatures above 40 C, the e enzymes denature changing the active site.
Enzyme-substrate complexes do not form, metabolism slows down and eventually
stops.
ii) Explain how this range of temperatures has been maintained by the presence of carbon dioxide
and methane in the upper atmosphere. (3)
• Carbon dioxide and methane are greenhouse gases which absorb infra-red radiation
reflected / reradiated from the earth’s surface.
• This prevents infra-red radiation from escaping into space.
• The heat is retained on the earth’s surface maintaining temperatures higher than they
would be.

3. Analysis of pollen in peat bogs can provide evidence for global warming. Peat is acidic and has
low levels of oxygen. As a result, pollen is preserved in the peat for many years.
The diagram below shows the structure of a pollen grain.

The inner cell wall contains cellulose and the outer cell wall contains sporopollenin.
Sporopollenin is chemically stable and very resistant to decomposition.

53
a) Describe the structure of cellulose in cell walls. (4)
• It’s a straight chain polymer of β glucose monomers, linked by β 1-4 glycosidic bonds
formed through condensation reaction.
• Every other glucose monomer is inverted at 180° angle.
• The cellulose molecules arranged are arranged in a parallel manner forming
microfibrils.
• The microfibrils are joined by hydrogen bonds.
b) Suggest why pollen in peat bogs is preserved for many years. (4)
• Because of very slow decomposition due to lack of oxygen.
• Due to lack of microorganisms such as bacteria and fungi involved in decomposition as
aerobic microorganism cannot survive. As a result there are fewer enzymes released
causing little decomposition.
• The low pH (acidic conditions) reduces enzyme activity slowing decomposition and may
results in death of microorganisms.
• Sporopollenin is very resistant to decomposition and most bacteria cannot produce
enzymes to breakdown sporopollenin.

54
CORE PRACTICAL 11:
CARRY OUT A STUDY OF THE ECOLOGY OF A HABITAT, SUCH AS USING QUADRATS AND
TRANSECTS TO DETERMINE THE DISTRIBUTION AND ABUNDANCE OF ORGANISMS, AND
MEASURING ABIOTIC FACTORS APPROPRIATE TO THE HABITAT.

This involves investigating ecosystems

Objective:
Describe how to carry out a study on the ecology of a habitat to produce valid and reliable data
(including the use of quadrats and transects to assess abundance and distribution of organisms and
the measurement of abiotic factors, e.g. solar energy input, climate, topography, oxygen availability
and edaphic factors).

• Studying ecosystems is an important but a difficult task. Unlike a highly controlled laboratory
investigation, ecological investigations often involve many variables that cannot all be
adequately controlled.
• This means they need even more careful planning and cautious interpretation.
• Two important variables which ecologists investigate are:
a. Abundance of organisms: Number of individuals of a species in a particular area.
b. Distribution of organisms: refers to the location of organisms in an area i.e.
where a particular species is within the area being investigated.
• When ecologists study habitats, they try to account for plant and animal abundance and
distribution, correlating them to the abiotic and biotic factors affecting the habitat.
- Abiotic means non-living and examples of abiotic factors include light intensity,
slope, humidity, wind exposure and edaphic characteristics such as pH and soil
moisture.
- Biotic means living and examples of biotic factors include competition, grazing and
predation.

SAMPLING
• It is normally impossible to measure everything in a whole habitat or ecosystem, therefore
samples of smaller areas are taken from which conclusions can be drawn. e.g. other than
counting every single tree in a 400 acre forest, one can sample the forest to get a
representative idea of how many trees there are in that forest.

• A sample is a small representative group of individuals obtained from a population.

• The manner in which a sample is obtained is called sampling method:
• Sampling is useful because it makes the investigation possible, but the final answer reached
is just an estimate. Sampling can also introduce bias.
There are two types of sampling methods / sampling techniques:

55
1. Systematic sampling method-
- Carried out where there is a change across the area / long environmental gradient
and the sampling technique is transect.
- A transect is effectively a line laid out across the habitat, usually using a tape
measure, along which samples are taken
- In transect, a line is laid out along an environmental gradient. This could be down the
slope of a hill, along the edge or along a stream.
- The transect can be either a line transect or a belt transect
- Once the transect is identified, a tape measure is laid and a quadrat is placed one
after the other along the tape measure, at regular intervals. Everything that is within
and touching the line gets counted.
- Systematic sampling is useful because if conditions change across a habitat, for
example across a rocky shore, then systematic sampling along a transect allows the
changes to be studied.
- Line transects and belt transect can be used when:
▪ When there is tall vegetation in an environmental gradient
▪ When individuals are widely spaced in an environmental gradient
▪ When individuals are closely spaced in an environmental gradient

56
 2. Random sampling method-
▪ Carried out in a uniform area or when comparing two areas that appear different and the
sampling technique is quadrat.
▪ Random sampling is usually carried out when the area under study is fairly uniform, very
large, and or there is limited time available.
▪ When using random sampling techniques, large numbers of samples are taken from different
positions within the habitat.
▪ The sampling technique is quadrat which can be randomly placed within the area under
investigation.

▪ The quadrats may be divided into 25 smaller sections when estimating abundance using
percentage cover in order to;

➢ make it easier to estimate /measure / calculate

➢ make it more precise.
➢

Methods of assessing abundance

• Individual counts /Density:

The simplest method to use provided numbers are reasonably low and each individual is
easily distinguished. Count the number of individuals in several quadrats and take the mean
to give number per unit area, for example per meter squared
(m-2)
• Percentage cover:
This is the percentage of the ground covered by a species within the sampling unit. Count the
number of squares within the quadrat that the plant completely covers, then count those that

57
 are only partly covered and estimate the total number of full squares that would be completely
covered by that species.
Density:
• Count the number of individuals in several quadrats and take the mean to give number per
unit area, for example per meter squared (m-2)

MEASURING ABIOTIC FACTORS WHEN SAMPLING THE ENVIRONMENT

Abiotic factors affect the distribution and abundance of organisms in a habitat. Therefore, they should
be measured when sampling the environment to establish any relationship.

ABIOTIC FACTORS, HOW THEY ARE MEASURED AND HOW THEY CONTROL DISTRIBUTION
AND ABUNDANCE OF ORGANISMS

1. Solar energy input (amount of light intensity)

a) Measurement technique – light probe/ light sensor
Take several measurements at different times of the day at different places.
Calculate and record the mean.
Its light-sensitive panel contains sensors that trap the light to measure it, displaying the reading on
its screen.
In order to work properly, care must be taken to keep the sensor steady, keep the meter out of shade
and to make sure that the panel is facing the maximum sunlight available.

The light meter is used by pointing in the direction of the maximum light intensity

b) Limitation
Light intensity changes with time of the day, different days and cloud cover

c) Its role in primary productivity;

(i) Needed in light dependent reaction of photosynthesis where it splits water into
protons and oxygen. H+ and electrons synthesize NADPH and ATP in light
dependent reactions to be used in light independent reaction to synthesize
carbohydrates and other biological molecules.

58
 (ii) It causes excitation of electrons from photosystems in light dependent reactions to
synthesize NADPH and ATP in light dependent reactions to be used in light
independent reaction to synthesize carbohydrates and other biological molecules.
(iii) Controls the opening and closing of stomata for gas exchange needed for
photosynthesis.

2. Temperature
a) Measurement technique – Using a thermometer
Take several measurements at different times of the day at different places.
Calculate and record the mean.

b) b) Limitation
Temperature changes with time of the day, different days and cloud cover
c) Its role in primary productivity
➢ It controls enzyme-controlled reactions e.g. it controls the activity of enzymes in
light dependent, light independent reactions of photosynthesis and in respiration. Low
temperature inactivates enzymes, high temperature denatures enzymes and
therefore the best temperature is optimum.
3. Rainfall
a) Measurement technique – Using a rain gauge
Take several measurements.
Calculate and record the mean.
b) Its role in primary productivity;
(i) It is split by light into protons and oxygen releasing free elctrons. Electrons and protons
synthesize reduced NADP and ATP in light dependent reactions to be used in light
independent reaction to synthesize carbohydrates and non-carbohydrates.
(ii) Provides turgidity to plant cells to give support.
(iii) It’s as a medium for chemical reactions.
(iv) It’s a medium for transport.
(v) Water is a solvent.

4. Topography (shape of the land)

➢ This refers to aspect. This is the direction the land is facing, either north or south.
Measurement techniques for aspect;
(i) Topographical surveys
(ii) Compass.

59
- Hold the compass flat on the palm of your hand .
- Wait for the magnetic north needle to stop moving. and the needle points to N.
- Record the direction,eg, NW, S, SE. This is the aspect of your slope.
➢ Topography also refers inclination. This is steepness of the land i.e. sloppy, gentle
sloping or flat.
a) Measurement technique of inclination. This is by use of clinometers and ranging poles.
A standard clinometer is bent into an arc which marks every degree.
- A clinometer is essentially a protractor with a marker to indicate the angle of the slope in degrees.
Record the gradient (slope) in degrees.

Clinometer

Roles of topography in primary productivity;

c) Aspect
➢ Land facing direction of the sun is warmer hence productivity is higher due to more
active enzymes as a result of relatively high temperature.

d) Inclination
(i) Very sloppy land encourages loss of top fertile soil and less retention of water (high
drainage) hence less productivity.
(ii) Gentle slope – due to moderate drainage there is enough water and the top fertile soil
is retained hence has high productivity.

60
 (iii) Flat area has poor drainage hence flooding that causes water-logged soil that
decreases productivity due to lack of oxygen needed for respiration.

5. Oxygen availability
a) Measurement technique: Oxygen probe. A colorimeter can also be used. - explain
If in a water body, take several samples at different parts of the water body.
Calculate and record the mean.

c) Its role in primary productivity: Needed for aerobic respiration to produce ATP needed for
energy hence increases growth.

5. Edaphic factors (soil conditions)

i) Soil pH
a) Measurement technique – pH probe or soil pH meter.
The pH of soil, rainwater, and water in rivers and ponds can be measured using a soil pH meter.
The probe is pushed into the soil and the reading is displayed on its screen.

Place the probe in the area to be measured.

Read the scale
Wipe the probe and repeat a number of times.
Take several readings.
Calculate and record the mean.

b) Errors can be made when the pH meter is not wiped between readings.

c) Its role in primary productivity –

Alkaline soil has more calcium ions which encourages availability of other important minerals which
are needed for growth. Acidic soil has less calcium ions hence less of important
minerals and this leads to less productivity.
However, some plants are adapted to survive in acidic soil while most of them are adapted to survive
in alkaline soil.

61
ii) Soil Minerals
a) Measurement technique – Gardener’s test kit.

b) Role in primary productivity;

1) Phosphates and nitrates are used to synthesise nucleic acids (DNA and RNA).
2) Nitrates and sulphur are used to synthesise amino acids.
3) Magnesium is needed for are used to synthesise chlorophyll for trapping light energy for
photosynthesis.
4) Calcium is needed in to synthesise the middle lamella in plant cell wall.

ii) Soil Water

a) Measurement technique;
Soil moisture levels can be measured using a soil moisture meter.
The probe is pushed into the soil and the reading is displayed on its screen.
Place the probe in the area to be measured.
Read the scale
Wipe the probe and repeat a number of times.
Take several readings.
Calculate and record the mean.
OR
- Soil sample is weighed, dried in an oven at a constant temperature until constant
mass is obtained and then re-weighed and the difference is the mass of water.
- To calculate the percentage mass of soil water, the following equation is used;

original mass−final mass

% mass of soil water = × 100
original mass

b) Role in primary productivity;

(i) It’s absorbed by the root hair cells. It’s then split by light energy into protons and oxygen
releasing free electrons. Electrons and protons are needed in light dependent reactions of
photosynthesis to generate NADPH and ATP.
(ii) Provides turgidity to plant cells to give support.
(iii) It’s as a medium for chemical reactions.
(iv) It’s a medium for transport.
(v) Water is a solvent.

iv) Soil organic matter

When organic matter decomposes, it releases nutrients such as carbon, nitrogen and
phosphorus.
a) Measurement technique;

62
 - Dry soil sample is weighed, burnt in a crucible at a constant temperature until a
constant mass is obtained and then re-weighed. Any organic matter is burnt off which
accounts for any difference in mass.
- To calculate the percentage mass of soil organic matter, the following equation is
used;
original mass of dry soil−final mass
% mass of soil organic matter = × 100
original mass of dry soil

b) Role in primary productivity;

These nutrients are needed to synthesize biological molecules such as nucleic acids and
amino acids.

v) Soil texture
Texture refers to the size of the particles that make up the soil. The terms sand, loam and
clay refer to relative sizes of the soil particles.
a) Measurement technique;
Use of soil texture chart to assess whether the soil is clay, loam or sand.

b) Productivity
Clay soil
Very low productivity because its easily water-logged reducing oxygen in the soil and it takes
longer time to warm up.
Loam soil
It supports a lot of vegetation hence high productivity because it has moderate leaching, its well
aerated and warms up easily.
Sand soil
Very low productivity due to lack of water and minerals.
They are well aerated, have highest drainage hence very little water is retained and are heavily
leached (very little minerals if any).

COMMONLY TESTED QUESTIONS

1- ) Comparing the distribution of a plant on two different parts of land

- Measuring off two areas of the same size
- Use of a quadrat
- Use of random sampling
- Estimation of abundance e.g. number of plants, percentage cover
- Use of several sample sites
- Method of recording quantitative data e.g., table,

63
2-) Investigating the distribution of aquatic animals
- Taking sample of water or using a net
- Details of sampling method; same volume of water.
- Systematic sampling / sampling at regular intervals along the river
- Counting the numbers of aquatic animals
- Measuring other abiotic factor; temperature / oxygen / depth – this determines light
penetration
- Method of recording quantitative data; e.g. table

3-) Measuring an abiotic factor

- Use of appropriate apparatus
- Taking several measurements – to calculate man – for reliability

64
MUTATION AND SPECIATION AND EVOLUTION
SPECIATION
• This is the formation of a new species from the existing one.
• A species is a group of individuals that freely interbreed and give rise to fertile offspring.
• So, the only barrier between different species is reproductive barrier or reproduction isolation
• The reproductive barriers ensure that there is no successful reproduction hence no gene flow.
Instead, they ensure that there is accumulation of gene pools.
• Reproductive barriers occur due to behavioural and/or geographical isolation mechanisms.

There are two types of speciation:

1. Allopatric speciation-
Refers to formation of a new species from an existing one but in different places due to
factors such as geographical barriers.
In allopatric speciation, a population is reproductively isolated by a geographical barrier such as
ocean. The two populations are subjected to different environmental conditions.
Gene mutations occurs resulting in new alleles. These alleles code for adaptive features with survival
advantage. The individuals with the advantageous alleles are selected for, they survive, reproduce
and pass on these advantageous alleles to the offspring. Over many years, two populations become
genetically different forming two different species. Individuals from the two populations cannot mate
hence there is no gene flow.

2. Sympatric speciation-
Refers to formation of the new species from the existing one in the same habitat/area due to
factors such as change in reproductive behaviour, such as courtship behaviour.
Gene mutations occurs by chance giving give rise to new alleles within a population that is living in
the same area.
This may lead to a group of organisms expressing a different reproductive behaviour e.g a song or
a dance that is not recognised by other organisms of the same species, production of a
chemical substance by the stigma that cannot interact with the chemicals in the pollen grain.
The organisms survive, reproduce and pass on these advantageous alleles to the offspring.
Over many years, two populations become genetically different forming two different species in which
individuals from the two populations cannot interbreed because they cannot attract each other hence
there is no gene flow.

REPRODUCTIVE BEHAVIOUR ISOLATION MECHANISMS

• Reproductive isolation refers to a set of mechanisms that prevent species or the members of
the same group from breeding or mating with each other.
• Thus, it prevents the production of fertile offspring. Several mechanisms are responsible for
reproductive isolation. Among them, prezygotic and postzygotic are two main mechanisms.
• Prezygotic and postzygotic are two main reproductive isolation mechanisms.

65
 • The reproductive isolation that occurs before fertilization is the prezygotic isolation.
while, the reproductive isolation that occurs after fertilization and prevents the fertilized egg to
become a fertile offspring is the post-zygotic isolation.

Prezygotic isolation mechanism - Fertilization is prevented

They are;
✓ Behavioural isolation;
Occurs when the behaviour of animals changes and they do not recognize the other members
as mating partners OR behaviour of pollinators change and do not pollinate some plant
species Behavioural isolation occurs when the species are not aware of the mating rituals or
when there is no sexual attraction, etc.

✓ Habitat isolation,
Organisms occupy different habitats in the same area and do not come into contact during the
reproductive season.
✓ Seasonal isolation;
Occurs when organisms mature at different times e.g anther mature earlier than stigma
✓ Gametic isolation,
Occurs when the female gamete fails to attract the male gamete or the male gamete cannot
penetrate the female gamete or a pollen tube may also not grow down the style.

Post zygotic isolation mechanisms - Zygote fails to grow and develop or is unable to breed
They are;
✓ Low hybrid zygote vigour / The Zygote Is Not Viable
The zygote fails to develop properly and die during embryonic development
If the hybrid does manage to make it to birth, it often has at least one, and more likely multiple
defects that keep it from becoming a healthy adult Low hybrid adult viability / Adults of the
✓ Hybrid Species Are Not Viable
The zygote develops but the offspring produced fail to grow properly.
✓ Hybrid infertility / Adults of the Hybrid Species Are Not Fertile
Offsprings appear healthy but infertile. E.g. a mule is a hybrid of a donkey and a horse.
However, mules are sterile and cannot produce offspring, so the only way to make more
mules is to mate more donkeys and horses.

Molecular biology techniques that lead to the new molecular evidence of evolution.
i) DNA hybridization
➢ DNA from individuals of different species are gently heated separately to obtain individual
strands of DNA and then mixed to get hybrid DNA molecules. When heated, the hybrid DNA

66
 from closely related species separate or denature at higher temperature than hybrid DNA
molecules from less closely related species.
(ii) DNA profiling
➢ DNA obtained from individuals of different species are mixed with the same restriction
enzymes that cut them into different sizes. These DNA fragments are placed on the gel
electrophoresis (separates DNA fragments into different sizes) and the DNA fragments of
closely related species are more or less of the same size and occupy the same position on
the gel.
(iii) DNA sequencing
➢ From different individuals of different species, sequence of bases in the DNA is analyzed and
the closely related organisms have more or less the same sequence i.e. they evolved from a
common ancestor more recently.

iv) Protein sequencing

➢ Proteins obtained from individuals of different species are analyzed to get the sequence of
amino acids. If there are very few differences in amino acid sequence, then the organisms are
closely related hence they shared the common ancestor more recently.
v) DNA molecular clock (agreed mutation rate).
➢ As species evolve, they accumulate random mutations at a regular rate becoming
genetically more different.
➢ By counting the number of differences (mutations) between species and by using an agreed
mutation rate as a kind of molecular clock, one can find out how closely related the species
are because the fewer the differences the more closely related they are and vice versa. One
can also estimate how long ago they shared a common ancestor (number of
differences/mutations x number of years).

➢ This method has limitations because:

(i) DNA starts to degrade immediately after death
(ii) The rate at which different parts of the DNA mutate can vary

Q. Give reasons why the theory of evolution is controversial for some people
(i) conflicts with religion as it excludes presence of God
(ii) it suggests that the world is older than the period given in the Bible, hence appears to
contradict Genesis
(iii) some people believe that the evidence of evolution is unsatisfactory i.e. not enough
evidence
(iv) The ideas that humans evolved from apes is considered to be offensive as it challenges
the idea that humans are God’s special creations.

67
Validation of new evidence
➢ Any new evidence must be carefully studied before it is accepted
➢ The potential paper/article is submitted to a scientific journal where it undergoes 3 key
stages of scientific process;
(i) Peer review
(ii) Publishing in the scientific journal
(iii) Scientific conferences

Peer review process

➢ The article is sent to 2 or 3 other scientists in the same profession to verify if:
(i) The paper is valid – whether the conclusions are based on good methods
(ii) The paper is reliable – it can be replicated
(iii) The paper is original – to prevent plagiarism
(iv) The paper is significant – the paper must make a useful addition to the existing body of
scientific knowledge.
➢ The identity of the reviewers is not given to the author of the paper for 2 reasons
(i) To give the reviewers freedom to critically examine the work without offending the author
(ii) To prevent the author from influencing the reviewers.
➢ The outcome of the peer review process could be one of the following three;
(i) The paper is accepted as it has met all the requirements hence it should be published.
(ii) The paper is accepted but with modification and therefore it should be published after
modification
(iii) The paper is rejected.

Once the paper is published in a journal, it gives room for critical evaluation of the data by the
scientific community. “Critical evaluation of the data by the scientific community” means that :
➢ The experiment can be repeated to verify it and to prove reliability
➢ More data can be collected
➢ Validate the data by comparing with other scientists’ work.

Scientific conferences
➢ These conferences allow scientists to share ideas with other scientists in the same field or
related fields.
➢ The suggestions from other scientists can be deliberated on but there is no need to go
through the peer review process and these suggestions are not included in the journal.
➢ The advantages of scientific conferences are;
(i) Provides opportunities to challenge the validity of results
(ii) They come up with new ideas that are debated on and that will be useful in future
research.
(iii) There is exchange of different scientific techniques

68
 ➢ The disadvantages of scientific conferences are
(i) There is pressure to publish the paper so that one can attend the conference hence the
paper may not be scientifically conclusive
(ii) Vested interests and large funding groups can easily throw out some papers that might
be useful
(iii) Timing of the paper can influence audience
(iv) New ideas that go against the accepted view can be harshly criticized and this slows
down the development of new ideas
(v) Attendance could be very expensive especially in poor countries.

Online publishing without peer review

➢ Many scientific ideas are now published online without peer review
➢ The advantages of this include:
(i) It is much quicker
(ii) It can lead to quick exchange of ideas
(iii) It is less expensive
➢ The disadvantages of this include – poorly conducted experiments or investigations can be put in
public domain and can mislead people hence affecting future scientific research.

69
TOPIC 5: MICROBIOLOGY, IMMUNITY AND FORENSICS

TOPIC 5: MICROBIOLOGY, IMMUNITY AND FORENSICS

MICROBIOLOGY
It’s the study of microscopic organisms like bacteria, viruses, fungi and bacteria.
CULTURING MICROORGANISMS

❖ To study microorganisms, they must first be grown in the lab on one of two types of culture - a
pure culture containing one type of microorganism only, or a mixed culture containing a mix of
species.
❖ The microorganisms can be obtained from the environment e.g. pond water, blood of a
patient with bacterial infection.
❖ Growing microorganisms requires aseptic technique which means free from contamination.
Various types of bacteria and fungal spores are present in the air and are an unwanted when
trying to grow a specific microorganism only, since they will compete for nutrients, space and
oxygen and reduce the yield of the desired culture.
❖ Various aseptic techniques have been developed, such as:
● Buying sterile equipment or sterilising reusable equipment with a Bunsen burner flame
and ethanol.
● Cleaning surfaces before and after with ethanol.
● Using a bunsen burner flame to heat the air, causing it to rise and carry away airborne
microorganisms. The same can be done to bottles or flasks to remove contaminants.

ASEPTIC TECHNIQUES
a) Preparation of the work area
1. Clear the work space of all non-essential items.
2. Wipe the benches / work area with alcohol e.g.ethanol.
o Reason - this kills all unwanted bacteria and so decreases the chance of the agar plate
becoming contaminated.
b) Preparing the culture medium for growth of a colony of bacteria

• A culture medium (growth medium) is a special medium used in microbiological laboratories

to grow microorganisms.
• It’s a nutritive substance composed of different nutrients that are essential for microbial
growth.
• Different nutrients and chemicals are added to it to allow the growth of different
microorganisms.
• A culture may be solid or liquid medium.
The solid culture media (plu.) is composed of agar jelly.
• The agar jelly is prepared on agar plates.

70
In a liquid culture medium (broth culture) the desired bacteria are suspended in a liquid nutrient
medium, such as in a flask. It’s prepared by dissolving known amounts of nutrients in distilled water;
the pH is then adjusted.
• When preparing the culture medium the;
Conical flasks for the culture medium or glass petri dishes and agar gel must be sterilised before use
by using an autoclave, or pre-sterilised plastic petri dishes can be bought.
o Reason - this will kill any unwanted bacteria that are present in the solution or on the petri
dishes.
Conical flasks for the liquid culture medium, petri dishes and agar gel must be sterilised before use in
an autoclave.
o Reason - kills unwanted bacteria microorganisms.

c) Plating the bacteria

• This involves transferring bacteria to the agar plates.
This should be done beside a blue Bunsen flame.
o Reason - to prevent the agar media getting contaminated with unwanted bacteria from the
air.
To do this;
✓ Swirl the bacterial suspension to make sure that the bacterial suspension is well mixed.
o Reason - to make sure that the bacteria aren't all at the bottom of the bacteria bottle.
✓ Sterilise the inoculating loop, by placing it in pure alcohol for a few seconds then, heating it
over Bunsen burner flame. Leave it to cool.
o Reason – this kills any unwanted bacteria that are present on the loop.
Allow the inoculating loop to cool for approximately 10 seconds.
Reason - so that the heat does not kill the microorganisms.

✓ Remove the lid from the bacterial bottle and put the mouth of the bottle in the Bunsen flame.
o Reason - to kill off any unwanted bacteria that could be on the bacterial bottle.
✓ Dip the inoculation loop into the microorganism solution and transfer on the surface of the
agar plate.
o Reason - this allows the bacteria to spread out and to grow in individual colonies on the agar
plate. A sterile spreader to evenly spread the bacteria across the whole of the plate.
✓ Replace the lid of the petri dish as soon as possible and secure with tape. Allow the plate to
dry then label the half of the petri dish containing the media (do not label the top). Invert the
plate and store it upside down.
o Reason - The lid stops additional unwanted bacteria in the air contaminating the plate. Do
not fully seal the lid, as this will stop oxygen reaching the bacterium, and this may encourage
harmful anaerobic bacteria to grow. Labels are important, as this identifies the growing
bacterium. If the lid is separated from the petri dish for some reason, the label will stay with
the part that has the bacteria on it, so it can be identified.

71
 ✓ Incubate at a temperature of 25° - 30oC.
o Reason - this reduces the chance of growing harmful pathogens, which would grow at 37°C
in a human body. Hospital laboratories would incubate plates at 37°C (body temperature) to
allow quick growth and identification.
NB: When a culturing in a liquid nutrient broth, mix known volume of the suspension of the
microorganism to be grown with the sterile nutrient broth in the conical flask then incubate at a
temperature of 25° - 30oC.

d) Clearing up after
1. All contaminated materials need to be disposed of either in autoclave bags (for disposable
materials that need to be sterilised, eg spreaders/Petri dishes) or pots (for items that are to be
washed, sterilised and then reused).
2. It is essential that all work surfaces need to be thoroughly disinfected at the end of the activity.
Ensure that hands are washed with soap and water at the end of the activity.

There are 2 different types of culturing:

1. Batch culture.
• In batch culture, the fermentation is carried out in a closed fermenter.
• The microorganisms and nutrients are added and then left to grow for a particular period of
time.
• No further nutrients are added, and products are removed at the end of the period.
Continuous culture.
• Continuous culture takes place in an open fermenter, where nutrients are continuously added
and products are removed at a steady rate.
• Even though the batch culture is easier to set up and maintain that the continuous culture, the
growth rate isn’t as fast.
• However, in the case of contamination of batch culture, only a single batch is lost whereas in
the case of continuous culture, it can lead to huge amount of product lost.
To maximise growth of a pure culture during culturing;
❖ Provide the microorganisms with the right nutrients. This is done using a nutrient medium,
either
i) In form of nutrient broth where the nutrients are in added into a liquid medium in a liquid
culture in a test tube or flask
ii) In a solid form usually nutrient agar where the nutrients are in added into a liquid agar
which solidifies as a jelly in agar plates.
❖ The temperature needs to be maintained at the optimum so that the enzyme activityis not
affected.
❖ A sufficient nutrient supply should be maintained

72
 ❖ Depending on the type of microorganisms grown, either aerobic conditions or anaerobic
conditions should be maintained to prevent growth of a combination of both aerobic and
anaerobic microorganisms as well as the formation of undesired products.
i.e. Growing a culture under anaerobic conditions will ensure that only anaerobic
microorganisms survive and grow. Similarly, growing a culture under aerobic conditions will
ensure that only aerobic microorganisms survive and grow. However, some bacteria will
grow under both conditions therefore by controlling the conditions, the varieties are reduced
considerably.
❖ The pH needs to be kept constant to ensure that the enzyme activityis not affected.

MEASURING THE GROWTH OF CULTURES

There are several ways to measure / monitor growth:
a) Cell count
b) Dilution plating
c) Area and Mass
d) Turbidity (Optical method)

A) Cell counts

• This can be done at regular intervals to measure growth rate.

• It can be done e.g., using a hemocytometer (haemocytometer).
• A hemocytometer is a counting-chamber used to calculate the density of cells in a liquid sample.
• It can also be described as a specimen slide.
• It consists of a thick glass microscope slide with a grid of perpendicular lines etched in the middle
forming squares.
The squares are of different sizes, to allow easy counting of cells. This way it is possible to determine
the number of cells in a specified volume.
• The cover slip, is held in place at a specified height (usually 0.1mm) by cover slip support.
• It can hold a standard volume of liquid of 0.1mm3 . Thus, the hemocytometer is designed such that
each of the volume of the squares is constant.
• The process involves pipetting a small sample of the liquid culture onto a slide with a grid on it.

73
 Above and side view.
Note the cover slip support.

Each corner of a hemocytometer grid has a square divided into 16 smaller squares as shown below.

✓ The corner squares have large subdivisions, hence, can be used to count larger cells.
✓ Cells that are 10 μm or more are appropriately counted in the corner squares e.g. bacteria
and white blood cells.
✓ The central square, has smaller subdivisions.
✓ Cells that are less than 10 μm should be counted in the central square e.g platelets.

74
• To distinguish between dead and viable cells / living cells, the sample is diluted with trypan
blue. This stain selectively penetrates cell membranes of dead cells, coloring them blue,
whereas it is not absorbed by membranes of live cells, thus when viewed under a
microscope, dead cells would appear as dark blue.
• The hemocytometer is standardised so that the number of cells in one set of 16 squares is
equal to; the number of cells counted x 104 per cm3 of liquid culture

• Note; A hemocytometer is a modified and calibrated microscope slide designed to allow

operators to quickly estimate the concentration of cells in a sample.

• To count bacteria cells;

- The cell suspensions should be dilute enough so that the cells do not overlap each
other on the grid, and should be uniformly distributed.
- First place the coverslip over the counting surface before loading the cell suspension.
- The loaded hemocytometer is then placed on the microscope stage.
Allow the sample to settle for a couple of minutes and avoid moving the coverslip as it
might introduce air bubbles and make counting difficult.
- Pipette 10µL and deliver into the gap between the coverslip and the counting
chamber.

• Count the total number of cells found in the 4 large corner squares i.e. the number of cells in
each of the four sets of 16 squares), and calculate the mean.
• Count the live cells (without trypan blue)

75
 • For large cells, the cells inside the four large corner squares are counted and the middle one.
• For small cells the cells in the four outer and middle one are counted.
• Decide how the cells touching the line will be counted e.g. count the cells on or touching the
bottom and right lines and do not count the cells on or touching the top and left lines or vice
versa.

• Once you have obtained the total cell count, cell concentration can be calculated from the
following formula:

So, for example, if you diluted your sample 1:1 with Trypan blue, and you counted
325 cells in 4 corner squares plus the central big square, total cells per ml =

76
What is the dilution factor?

Represents how much more volume there is in your mixture in addition to the original sample you
had.

To calculate the dilution factor; divide the final volume by the initial volume.
DF = Vf / Vi
Where Vf = Final volume
Vi = Initial volume

Example 1

A dilution is made where 10mL of the sample is obtained and 40mL water is added as shown below.

The sample is 10mL and the final volume is 50mL.

Dilution factor = 50/10 = 5

Possible calculations

1. Average number of viable cells per square

2. Percentage of viable (living) cells

3. Dilution factor

4. Concentration of viable cells per ml

5. Concentration of total cells per ml

77
Example;

100ul of trypan blue was added to 100ul of cell suspension to make a volume of 200ul. 10ul was
obtained using a pipette and transferred to the hemocytometer.

Cell counting was done using haemocytometer in 5 squares . The cells counted were as follows;

Total viable cells = 60

Total non viable cells = 3

Calculate:

1. Average number of viable cells per square

Number of viable cells

Total number of cells

= 60 / 5 = 12 cells

2. Percentage of viable cells (percentage of living cells)

Number of viable cells X 100

Total number of cells

= 60/63 * 100 = 95.2%

3. Dilution factor

Final volume

Volume of cells

= 200 / 100 = 2

4. Concentration of viable cells per ml

Average number of viable cells per square X Dilution factor X 104

12 X 2 X 104 = 240000 cells / ml

= 2.4 X 105 cells / ml

Remember; The hemocytometer is standardised so that the number of cells in one of the corner
squares is equal to; the number of cells counted x 10 4 per cm3 of liquid culture

5. Concentration of total cells per ml

Average number of total cells per square X Dilution factor X 104

12.6 X 2 X104 = 25.2 X 104 = 252000

= 2.52 X 105

78
B) DILUTION PLATING
This procedure is used to identify the number of micro-organisms in a fixed amount of a liquid.

• It involves mixing known volumes of a culture with a sterile liquid at different ratios to produce
a series of dilutions until you reach a point when you can count the cells. The resulting
dilutions are used to count the number of cells present.
• Using the values counted, you can work back using its dilution factor. The original number of
microorganisms in the sample is calculated by multiplying the number of cells counted by the
dilution factor.
This can be done at regular intervals to measure growth rate.

The process involves;

1. Prepare a series of diluted bacterial cultures in a liquid culture

2.Spread a sample of each diluted culture on nutrient agar plates.

(Each single bacterium divides to form a colony which is large enough to be seen)

79
3. Count the number of colonies in the agar plate. Use this to calculate the number of bacteria present
in the original culture tube. The values counted are used to work the original number of
microorganisms in the sample back from the counted plate using its dilution factor. Since each colony
arose from a single living cell, this is the number of viable cells in the initial small volume.
• Example;
• If a plate containing a 1:1,000,000 dilution of the original ml of sample shows 150 colonies,
then the number of cells per ml in the original sample is calculated by:
The number of colonies per ml X The dilution factor of the plate counted
In this case 150 x 1,000,000 = 150,000,000 cells per ml
1.5 X 108 cells / ml

80
NOTE: This method is used to count only live (viable) cells while the haemocytometer counts living
and dead cells

C) Mass
This involves measuring the dry mass of the cells.
The method is suitable by using a liquid growth medium

• A known volume can be sampled from a liquid culture, placed in a centrifuge and spun till only
the solid mass of cells settle at the bottom.
• The cells can also be obtained by filtering.
• The dry mass of the cells is obtained by heating the cells at constant temperature to obtain a
constant mass e.g. in a oven at 100oC for 12 hours.
• This gives the dry mass of the cells in a culture medium at a particular time.
• This can be done at regular intervals to measure growth rate.

When culturing bacterial and fungi, growth can also be determined by measuring the diameter of a
bacterial colony or a fungal mycelium at specific time interval.

D) Turbidity (Optical methods)

• This is the most common method of measuring the growth of cells.

• Turbidity describes how light passes through a sample of liquid as a measure of how many
particles are suspended in that liquid.
• Involves measuring the amount of light transmitted or optical density (amount of light
absorbed and scatterered) by a sample of cell suspension in a culture medium.
• As cultures grow, the solution in the flask or test tube becomes more and more turbid (cloudy
and increasingly opaque).
• A turbid solution absorbs more light, so less light can pass through it.
• So as more cells grow,less light can pass through.
• Particles in solution scatter light and the more particles (microorganisms) found in a solution,
the more light is scattered by them. Therefore, a replicating population of microorganisms
increases light scattering and measured absorbance values.
• The degree to which a medium retards transmitted rays of light is called optical density (OD).
• OD value can be directly related to the number of microorganisms in very low-density
suspensions.
• Small samples can be taken at regular intervals and placed in a cuvette where a colorimeter
records the amount of light passing through, giving a measurement of the number of cells in
the culture.

81
• The less the light that passes through the higher the number of cells in the sample.
• The colorimeter also can be used to record the optical density giving a measurement of
number of cells in the culture.
• The higher the optical density values, the higher the number of cells in the sample.

82
NOTE:
Measuring growth of cultures by cell count, dilution plating, Mass or area and by measuring turbidity
can be used to compare growth rates in different conditions e.g. different pH values or different
temperatures.

NB: Continual measurement of growth and recording of the results can be used to plot a bacterial
growth curve.

PHASES OF A BACTERIAL GROWTH CURVE

1. Lag Phase
When a microorganism is introduced into the fresh medium, it takes some time to adjust with the new
environment. It’s the first phase of microorganism growth is the lag phase where microorganisms are
adjusting to the environment before starting to reproduce, thus during the lag phase the population
remains constant.

2. Log Phase / Exponential Phase

The next part of the growth curve is the log phase where the population size grows exponentially
meaning that every round of division doubles the population size, so long as the dividing organism
has a sufficient amount of nutrients.
During this phase, the culture has the maximum growth rate and the number of bacteria increases
logarithmically (exponentially). A single cell divide into two, which replicate into four, eight, sixteen,
thirty two and so on (That is 20, 21, 22, 23.........2n, where n is the number of generations)
The time taken by the bacteria to double in number during a specified time period is known as the
generation time.
The generation time tends to vary with different organisms. E.coli divides in every 20 minutes, hence
its generation time is 20 minutes, and for Staphylococcus aureus it is 30 minutes.

3. Stationary Phase
The stationary phase is where the population size reaches its maximum due to decreasing nutrient
levels and build of up toxic substances.
As the bacterial population continues to grow, all the nutrients in the growth medium are used up by
the microorganism for their rapid multiplication. This result in the accumulation of waste materials and
toxic metabolites. This shifts the conditions of the medium such as pH and temperature, thereby
creating an unfavourable environment for the bacterial growth. The reproduction rate will slow down,
the cells undergoing division is equal to the number of cell death, and finally bacterium stops its
division completely. The cell number is not increased and thus the growth rate is stabilised. If a cell
taken from the stationary phase is introduced into a fresh medium, the cell can easily move on the
exponential phase and is able to perform its metabolic activities as usual.

83
4. Death phase / Decline Phase
The stationary phase is followed by decline phase where depletion of nutrients and accumulation of
metabolic wastes products causes death of organisms.
During this, the bacteria completely loses the ability to reproduce and begin to die due to the
unfavourable conditions. The number of dead cells exceeds the number of live cells.

NOTE:
In ideal conditions and unlimited space, bacteria reproduce very rapidly resulting to very large cell
numbers, which are difficult to represent.
Therefore, growth of bacterial colonies is represented using L = log(numbers) on the y axis, versus T
(time) – on the x axis.
A log10 scale is used meaning that every number on the y axis represents a power of 10 e.g. 2 is 10 2
which is 100.

84
EXPONENTIAL GROWTH RATE

❖ During exponential growth phase, the number of microorganism doubles exponentially at a

constant rate.
❖ Since the values obtained on counting the cells could be very large, a logarithmic scale is
used which is easier to manage.

❖ The log10 values of number of microorganisms are plotted against time.

Possible calculations:

❖ Exponential growth rate constant

❖ Number of bacterial in a population
❖ Generation time

Exponential growth rate constant

❖ The exponential growth rate constant k equals the number of times that the population will
double in a unit time. Its calculated using the formula;
K = log10 Nt – log10 No
log102 x t
OR

Where the value of 0.301 in the formula is log102

85
Example
Microorganisms were grown in a culture, the growth was measured at regular intervals and the
following graphs were plotted

What is the exponential growth rate constant from the graph between 5 and 15 hours?

k is also called the generation rate constant because it is the reciprocal of the generation or doubling
time T, i.e. k = 1/T

NB: In this case we don’t need to take the logs of 4.1 and 4.95, because they are
already logs.

Note: Sometimes we are given actual cell counts, so we do need to take logs.

86
Number of bacterial in a population
To calculate the number of microorganisms in a population at different time of growth, the following
formula can be used;

Nt = No X 2kt

Where,

Nt = The number of organisms at time t

No = The number of organisms at the beginning of the
experiment (time o)
k= Exponential growth rate constant
t = The time the colony has been growing

Example

Calculate the number of bacterial cells over 5 hours space interval, if the number of bacteria at the
beginning is I and the growth rate constant is 2.

Nt = No X 2kt
N5 = 1 X 2(2)(5)
= 1 X 210 = 1024 cells
Generation time or Doubling time:
Generation time the time taken for one generation, or for the population to double in size. A feature of
exponential growth is that it has a constant doubling time.
It is the time required for a bacterium to give rise to two daughter cells under optimum conditions. The
generation time for most of the pathogenic bacteria, such as E. coli, is about 20 minutes.
G (generation time) = t /n
Where, t = time interval in minutes or hours
n = number of generations
i.e. G = t/n
n = logNt -logNo
log2
where Nt = Number of bacteria at the end of a time interval
No = Number of bacteria at the beginning of a time interval

87
Example
What is the generation time of a bacterial population that increases from 10,000 cells to 10,000,000
cells in four hours of growth as shown in the graph below?

G (generation time) = t /n
Where, t = time interval in minutes or hours
n = number of generations (number of times the cell population doubles during the
time interval)

i.e
G = t/n
n = logNt -logNo
log2
where Nt = Number of bacteria at the end of a time interval
No = Number of bacteria at the beginning of a time interval
n= 7–4 = 9.967
0.301
In this case; G = t/n
= 4 hours = 0.401 hours = 24.079 minutes
9.967

k is also called the generation rate constant because it is the reciprocal of the generation or doubling
time T, i.e. k = 1/T
so, T = 1/k
Example
What is the exponential growth rate constant and generation time (in minutes) if a cell count increases
from 2.5 × 103 to 4.0 × 106 over the space of 8 hours?

88
Answer
Using a calculator; log10 (2.5 × 103) = 3.40 and log10 (4.0 × 106) = 6.60.

In some cases the generation time can be read directly from the graph as shown in the graphs below.

The exactly doubled points from the absorbance readings are taken and, the points extrapolated to
meet the respective time axis.

Generation Time = (Time in minutes to obtain the absorbance 0.4) – (Time in minutes to obtain the
absorbance 0.2)
= 90-60
= 30 minutes

89
BACTERIA AND VIRUSES AS AGENTS OF DISEASE
Bacteria and viruses are some of the main disease causing pathogens in humans. Even though they
both cause disease, they vary in many ways.

DIFFERENCES BETWEEN BACTERIA AND VIRUSES

• Bacteria can reproduce independently, whereas viruses can only reproduce inside a host cell
• Bacteria contain ribosomes, viruses do not.
• Bacteria are living single-celled microorganisms, while viruses are technicallynot living as
they require other living cells to function.
• In bacteria the genetic material is circular loop of DNA while in viruses the genetic material is
DNA or RNA.
• Bacteria have a cell surface membrane, cytoplasm and cell wall while viruses lack a cell
surface membrane, cytoplasm and cell wall
• Bacteria often have a capsule while viruses have a capsid.
• The average diameter of a bacteria is 0.5u to 5um while viruses are 20nm to 40nm in size.

Bacteria Viruses

Bacteria can reproduce independently, viruses can only reproduce inside a host cell

Bacteria contain ribosomes viruses do not contain ribosomes

In bacteria the genetic material is circular loop of In viruses the genetic material is DNA or RNA.
DNA

Bacteria have a cell surface membrane, viruses lack a cell surface membrane,
cytoplasm and cell wall cytoplasm and cell wall

Bacteria often have a capsule Viruses have a capsid.

VIRUSES
VIRAL STRUCTURE
They consists of;

• Genetic material - DNA or RNA.

The genetic material can be circular or linear.
The RNA genetic material can be single stranded or double stranded.
Some viruses contain single stranded RNA that can be read directly by the host ribosomes
as a mRNA while some viruses contain single stranded RNA that cannot be read directly by
the host ribosomes as a mRNA.

90
 • Capsid - A protein coat surrounding the genetic material. The capsid consist of repeating
protein units called capsomeres.
The repeating units:
i) Reduces the amount of genetic information needed to code for coat production.
ii) Simplifies the assembling of the protein coat in the host.
• Some have a lipid envelope acquired from the previous host cell.
The presence of the lipid envelope makes it easier for the for the virus to enter the host cell
through fusion of the phospholipids
• Viral Attachment Proteins – These stick out from the capsid and are used to attach to host
cells. The Viral Attachment Proteins are specific to the host cells they attack hence viruses
are specific to the host cells they invade.
• They project from their capsid or envelope. These bind to extracellular molecules on the
surface of host cells, and assist the virus in attachment to and and entry specifically into the
host cell (and in exit after infection).
• Enzymes - Some viruses contain an enzyme called reverse transcriptase which is used to
convert their RNA into DNA, which can be inserted into the host cell’s genome so that viral
proteins are transcribed for translation.

Viruses attack the host cells in several ways;

(i) Some enter taken into the cell by receptor mediated endocytosis
(ii) For those that have an envelope, the envelope fuses with the host cell membrane
releasing the virus inside the host cell.
(iii) Some plant viruses use vectors to enter plant cells
(iv) Lambda phage (bacteriophage) that infect bacteria use structures called pili to enter
bacterial cells.

LIFE CYCLE OF A VIRUS

There are 2 different life cycles of viruses that infect bacteria;

a) The lytic cycle
b) The lysogenic cycle.
THE LYTIC CYCLE

• The lytic cycle is where viruses infect their host cells and their viral genetic material is
replicated immediately after infection.
• The replication of the viral genetic material occur independently of the host DNA or they can
insert their genetic material into the host’s DNA.
• Viral proteins are synthesized from viral genetic material
• So new viral particles are made.
• As more and more viral particles are made they eventually burst from the cell in a process
known as lysis, where the cell membrane is ruptured and the cell destroyed.

91
 • The new viral particles are released from the cell and can infect more host cells.

LYSOGENIC CYCLE

• In the lysogenic cycle, the virus enters the host cycle and incorporates its genetic information
into the host genome, but remains dormant and latent without causing disease. The inserted
genome is called a provirus.
• As the host cell replicates its DNA, the viral DNA is replicated and many copies of host cells
containing the viral DNA are produced.
• No viral mRNA is produced, no viral proteins are synthesised hence no new viral particles are
synthesised and no lyses of the host cell.
• However, the virus can become virulent under certain conditions such as poor diet and
psychological stress that lowers body immunity. This causes the amount of repressor protein
to be produced to decrease and the virus enters the lytic pathway.
• In certain conditions, the viral DNA leaves the main genome to form a plasmid which is only
then translated and transcribed, from which lysis later occurs.

CLASSIFICATION OF VIRUSES ACCORDING TO THEIR GENETIC MATERIAL

a) DNA viruses
They possess DNA as their genetic material which can be double stranded or single stranded.
b) RNA viruses
They possess RNA as their genetic material which can be double stranded or single stranded.
The RNA can positive strand (can be read as a mRNA by the ribosomes) or negative strand
(cannot be read as mRNA by the ribosomes).
c) Retroviruses
They possess;
i. Single stranded RNA
ii. Two protein coats
iii. A lipid envelope
iv. Reverses transcriptase – an enzyme used to convert the single stranded
RNA to double stranded DNA copy. e.g. Hepatitis B virus and HIV

VIRAL REPLICATION (VIRAL REPRODUCTION)

• Viral replication is the formation of new viruses in the host cells.

• Viruses must first get into the cell for viral replication to occur.
• Replication of viruses greatly varies between viruses and depends on the type of genetic
material either DNA or RNA as well as the genes involved in them.
• Generally, it involves generation of many copies of its genome, viral messenger RNA (mRNA)
and synthesis of viral proteins.
• This is followed by assembly of viral proteins around the viral genome to form new viral
particles

92
 • The new viral particles are eventually released out of the infected cells by lysis, a process that
kills the cell by bursting it.
• The virus continues infecting new hosts cells.

Steps of virus replication cycle

KEY EXAMPLES

A) EBOLA VIRUS

• The Ebola virus causes a severe and often fatal disease called Ebola virus disease (EVD).
• The Ebola virus has RNA as the genetic material, a matrix, a capsid and a lipid bilayer
envelope.
• The RNA codes for seven structural proteins of the virus.
• The viral genome cannot be read directly by the host ribosomes therefore it needs to be first
transcribed to produce mRNAs that can be translated by the host ribosomes hence it contains
its own RNA polymerase to synthesise a corresponding strand of its RNA.
• A glycoprotein (GP) projects as long spikes from its lipid bilayer envelop and it’s used for
attachment and entering new host cells. The glycoprotein spikes bind to cells and mediate
fusion between the viral envelope and the host cell membrane, enabling the virus to release
its contents into the host-cell cytoplasm.

93
Symptoms of EVD

They appear between eight and 10 days after exposure.

• unexplained bleeding and bruising

• Fever, weakness .
• diarrhea fatigue
• vomiting stomach pain

HOW EBOLA CAUSES SYMPTOMS THAT CAN LEAD TO DEATH

94
 • Upon entering the body, the virus targets specific cell types, including liver cells, cells in the
immune system, and endothelial cells, which line the inside of blood vessels.
• The viral glycoproteins interacts with receptors on the surface of the body cells and its taken
up into the cell.
• Once inside the cells, one of the proteins made by the virus disrupts cell adhesion, so that
cells have do not adhere to each other the normal way and to the attach to the extracellular
matrix, which helps to hold the cells together.
• Due to loss of cell adhesion and infection of blood vessel cells, the vessels become leaky,
leading to blood loss and internal bleeding.
• By targeting liver cells, the detoxification body’s ability to clear toxins out of the bloodstream is
affected, and by infecting the immune system, whose cells travel to many body parts, results
in virus spreading quickly in the body.
• Over time, infection of cells throughout the body can cause organ failure, while fever, internal
bleeding, diarrhea and vomiting can cause severe loss of ions (affecting transmission of
impulses) and loss of body fluids.. Ultimately, organ failure due to internal bleeding, leading
to death.
• Transmission
• Its transmitted through the blood, the exchange of bodily fluids, contact with infected humans,
and animals such as primates, bats, and pigs and consumption of infected meat.

HUMAN IMMUNODEFICIENCY VIRUS (HIV)

• It consist of a genome made up of 2 single stranded RNA molecules, 2 protein coats, reverse
transcriptase enzyme, intergrase enzyme, protease enzyme and a lipid envelope with surface
glycoproteins namely glycoprotein 120 (gp 120) as well as glycoprotein 41 (gp 41).
• It infects immune cells such as T helper cells, (mostly) macrophages and dendritic cells.
These cells have CD4 receptors to which gp 120 binds.

95
HIV INFECTION

• The HIV virus targets CD4 receptor bearing cells such as T helper cells and macrophages
which are part of the immune system cells.
• The virus attatches to the CD4 receptor on these cells using gp 120.
• The binding triggers a change in HIV gp 120 / gp 41 complex that causes the gp 120 to
interact with other receptors on the host cell membrane called CCR5.

96
 • This exposes the gp 41 allowing it to initiate fusion of the viral envelope with the host cell
membrane and the virus enters the host cell.
• Upon entry, uncoating (removal of the protein coat) occurs releasing the viral RNA into the
host’s cell cytoplasm.
• Once inside the cytoplasm of the infected cell, reverse transcriptase is used to synthesis viral
DNA.
• The viral DNA then moves to the nucleus of the host cell.
• In the nucleus the viral DNA is inserted / incorporated into the host DNA by enzyme
intergrase.
• As the host cell replicates and transcribes its DNA, the viral RNA is also replicated and
transcribed into viral RNA genetic material and also into mRNA.
• The viral mRNA is translated into viral proteins.
• The viral proteins are assembled around the viral RNA genetic material to make new viral
particles.
• Once many viral particles are made, they cause the infected host cell to lyse releasing new
viruses that infect more cells.
• This leads to death of infected immune cells, resulting into a weakened immune response and
HIV develops into AIDS where the body can’t defend against simple infections.

Symptoms of AIDS

include weight loss, diarrhoea, dementia, cancers and opportunistic infections such as TB and
can result in death.

TOBACCO MOSAIC VIRUS (TMV)

• This was the first plant virus to be discovered and infects the chloroplasts, reducing
chlorophyll formation.
• It also causes the leaves to curl up, reducing their surface area.
• This reduces the leaves’ ability to photosynthesise, thus reducing the plant’s growth.

Structure of Tobacco Mosaic Virus

• It’s a simple rod shaped RNA containing virus.

• It consists of centrally located single stranded RNA surounded by a protein coat (capsid).

97
 • It infects tobacco and other closely related species.
• It infects the chloroplasts, reducing chlorophyll formation.
• It also causes the leaves to curl up, reducing their surface area.
• This reduces the leaves’ ability to photosynthesise, thus reducing the plant’s growth.
• It infects the chloroplasts reducing chlorophyll formation.
• It can also make leaves curled up reducing their surface area.
• This reduces the plant's ability to photosynthesise and grow properly, which can
reducing crop yields.
• It enters the plant naturally through wounds and spreads from cell to cell through
plasmodesmata.
• The virus attaches to the cell wall, injects its RNA into the host cell.
• Inside the host cell, the protein coat dissociates, and viral nucleic acid becomes free in the
cell cytoplasm.
• The viral RNA first induces the formation of specific enzymes called “RNA polymerases”.
• The single stranded viral RNA synthesizes an additional RNA strand called replicative RNA.
• This RNA strand is complementary to the viral RNA genome and serves as a template for
producing the new RNA single strands which are copies of the parental viral RNA.
• The RNA strand also serves as a template for the synthesis of viral mRNAs for synthesis of
viral proteins.
• Each mRNA attaches to the host’s ribosomes and its translated using the host’s tRNAs and
amino acids to synthesise viral RNA protein subunits.
• The protein sub units are assembled to form the viral capsomeres that surround the viral RNA
to form new viral particles.
• The new viral particles infect other cells by passing through plasmodesmata that connect
adjacent plant cells. This process allows the virus to take over metabolic processes without
killing cells.

98
QUESTION

1. TMV can cause plants to produce less chlorophyll. This causes leaf discoloration. Explain why
plants with TMV have stunted growth.
Discoloured plant would lack chlorophyll. The leaves also curl reducing the surface area
reducing the surface area exposed to light. This means that less photosynthesis would occur
in the leaves of the plant, so less glucose is made as a result. Therefore there is less energy
released for growth as glucose is needed for respiration. As glucose is also needed to make
amino acids and proteins, there would be less amino acids and proteins for growth.

LAMBDA PHAGE

• A lambda phage is a type of bacteriophage - a virus that infects bacterial cells.

• This bacteriophage infects bacteria such as E. coli, which is a bacteria found in human
intestines, normally harmless but can cause food poisoning.
• The lambda phage structure consists of a head, tail and tail fibres,
• The bacteriophage shape differ from the shapes of viruses that infect humans and mammals.

99
• Upon interaction of the lambda phage receptors and the host cell, lambda phage DNA is
injected through into the cell.
• The phage λ leads two life cycles, the lytic cycle and the lysogenic cycle after injecting its
DNA into E.coli cell.
• In the lytic cycle, phage DNA is replicated and the phage genes are expressed resulting in
production of several phage particles.
• The lytic cycle ends with lysis of E.coli cells and release of phage particles.
• The lysogenic cycle results in integration of phage DNA with bacterial chromosome and
becomes a part of host DNA.
• It replicates along with bacterial chromosome and is inherited into new bacterial cells that are
produced by binary fission.
• The phage DNA integrated with bacterial chromosome is called prophage.
• The prophage is non-virulent.
• The bacteria containing prophage are called lysogenic bacteria, and the prophage stage of
viruses as lysogenic viruses.
• Lambda phage can also infect bacterial through pili.

100
 BACTERIA
All bacteria have;
(i) Cytoplasm
(ii) Circular DNA
(iii) Cell wall
(iv) Cell surface membrane
(v) 70s ribosome
(vi) Energy stores
1. Lipid droplets
2. Glycogen granules
Some bacteria have the following in addition to the above
(i) pilli
(ii) fimbriae
(iii) mesosomes
(iv) photosynthetic membrane
(v) plasmids
(vi) flagellum
(vii) slime layer and capsule

CLASSIFICATION OF BACTERIA
They can be classified according to;
1. Their shapes
2. Their structure of cell wall
3. Their respiratory requirements
According to their shape
a) Spherical –cocci
b) Bacillus- (rod shaped)
c) Spiral - (spirilla) – twisted
d) Vibrio - (comma shaped)

According to respiratory requirement

i) Obligate aerobes
Need oxygen for respiration e.g. Mycobacterium tuberclosis
ii) Facultative aerobes / anaerobes
Can survive in presence or absence of oxygen e.g. is E.coli and Salmonella
iii) Obligate anaerobes
Can only respire in absence of oxygen e.g. Clostridium tetani that causes tetanus.

101
According to structure of cell wall
- There are two types of bacterial cell walls which can be distinguished by Gram staining
depending on presence or absence of teichoic acid
- All bacteria are colourless before staining.
- Based on their cell walls bacteria can be divided into two;
1. Gram positive bacteria e.g. Staphylococcus aureus and Lactobacillus bulgaricus
These have teichoic acid and stain purple / blue with gram stain
They have thicker cell walls. The cell wall has an overall thickness of 20-80nm.
2. Gram negative bacteria e.g. Salmonella enteritidis and E.coli.
These lack teichoic acid and stain red with gram stain
They have thinner cell walls. The cell wall has an overall thickness of 8-11nm.

Key Example

Mycobacterium tuberculosis

• Causes tuberculosis

Structure of Mycobacterium tuberculosis

• Mycobacterium tuberculosis is an obligate aerobe that infects the well-aerated upper lobes
of the lungs.
• The bacterium can also survive as a facultative intracellular parasite, usually of
macrophages.
• It has slow generation time, 15-20 hours, a physiological characteristic that may contribute
to its virulence.
• The cell wall contains peptidoglycan, and complex lipids such as mycolic acids, cord factor,
and wax-D.
• Its encapsulated.

102
 • It infects phagocytes mainly the macrophages in the lungs.
• The first infection is symptomless as the infected phagocytes are enclosed in tubercles as a
result ofinflammatory response in the lungs.
• However, the bacteria lie dormant inside the tubercles as they are not destroyed by the
immune system as tubercles are covered with athick waxy coat.
• When the immune system becomes weakened, the bacteria become active again and slowly
destroy the lung tissue thus leading to breathing problems, coughing, weight loss as well as
fever. TB can potentially lead to death.

• Mycobacterium tuberculosis is an obligate aerobe. For this reason, it’s found in the well-
aerated upper lobes of the lungs.
• However it can live as facultative intracellular parasite, usually of macrophages.
• It multiply slowly (has a slow generation time, 15-20 hours) a physiological characteristic that
may contribute to its virulence.
• Although TB usually infects the lungs, it can infect any part of the body,
• It is commonly spread through droplet infection i.e. inhaling cough, talk and sneeze droplets
that contain bacteria. These droplets enter the lungs.
People who are vulnerable to TB are:-
(i) People with weakened immune system
(ii) Living or working in crowded places because people breath, cough, sneeze near to each
other
(iii) Malnourished (lack of balanced diet)
(iv) Drinking infected cattle milk
(v) Living or working in close contact with infected cattle
Symptoms of lung TB
(i) fever
(ii) loss of appetite
(iii) weight loss
(iv) persistent cough
(v) sputum stained with blood
(vi) tubercules- swellings in the lungs

INFECTION WITH TB
Involves two phases/stages
(i) primary infection
(ii) secondary infection (active TB)

1. Primary infection
• The bacteria enters an individual e.g. through inhalation of droplets from infected person

103
 • Once in the lungs they multiply slowly and sometimes there may be no symptoms.
• A localized inflammatory response of the immune system occurs causing the formation of
tubercles in the lungs, with the bacteria enclosed in the tubercles.

Phagocytes called macrophages surround and engulf the bacteria, and produce enzymes
from the lysosomes to kill the bacteria.
• In about 8 weeks, the immune system controls the infection.
• However, once inside the cell, some bacteria produce a thick waxy coat to evade the actions
of enzymes in the macrophages allowing them to survive inside the tubercles.
• This stops the macrophages from breaking the bacterial down and they become dormant or
grow very slowly.
• They lie dormant but if the immune system becomes weak they are re-activated.
• In some cases they burst out of the cells

1) Secondary infection (active Tuberculosis)

❖ 80% of secondary infection results due to activation of the dormant bacteria in the
tubercles when the immune system is weakened.
❖ While in the tubercles, the bacteria may undergo mutations and therefore not easily
destroyed by memory cells developed during the primary infection.
❖ 20% of secondary infection results due to severe first attack that overwhelms the
immune system whereby the immune system cannot destroy them.
❖ Lung tissue is slowly destroyed by the bacteria, causing breathing problems.
❖ The patient develops fever, persistent cough, produces sputum stained with blood, loses
weight, and loses appetite among other symptoms.
❖ In some case the bacteria invades other parts like the CNS and the glands.
❖ The bacteria target the cells of the immune system such as macrophages (lyse them by
producing toxins) and T-cells (.e.g. suppress the production of cytokines by T helper
cells).
❖ Due to damage to immune cells like macrophages, the immune system is weakened and
the patient becomes vulnerable to other opportunistic infection.
❖ This eventually leads to death.

104
Diagnosis of TB
a) Skin test
Small amounts of extract of M. tuberculosis are injected under the skin of the fore arm. A positive test
shows inflamed area of the skin around the site of the test injection. This shows presence of
antibodies specific to the TB antigens. It indicates that the TB bacteria are already present in the
blood that have caused the production of antibodies that cause the swelling.
b) Identification of bacteria in the sputum
c) Identification of bacteria in the blood.
d) Chest X-rays- this discovers the extent of the damage and extent of the disease in the lung.
Treatment of TB
• It is treated using antibiotics in two phases.
• The first phase involves a combination of 4 antibiotic drugs so that the bacteria will not resist
all.
• It takes 2 to 3 months
• The second phase involves 2 antibiotics and it takes 4 months.
• Treatment period ranges from 6-18 months depending on the intensity of infectionand the
patients immune system.

Some of the reasons why TB has not been eradicated are;

1. High mutation rate of the bacteria.
2. Incomplete course of treatment due to the long period of medication which makes
treatment expensive.
3. Increase in HIV case which weakens the immune system leading to occurrence of TB as
an opportunistic disease.
4. Increase in more foreign travel leading to transmission and spread between countries.
5. Its easily transmitted through droplets
6. Increase in population leading to crowded areas allowing easy transmission.
7. Increase in infection in cattle with Mycobacterium bovis that lead to infections to humans
through infected milk.
8. Limited medical resources that may lead to inappropriate diagnosis hence patients keep
spreading the bacteria.

Control of TB
1. The BCG vaccine – very effective in reducing effects of TB
2. Improving living standard i.e. less crowded housing and good nutrition
3. Preventing and treating the disease in cattle
4. Proper treatment of milk before consumption.
5. Prevention of HIV infection because TB is an opportunistic infection in AIDS

105
IMMUNITY

HOW PATHOGENS CAN ENTER THE BODY

There are many ways disease-causing microorganisms can enter the body.
Ways pathogens could enter include:

• Inhalation - Coughing, talking and sneezing are all means through which pathogens can pass
into the respiratory tract and further.
• Ingestion - Eating food contaminated with pathogens can cause illnesses like salmonella.
• Direct contact - By touching skin to skin or through bodily fluids like blood of an infected
Person e.g. HIV
• Vector - This is an organism that carries infection, such as mosquitoes which carry malaria
• Fomites - These are inanimate objects like dust that might land on the skin, eyes, mouth etc
that are contaminated with pathogens contaminated bed linens like in hospitals
• Inoculation – Introducing the pathogen directly through broken skin either through injury or
infected instruments e.g HIV.

Major routes pathogens take to enter the body and the role of barriers to protect the body
from infection.

1. Eye Tears contain the enzyme lysozyme which destroys bacterial cell walls
hence kill them.
2. Respiratory Mucus produced by goblet cells traps bacteria and then swallowed or
tract coughed up.
Mucus has lysozyme which kills pathogens.
Mucus provides physical barrier against pathogens
Cilia beat dirty mucus to pharynx to be swallowed or coughed out.
Epithelial surfaces have phagocytic WBCs which engulf and digest
pathogens.
Lung surfactant is antibacterial
3. Gastro- Stomach acid – the HCL with a pH of around 2 destroys most pathogens
intestinal Gut flora (bacteria);
tract (i) Outcompete pathogens for nutrients
(ii) Outcompete pathogens for space
(iii) Excrete lactic acid that kills pathogens
Mucus lining the gut:
(i) Physical barrier to infection.
(ii) Has lysozyme enzyme that kills pathogens
Vomiting reflex ejects bacteria and viruses from the body before the infection
can spread.

106
 4. Skin Tough physical barrier for pathogens unless it is cut or broken. Toughness is
due to keratin in the skin epidermis which forms a physical barrier.
Skin flora (bacteria);
a) Outcompete pathogens for nutrients
b) Outcompete pathogens for space
c) Produce chemicals that inhibit growth of pathogens.
Sebum – an oily fluid (lactic acid and fatty acids) which is made by the skin
and its roles include;
a) To kill pathogens
b) To enhance the growth of natural skin flora

However, the body has a number of physical and chemical barriers to entry of pathogens.

Physical barriers
Physical barriers that protect the body from infection include:

• Skin is a tough physical barrier consisting of keratin that makes it tough and water proof
• Internal tubes produce mucus which traps pathogens and prevent them from infecting cells.
They also have cilia that trap pathogens.

Chemical barriers

• Stomach Acid (hydrochloric acid) which kills bacteria due to the low pH. The low pH inhibits
enzymes of most bacteria interfering with DNA replication.
• Sebum produced by the skin is antiseptic and contains chemicals which inhibits growth of
microorganisms.
• Saliva has bactericidal and bacteriostatic properties.
• Lysozymes in tears, milk, saliva and mucus destroys microbial cell walls by breaking down
their glycosidic bonds leading to their death.
• Vomiting that ejects pathogens from the body before causing an infection or before an
infection spreads.
• Gut and skin flora – natural bacterial flora competes with pathogens for food and space and
out competes invading pathogens preventing them from causing infection.
• Cough reflex that removes pathogens from the body before causing an infection or before an
infection spreads.

Methods of transmitting pathogens and the natural barriers of the body that prevent infection
Method Description Barrier
Vector A vector is a living organism that carries a pathogen from one Tough skin due to
host to another e.g. malaria and yellow fever are transmitted by keratin
vectors. Blood clotting

107
 Fomites These are inanimate objects that carry pathogens from one host Natural skin flora
to another e.g. hospital towels and bedding e.g. Staphylococcus Sebum
infections
Direct Skin disease in children Tough skin due to
contact Sexual diseases e.g. HIV keratin
Skin flora
Sebum
Inhalation Through cough, sneeze or talk droplets which are infected with Mucus (Lysozyme
pathogens e.g. influenza, measles and TB & physical
bacteria)
Phagocytes
Lung surfactant
Ingestion Through infected food and drinks e.g. diarrhoea, hepatitis A, Mucus (Lysozyme
salmonella poisoning & physical
bacteria)
Stomach acid
Inoculation A pathogen is introduced or inoculated into the body directly Clotting of blood
through a break in the skin e.g. cut by infected knife, needle or
bite by infected animals e.g. HIV, hepatitis B, rabies and
tetanus.

However pathogens overcome through the barriers to entry invade the body of the host, causing
infection.
The host in turn responds through immune responses.

RESPONSE TO INFECTION
This is by various reactions mediated by immune cells and substances secreted by immune cells.
The immune system of the body has 4 main features;
(i) Can distinguish self from non-self-cells.
(ii) It is specific – response to specific foreign cells
(iii) It is diverse – can recognize many different antigens.
(iv) It has immunological memory i.e. after the first response to an infection (primary
response) due to a primary infection, any other response (secondary response) due
to a secondary infection is rapid and strong due to presence of memory cells.

Key Terms
• Cytokines – chemicals that T helper cells release that stimulate division and differentiation of B cells
• Antibodies – Y shaped protein molecules that belong to a class known as immunoglobulins.
Antibodies bind to antigens and act as labels, allowing phagocytosis to recognise and destroy the cell

108
• Antigen – any substance foreign to the body recognised as non-self that causes an immune
response and is capable of binding with an antibody or T cell. They produce antibodies.
• Major Histocompatibility Complex (MHC) Molecules are proteins which are specialized for displaying short
peptide fragments on the surface of cells. MHC molecules along with their bound peptides are detected
by T cell receptors and this interaction plays a major role in cell mediated immunity.

• Lysis – disintegration of cell membrane

• APC (Antigen Presenting Cell) – A cell displaying foreign antigens with MHC on their surfaces for
T cells to recognise them.

Immune Cells
Some of the immune cells are;
1. Neutrophils - Involved in phagocytosis and antigen presentation.

They are stimulated mainly during bacterial infection

2. Basophils - Involved in defense against parasite infection and during allergic reactions

3. Eosinophils - Involved in defense against parasite infection like worms

NB; Neutrophils, basophils and eosinophils have granules in their cytoplasm hence called
Granulocytes
4. Mast cells – Cause inflammation and Involved during allergic reactions

5. Macrophages - Involved in phagocytosis and antigen presentation

6. Dendritic cells - Involved in phagocytosis and antigen presentation

7. B cells / B lymphocytes – Lymphocytes involved in the humoral response. -

They produce specific antibodies against antigens, perform the role of APCs and develop into B
memory cells.
Each has a unique receptor protein on its surface. These receptors are Ig M, Ig G, Ig A, Ig D and
Ig E ( Ig stands for immunoglobulin – which is another word for antibody.)
They produce antibodies.
They originate in bone marrow.
8. T cells / T lymphocytes
T cells are involved in cell mediated immunity.
They have T cell receptors on their surface. T cells originate and mature in the thymus.
They are classified as;

i. T helper cells (Th) – They display CD4 receptors.

They carry out multiple functions in coordinating the other immune cells e.g when
activated, they stimulate B cells to divide and differentiate to produce antibodies and T
killer cells to divide and produce chemical substances
ii. T killer cells (Tk) / T cytotoxic cells (TC) – They display CD8 receptors.

109
 They kill virus infected cells and cancer cells.
iii. T suppressor cells (Ts) – They regulate the immune system and prevent the immune
cells from destroying their own body cells.

• T memory cells (Tm) – They are formed after a primary infection. They are long lived T cell that has
receptors for an antigen due to its encounter with a prior infection or vaccination

• Note;- Memory cells can be Th memory, Tk memory or B memory cells. They remain in the body for
months or years, enabling an individual to respond quickly to the same antigen. They are specific to
the antigen encountered during the primary immune response – involved in the secondary immune
response

❖ Immune cells communicate in a number of ways; either by cell to cell contact through
receptors or through secreted signaling molecules.
❖ Most cells have a protein called Major Histocompatibility Antigen protein (MHC protein) also
known as Human Leucocyte Antigen (HLA) that signal whether a cell is host or foreign
triggering an immune response or not.
❖ Different individuals have unique MHC proteins

TYPES OF IMMUNE RESPONSES

a) Non - specific immune response

b) Specific immune response

NON - SPECIFIC IMMUNE RESPONSE

• These are immune responses to any foreign antigens

• Also called innate immune responses.

Examples

a) Inflammation
✓ When a tissue becomes damaged, for instance by bacteria due to infection, mast cellsand
damaged basophils release chemicals such as histamines.
✓ This causes vasodilation of blood vessels which increases the flow of blood to the infected
area and increases permeability of blood vessels.
✓ As a result of thatantibodies, phagocytes and plasma leak out into the infected tissue and
destroy the pathogen. This cause inflammation.
b) Lysozyme action

110
 ✓ Lysozyme is an enzyme found in secretions such as tears and mucus which kills
bacterial cells by damaging their cell wall by breaking down their glycosidic bonds
leading to their death.
c) Interferon
✓ Interferons are a group of protein molecules produced by virus infected cells.
✓ They prevent viruses from spreading to uninfected cells by stopping protein synthesis
in viruses, so stopping their replication.
✓ They inhibit viral replication in the cells that produce them
They bind to receptors on the infected cells and this activates an enzyme called
Protein Kinase R (PKR).
Activated PKR inhibits translation process inhibiting protein synthesis thus preventing
production of new viruses.
Activated PKR is also induces apoptosis preventing further synthesis of viral particles
✓ Interferons also diffuse from the cells that produce them and bind to receptors on the
surface of uninfected (healthy) cells. This prevents infection of more cells by viruses
released from infected cells when they lyse.
✓ Interferons also activate macrophages.

d) Phagocytosis

✓ It’s the process in which a type of white blood cell known as a phagocyte engulfs pathogens.
✓ The pathogen is enclosed in a phagocytic vesicle. Lysosomes in the phagocyte release
lysozymes in to digest the pathogen, destroying it.

e) Fever

✓ Infections by pathogens trigger the hypothalamus to set a high body temperature

resulting in fever.
✓ This is because presence of pathogens results in release of substances called pyrogens
into circulation.
✓ Examples of pyrogens are endotoxins & cytokines like lymphokines
✓ The pyrogens are detected by the hypothalamus and alter the body temperature set point
raising the core body temperature.
✓ The patient has a high fever.
✓ This raised temperature has the following advantages in the body
(i) Enhances immune function as the specific immune response works better at higher body
temperature
(ii) Enhances phagocytosis
(iii) Reduces the reproduction of bacteria and viruses in the body allowing the immune
system to destroy them.
NB: A pyrogen is a substance that induces fever.

111
SPECIFIC RESPONSE

✓ Also known as adaptive immune response.

✓ Involves mechanisms that target specific antigens.
✓ It’s mediated by B and T lymphocytes.

Two types of specific immune responses are;

(i) Humoral specific immune response

(ii) Cell mediated specific immune response

HUMORAL SPECIFIC IMMUNE RESPONSE

Humoral response involves two main stages;
a) T-helper activation stage whereby T cells are activated by APCs to form T helper memory cells
and T helper active cells
b) B-cells activation stage (Effector stage) whereby B cells are activated by cytokines from T cells
to from Plasma cells and B-memory cells

• Its mediated by antibodies

• When pathogens enter the body, immune cells detect them as foreign. Macrophages and
other phagocytes like dendritic cells take in a pathogen by engulfing them.
• B cells also take in a pathogen by endocytosis.
• The pathogen is enclosed in a vesicle, fuses with lysosomes and the enzymes from
lysosomes break down the pathogen into short peptide fragments referred to as antigens.
This is called antigen processing.
• The antigen combines with Major Histocompatibility Complex (MHC) proteins to form Antigen-
MHC protein complex.
• This complex moves to the outer surface of the cell surface membrane through exocytosis
and it’s presented on the surface and the cell (macrophage) becomes an Antigen Presenting
Cell (APC).
• The Antigen-MHC protein complex is the recognised by a T cell receptor and the T helper cell
bind to the Antigen-MHC protein complex by their CD4 receptors
• This activates the T helper cell to divide mitotically forming a clone of cells
• Some of these cells become T helper active and others become T helper memory.
• The T helper active cells release cytokines which stimulate B cells to divide mitotically forming
a clone of cells.
• Some of these cells become B active and others become B memory.
• The B active cells differentiate into B effector cells which in turn differentiate into B plasma
cells that produce antibodies with a shape complementary to the pathogen’s antigens.
• The antibodies move into circulation and defend the body in different ways.
• B Memory cells are long lasting and are involved in the secondary immune response

112
CELL MEDIATED SPECIFIC IMMUNE RESPONSE
This is the mediated by T cells and their products.
• When a pathogen infects a body cell, the pathogen is enclosed in a vesicle, fuses with
lysosomes and the enzymes from lysosomes break down the pathogen into short peptide
fragments referred to as antigens. This is called antigen processing.
• The antigen combines with Major Histocompatibility Complex (MHC) proteins to form Antigen-
MHC protein complex.
• This complex moves to the outer surface of the cell surface membrane through exocytosis
and it’s presented on the surface and the infected body cell becomes an Antigen Presenting
Cell (APC).
• The Antigen-MHC protein complex is the recognised by a T cell receptor and the T helper cell
bind to the Antigen-MHC protein complex by their CD4 receptors
• This activates the T helper cell to divide mitotically forming a clone of cells
• Some of these cells become T helper active and others become T helper memory.
• The T helper active cells release cytokines which stimulate T killers cells to divide mitotically
forming a clone of cells.
• Some of these cells become T killer active and others become T killer memory.
• The T killer active cells bind to the Antigen-MHC protein complex bound to the infected body
cells. The T killer active cells releases chemicals substances that create/ cause pores in the
infected body cell, the cell becomes permeable, so water and ions enter the cell, it swells,
bursts and dies.

Specific immune response glossary:

- Memory cells are cells which replicate themselves when exposed to an invading pathogen
and remain in the lymph nodes searching for the same antigen, thus resulting in a much
faster immune response. They allow long term immunity.
- Plasma cells are antibody producing cells. When the correct antibody is produced to fit the
antigen on the pathogen, the antibody divides by mitosis in order to multiply so that the
infection can be prevented. This is called clonal selection as the antibody clones itself.
- T helper cells stimulate B cells and T killer cells to divide, therefore activating the humoral
response and cell mediated response.
- T killer cells destroy pathogeninfected cells by creating holes its cell surface membrane,
causing it to burst.
- Memory B and T cells - remembers antigens for future infection (secondary infection). This
initiates a fast response if the same pathogen infects the body again in a secondary infection.

113
 B cells T cells

Produced and mature in bone marrow Produced in bone marrow but mature in
thymus gland

Involved in humoral response (involving Involved in cell mediated response

antibodies) (involving body cells)

Produces antibodies Does not produce antibodies

Responds to foreign cells outside body Responds to foreign material inside body
cells cells

Responds to bacteria and viruses Responds to own cells affected by virus,

cancer or response to a transplanted
tissue

A primary infection triggers a primary immune response.

• The primary immune response is slow because there are few cells which can make the
antibody needed to fight the pathogen. There is time needed for phagocytes like
macrophages and dendritic cells to process and present antigens, B cells to divide, and
differentiate into effector and to plasma cells to produce antibodies.
On the other hand, a secondary infection triggers a secondary response

• Secondary immune response involves memory cells.

• If infected by the same pathogen again, the immune system responds faster.
• There is a greater production of antibodies and it lasts longer.
• B memory cells differentiate immediately to produce plasma cells to release antibodies.
• T memory cells remember the specific antigen and will recognise it second time round.
• Faster because B memory cells already have complementary antigen receptors, memory
cells can divide rapidly into plasma cells and so there is quicker clonal expansion.
• The invading pathogens are often destroyed so rapidly that the person is unaware of any
symptoms – they are said to be immune

ANTIBODIES

❖ An antibody is a glycoprotein produced by plasma cells. Each plasma cell produces only one
type of antibody which binds to one antigen only.
❖ Antibody (Ab) also known as Immunoglobulin (Ig) is a large Y shaped protein produced by the
body’s immune system when it detects harmful substances, called antigens.
It can be described as having two main parts:

114
a) Fab region/ Fragment antigen-binding region:
The upper part which is V-shaped. It consists of a variable region that consists of antigen binding site
b) Fc region / Fragment crystallizable region: The tail of the Y-shape. It consists only of heavy chains.

The antibody can also be described as having:

a) Variable region- part of the Fab and consists of antigen binding site.
b) Constant region- part of the Fab and the tail that consists of heavy chain only.

• There are four polypeptide chains: two identical heavy chains and two identical light chains
connected by disulfide bonds.
• There are five types of Ig heavy chain (in mammal)
• The region that changes to various structures depending on differences in antigens is called
the variable region, and the region that has a constant structure is called the constant
region.

115
 ❖ They are produced by differentiated B cells called plasma cells.

Role of Disulphide bond in an antibody

• Holds polypeptides together; two long or heavy chains and two short or light chains
• Maintains 3D structure
Hinge region consist of disulphide bonds that join the two heavy chains. It gives flexibility in binding to
antigen.

An antibody has two binding sites

116
The role of antibodies in specific response to infection
1) Antibodies bind to pathogens causing opsonisation by labelling and marking the pathogen for
easy identification by phagocytes. This as a result enhancing phagocytosis where phagocytes
identify, engulf, digest and destroy them.
2) Causes agglutination (clumping together) of pathogens. This cause immobilization of
pathogens to prevent their spreading
3) Neutralizing toxins produced by pathogens. Antibodies can bind to the toxins produced by
pathogens. This prevents the toxins from affecting human cells, so the toxins are neutralised
(inactivated). The toxin-antibody complexes are also phagocytosed.
4) Preventing the pathogen binding to human cells
When antibodies bind to the antigens on pathogens, they may block the cell surface receptors
that the pathogens need to bind to the host cells. This means the pathogen can’t attach to or
infect the host cells.

IMMUNITY
The ability of an organism to resist a particular infection or toxin by the action of specific antibodies or
sensitized memory blood cells.

TYPES OF IMMUNITY
There are 2 types of immunity

(i) Active immunity

(ii) Passive immunity

Each can be obtained naturally or artificially.

Active Immunity

• Active immunity is where the body actively produces antibodies and memory cells.
• It takes a while for immunity to develop, requires exposure to antigens and the immunity
is long-term since memory cells will recognise antigens upon future infection.
Two types of active Immunity are;

a) Natural active immunity

This is where the person has come across antigens naturally, by being infected by the pathogen
i.e infection occurs, memory cells and antibodies are actively made in the body.

b) Artificial active immunity

An individual is exposed to weakened pathogens or toxins from the pathogen. The body of
individual responds by actively synthesising antibodies and memory cells.
It’s acquired after a vaccination

117
Passive Immunity
• Passive immunity occurs when the body doesn’t make antibodies against a pathogen but
has immunity by receiving immunity from another source.
• It doesn’t require exposure to antigens.
• it is short-lived as no memory cells are made.
• It gives immediate protection.

Two types of passive immunity are;

a) Natural passive immunity - antibodies being passed to a foetus / baby through the placenta /
breast milk. It’s short lived because there are no memory cells.
Infants begin to produce low levels of their own antibodies between 3 and 6 months before birth.
IgM antibodies production occurs early in life.
Levels of an infant's own IgG start to rise after birth, however don't reach a reasonable level until after
the child is arond 1 year old.

b) Artificial passive immunity - being injected with antibodies. It’s short lived because there are no
memory cells.

ANTIBIOTICS
Antibiotics are chemicals used to treat bacterial infection by killing the bacteria and stopping their
growth.
There are two types of antibiotics:
a) Bactericidal antibiotics kill bacteria by destroying their cell wall thus causing them to burst.
b) Bacteriostatic antibiotics which inhibit the growth of bacteria by stopping protein synthesis and
production of nucleic acids so the bacteria can’t grow and divide.

118
EVOLUTIONARY RACE BETWEEN PATHOGENS AND THE HOST
Evolutionary race means that as quickly as host evolve mechanisms to combat pathogens, pathogens
quickly evolve methods to overcome the host.
Evolutionary race between pathogen and host cell occurs whereby as organisms have evolved over
time to defend against infection from pathogens, the pathogens too have evolved to evade the
immune system.

For instance;

❖ The host have immune cells that function in diverse ways to prevent infection by pathogens.
However pathogens like HIV and Mycobacterium tuberculosis infects these immune cells
slowing down immune response.
❖ Mycobacterium tuberculosis in the tubercles develop thick waxy coat so that in the
macrophages the enzymes from lysosomes cannot destroy them. They remain dormant till
the immune system weakens.
❖ Pathogens like HIV incorporate their DNA into the host DNA therefore, not easily destroyed
by the immune cells.
❖ Most pathogens undergo high rate of mutation leading to development of new strains that are
not easily targeted by the immune cells and memory cells as well as circulating antibodies in
case of reinfection.
Examples of effects of mutation
- The virus HIV has a high mutation rate which cause its pathogens to change, meaning every
infection requires a new primary immune response even if the person has had it before, since
the antigens have changed so are not recognised by memory cells.
- Mycobacterium tuberculosis in the tubercles may also mutations while in the tubercles leading
to development of new strains that are not easily targeted the memory cells and circulating
antibodies produced during the primary infection
- Furthermore, mutations may arise in bacteria that make them resistant to antibiotics;
antibiotics provide a selection pressure, so bacteria that are resistant to them have a selective
advantage and are more likely to survive, reproduce and pass on the genes for resistance to
future generations. Over time the advantageous allele for immunity would increase in
frequency creating a resistant strain, this would happen relatively quickly in bacteria as they
reproduce up to once every 20 minutes, so advantageous alleles get passed on rapidly.
❖ Humans produce antibiotics but the pathogens develop resistance to the antibiotics e.g
MRSA was effectively destroyed by methicillin but it later became resistant to it. Humans
came up with vancomycin that effectively killed MRSA but later became resistant to it.
Humans also developed Linezolid and also became resistant to it.

HOSPITAL ACQUIRED INFECTIONS (HAI)

• These are infections spread and contracted within a hospital environment.

119
 • They can spread by means such as;
- Infected hospital bed linen
- Patient – staff contact
- Infected hospital toilets
- Air droplets
• Some HAIs are caused by bacteria that are resistant to all or most antibiotics. Such bacteria
are called hospital superbugs.
• They are common where antibiotics are commonly used e.g surgery rooms.
• Resistance to antibiotics results in antibiotic resistant to bacterial infections in hospitals such
as MRSA.

Examples of HAIs
1. Methicillin Resistant Staphylococcus aureus
• MRSA is a mutant type of the bacteria staphylococcus aureus which is resistant to the
antibiotic methicillin
Methicillin antibiotic was developed to kill Staphylococcus aurues. However, S. aureus
developed a gene mutation that enabled them to secrete an enzyme that breaks down
methicillin giving rise to MRSA. S.aureus was effectively destroyed by methicillin but it later
became resistant to it. Humans came up with vancomycin that effectively killed MRSA but
later became resistant to it. Humans also developed Linezolid and MRSA also became
resistant to it.
• S. aureus and MRSA are carried in the skin and nasal passages of healthy individual without
causing infections
• However, in hospital environment S. aureus and MRSA can cause infections in patients with a
weakened immune system
• Infection with S. aureus can be treated with methicillin and other antibiotics but infections with
MRSA cannot be treated with methicillin
• Therefore MRSA is commonly referred to as hospital ‘’superbug’’
Evolutionary race between bacteria and drug developers continues.
There are over 100 different types of antibiotic and in the 40years since their development 4 species
of bacterium have developed resistance against all of them.
E.g. Methicillin Resistant Staphyloccus aureus (MRSA) has been named the Superbug, because we
have no drugs left that can effectively kill it.

WHY MRSA IS A HOSPITAL SUPERBUG

• It’s resistant to many antibiotics like penicillin and methicillin
• It has a long life span
• It spreads very easily hence infects many patients
• It infects many body parts like skin, blood, urinary tract, genital e t c

120
2. Clostridum difficile
• This is anaerobic bacteria found in the gut of most people
• It doesn’t cause infections in healthy individuals as the gut flora out completes them
• However, use of blood spectrum antibiotics that destroy harmful and beneficial bacteria in the
gut may lead to rapid spread of C. difficile
• Recently, a mutant type of C. difficile has been identified which produce highly toxic
substance that lead to death in patients with a wakened immune system
• The mutant strain is highly spread causing infections in hospital environment in patients with a
weakened immune system

3. Escherichia coli [E. coli]

• Does not cause infections in individuals with a healthy immune system
• It lives within the gut

PREVENTION AND CONTROL OF HAIs

1. Controlling the use of antibiotics
• Antibiotics should be administered only when needed and the requested dosage
• The hospital staff should ensure that the course of treatment is completed e.g. by regular
check up visit to the hospital by the patient
• This reduces exposure of bacteria to many antibiotics reducing the chances of developing
resistant strains
• The patient should be advised on the importance of taking the full prescription
• Patients should also avoid self- prescription to ensure the right antibiotic is used for the right
treatment
• Antibiotics should only used when needed and their course is completed to ensure that all
the bacteria are destroyed and to minimise the selection pressure on bacteria to prevent
resistant strains from forming

2. Practising hospital code of practise

• Hospital code of practise is a collection of measures in hospitals to prevent the spread of
hospital acquired infections
• They include ;
- Hand washing by patients to prevent spread of infected between patients
- Isolation of patients with resistant infections and highly contagious diseases
- Screening of people coming to the hospital to identify infected individuals who have no
symptoms
- Controlling the use of antibiotics by administering them for the correct treatment and
not for prevention.
- Monitoring the level of HAIs to reduce spread among other patients

121
 - Use of gloves by hospital staff to prevent spread of infections between patients
- Use of alcohol based antibacterial gels which minimises the transmission of resistant
bacteria
- Ensuring proper clothing for hospital workers by avoiding jewellery, ties, and long
sleeved shirts to prevent picking pathogens from patients or contaminated surfaces
and transferring them to other patients
3. New patients should be screened at arrival, isolated and treated if they are infected to prevent the
spread of bacteria between patients

MICROORGANISMS, DECOMPOSITION AND RECYCLING OF CARBON

❖ Microorganisms play an important role in breaking down organic matter and returning
inorganic ions and carbon to the environment for use by other organisms.
❖ The inorganic ions released by the decomposition process are returned to the soil, where
they are assimilated into plants and the carbon taken in by decomposers is released to the
atmosphere as they respire. The CO2 released is then taken in by plants through
photosynthesis where the carbon is converted into biomass in the plant; this then moves
through the food chain as the plants are eaten by animals, which are in turn either eaten
themselves or die, where the decomposition cycle begins again.

FORENSIC INVESTIGATIONS

- It’s the application of science during criminal investigation, Forensic scientists collect,
preserve, and analyze scientific evidence during the course of an investigation.

DNA PROFILING (GENETIC FINGER PRINTING)

• It involves producing DNA patterns that can be used for individual or species identification
• Introns are used during DNA profiling
• Within the introns, there are short repeated nucleotide sequences called SHORT TANDEEM
REPEATS (STRs).This DNA is called satellite DNA
• If a two to four [2-4] nucleotide sequence is repeated between 5-15 times it is called a
microsatellite and if a 5-50 nucleotide sequence is repeated between 1-50 several 100 times
it is called a mini satellite
• Individuals vary in regard to the number of these repeats at each locus
• They also vary in length of the repeats
• This DNA is highly prone to mutation and therefore highly diverse
• No individuals have the same patterns of the repeats unless identical twins but more closely
related individuals are more similar in their patterns
• The restriction enzyme recognise a DNA sequence called recognition site
• They wrap around the DNA at that site and cause both strands to break at that point
• Different restrictions enzymes recognise different recognition sites (base sequences).

122
HOW TO PRODUCE A DNA PROFILE
A) Extraction of DNA

• The biological material which can be hair strands, blood, tissue etc is broken down in a
buffered solution that contains salts and detergents to disrupt the cell membranes
• Proteases are also added to digest the proteins
• Cold ethanol is added to precipitate the DNA
• The suspended particles which include DNA and proteins are separated by filtering or
centrifuging.

B) Purification of DNA

• Several stages of washing the DNA are carried out in buffered solutions to purify the DNA
• If the DNA sample is little it can be amplified (made into many copies) by polymerase chain
reaction [PCR]

C) Polymerase Chain Reaction [PCR]

This is carried out so there is enough DNA to work with. A common in vitro technique of amplifying
DNA is through the Polymerase Chain Reaction (PCR).
Requirements for PCR

• Automated thermocycler – programmable heating unit to maintain cyclic sequences of

temperatures for the PCR process
• PCR viral
• DNA sample
• Buffered solution
• Taq polymerase – a heat resistant DNA polymerase from a bacteria called Thermus
aquaticus
• Primers – short sequences of single stranded DNA complimentary to the DNA adjacent to the
STR. They are needed to initiate DNA synthesis in PCR
• DNTPs (Deoxyribonucleotide phosphates)

Procedure

1. The reactants (DNA sample, Taq polymerase, primers and DNTPs) are mixed together in a PCR
vial. They are placed in a thermocycler (PCR machine).
DNA primers are short, single-stranded lengths of DNA that are complementary to those at the
start of the STRs.

123
2. The mixture is heated to between 900 C and 950 C for 30 seconds. This is to break the
hydrogen bonds to separate the double helix into two strands
3. The mixture is then cooled to between 55-60 degrees for 20 seconds for the primers to
anneal (bind) to the start of the STRs in the separated DNA strands by formation of hydrogen
bonds between the base pairs.

4. The mixture is then heated to 700 C - 750 C for 1 minute for Taq polymerase to catalyse the
formation of complimentary strands as this is the optimum temperature for Taq polymerase to
work at.
5. DNA polymerase creates a copy of the sample by complementary base pairing using the
DNTPs added. DNA polymerases attach to the primers and extend them, replicating the STR
sequence and the adjacent DNA

124
 6. Step 2 to step 4 are repeated at least 30 times to produce enough DNA however, it can run
up to 3 hours to produce a mixture of DNA fragments unique to the individual

The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n,
where n is the number of cycles. Thus, a reaction set for 30 cycles results in 2 30, or 1,073,741,824,
copies of the original double-stranded DNA target region.

D) DNA digesting

• The sample DNA is cut into fragments using restrictions endonucleases

• This leaves fragments of DNA which are then separated and identified using gel
electrophoresis

125
E) Gel electrophoresis
After using PCR, gel electrophoresis can be used to separate them according to their size and an
image of the fragments produced
Gel electrophoresis is a process used to separate the DNA fragments according to their size using an
electric current.
The amplified DNA is placed in a slab of gel to which buffer solution is added and an electric current is
passed through.

• Agar gel medium is prepared in agar plate

• To the agar, a buffer solution is added to maintain pH constant
• A dye, Ethidium Bromide [EtBr] is also added that the binds to the DNA fragments and
fluoresce when placed under UV light making DNA bands visible
• Another dye is added that doesn’t bind to the DNA but moves through the gel faster than the
DNA fragments. This is to show the position of the DNA bands in the gel so that the current
can be turned off before the DNA runs out of the gel.
• DNA fragments are placed in wells in the gel alongside the unknown DNA in a controlled way
for identification
• Electric current is passed through the apparatus and the DNA moves towards the anode
(positive electrode) because DNA is negatively charged
• The fragments move at different rates depending on their mass and charge where shorter
fragments move faster and further so, mixture is separated out into a pattern of bands.
• The agar is placed under UV lights the DNA fluoresce and it’s identified against the unknown
DNA

• Comparison of the DNA profiles produced is done by comparing;

- Total number of DNA bands
- The position of DNA bands
- The size of DNA bands

❖ The position of DNA across the agar gel is unique for each person, giving a genetic
fingerprint. Genetic fingerprints are unique because people have different numbers of variable
number tandem repeats (VNTRs, these are repeats of certain sequences of bases – 1 person
may have 10 repeats of CAG over and over, another person may have 50 repeats) at various
positions across their genome, making parts of their DNA different lengths.
❖ The chances of 2 individuals having the same VNTRs at all locations is so slim that the
genetic fingerprint produced from electrophoresis and separation of the fragments can be
used to identify people, such as linking DNA found at a crime scene to a person.
❖ Comparing genetic fingerprints can also be used to determine genetic relationships, such as
with paternity tests.

126
 ❖ An individual will share roughly half of the same VNTRs as their parents or children, with less
and less VNTRs in common with more distant relationships, for instance cousins. Breeding
programmes can also compare genetic fingerprints to ensure they are not breeding closely-
related family members, as this can increase the risk of genetic disorders and reduce genetic
variation.

Uses of DNA profiling

1. Criminal investigations: such as murder cases.
2. Relationship disputes such as paternity disputes
3. Relationship identification - DNA fragments of closely related species or individuals are more or
less of the same size and occupy the same position on the gel.

ESTIMATING TIME OF DEATH IN A MAMMAL

Indicators of time of death in mammals include;

1; Body temperature
2; Rigor mortis (Degree of muscle contraction)
3; Extent of decomposition
4; Forensic entomology
5; Stage of succession

1. Body temperature
• Normal body temperature in mammals is 370 C
• The body begins to cool from 370 C after death as metabolic reactions slow down and finally
stops
• Heat loss from the body to the surrounding is at a slow rate during the first few hours after
death and then rapidly decreases to surrounding (ambient) temperature
• By the end of 24 hours the body temperature falls to sorroundin temperature therefore this is
useful for the first 24 hours
• Body temperature decreases over the first 24 hours in the following shaped curve.

127
 • The environment the body was found in must also be considered, since if found in water or
where there is air movement it speeds cooling while clothing and being indoors slows cooling.

Factors that affect how fast the body cools

a) Mode of death
* Whether the victim struggled or not before the death
b) Temperature of the surrounding
* This affects how quickly the heat is lost from the body
c) Body size
*This is because of surface area to volume ration
d) Clothing
* The less the clothes covering the body the more the heat loss and vice versa
e) Amount of stored fat
* Fat acts as an insulator

2. Degree of muscle contraction

• Rigor mortis is the process where the body muscles contract after death. The myosin and
actin cross bridges in muscle cells can’t break as this requires ATP, which is no longer made
after death. Small muscles contract first, followed by larger ones.
• 36 hours after death the muscles begin to unstiffen as muscle fibres are broken down, the
smaller muscles are the last to unstiffen. Upon death muscles are in a relaxed state, but then
stiffens
• This is because after death aerobic respiration stops hence no ATP is produced
• The stiffening effect is called rigor mortis
• Rigor mortis starts 2-4 hours after death taking full effect between 6-9 hours and wears off
after 36-48 hours after muscle fibres begin to break down due to anaerobic decomposition
• Under normal conditions ATP is needed to maintain muscle fibres in a relaxed state
• Therefore once the stored ATP is utilised and no more ATP is being produced the muscle
fibres remain contracted beginning from the face and neck region and finally to the large
muscles of the body e.g. leg muscles
• Rigor mortis is useful for the first 36-48 hours after death.

3. Extent of decomposition
• When death occurs, digestive enzymes in the gut start to breakdown the cell of the gut wall
• Also due to lack of oxygen anaerobic bacteria in the gut break down more cells starting from
the gut region spreading to the surrounding tissues releasing lactic acid
• Due to the stopping of metabolic processes , cells begin to die
• The dying cells release hydrolytic enzymes from their lysosomes and tis contributes to break
down of more cells

128
 • Body decomposition begins from the gut progressing outwards and most bodies decompose
in a similar manner in stages.
• Therefore extent of decomposition can be used to estimate time of death
• The appearance of the body can give an estimate to time of death as shown below.

4. Forensic entomology
• It’s the study of insect’s life cycle in relation to crime and how they vary at different
temperatures
• The most studied and used insect life cycle is that of blow flies
• Within minutes after death of a human , blow flies arrive on the body
• They are highly sensitive to the smell from body openings like nose and ears
• They lay eggs on body openings and forensic investigators can analyse their developmental
stages e.g. egg, larvae, pupae and adults to estimate the time of death
• Maggots
• Larvae can also be used.
• By observing their length and the temperature they’re found at, looking at what stage of their
life cycle they are and finally by taking some larvae and continuing to keep them in the lab
until they pupate, from there you can work backwards to estimate when they hatched.

5. Stages of succession
• There are 5 main stages of decomposition that occur after death, each attracting different
organisms that come and feed on the organism. The different stages can occur over days,

129
 weeks and years; meaning analysis of succession can aid estimating the time of death of
even long-dead organisms.
• Therefore as a body decomposes , there is a succession of species on the body .
e.g
• Anaerobic bacteria; [colonises] start decomposing the body from the gut

• Blow flies; Insects that are highly sensitive to smell of dead organisms

• Beetles; Lay eggs and their larvae and feed on the larvae of blow flies

• Parasitic wasps; Lay their eggs and they feed on the larvae of the beetles and on
harder body parts of the dead body

• Mites and moths; Feed on connective tissues [tendons , ligaments ]

130