Chapter 6 A Level

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YOUR NOTES
International A Level Biology Edexcel 
6. Microbiology, Immunity & Forensics
CONTENTS
Microbiology
6.1 Culturing Microorganisms
6.2 Measuring the Growth of Microorganisms
6.3 The Bacterial Growth Curve
6.4 Core Practical 13: Rate of Growth of Microorganisms
6.5 Comparison of Bacterial & Viral Structure
Immunity
6.6 Tuberculosis & HIV
6.7 Pathogens: Routes of Entry
6.8 Non-Specific Immune Responses
6.9 Specific Immune Responses
6.10 Lymphocytes: Types & Roles
6.11 Developing Immunity
6.12 Pathogens vs Hosts: An Evolutionary Race
Antibiotics
6.13 Antibiotics
6.14 Core Practical 14: The Effects of Different Antibiotics
6.15 Current Usage of Antibiotics
Decomposition & Decay
6.16 The Role of Microorganisms
Forensics
6.17 Polymerase Chain Reaction
6.18 Gel Electrophoresis in Forensics
6.19 DNA Profiling
6.20 Types of Data Provided by Forensic Analysis
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Microbiology YOUR NOTES

6.1 Culturing Microorganisms
Culturing Microorganisms
Most microorganisms are only visible using a microscope
For microbe investigations it is therefore necessary to culture microorganisms
This grows enough microorganisms to make measurements during investigations
E.g. bacteria reproduce by cloning themselves, so when they are grown on agar gel
they form a colony of identical individuals that is visible to the naked eye
Microorganisms must be provided with everything they need to grow such as
Nutrients
Oxygen
Note that anaerobic microorganisms would require the absence of oxygen
Optimum pH
Favourable temperature
Microorganisms should be cultured with great care
There is always the risk that a mutation could lead to the formation of
pathogenic strains
Pathogenic bacteria from the environment could contaminate the bacterial culture
being investigated
Remember to
Follow health and safety precautions
Ensure that all equipment are sterilised before culturing the bacteria
Sterilising involves killing microorganisms, e.g. by heating to a high temperature or
the use of antimicrobial chemicals
Keep the culture in the laboratory
Seal cultures in a plastic bag and sterilise at high temperature and pressure before
disposal
Culturing steps
Obtain a supply of the type of microorganism to be cultured
Provide them with the correct type of nutrients to facilitate growth
A nutrient growth medium (plural media) containing carbon, nitrogen, and minerals is
typically used
The medium could be in the form of a liquid culture or a solid nutrient agar, a type of
gel extracted from seaweed
Ensure that the nutrient medium is kept under sterile conditions until use
By adjusting the type of nutrients in the medium, conditions will be created for the optimal
growth of a certain type of microorganism; this is known as a selective medium
Selective media can be used to identify mutant strains of microorganisms or those
that are resistant to antibiotics
They are also useful for identifying genetically modified microorganisms
Microorganisms are introduced to a growth medium using inoculation with a sterilised
inoculation loop
Inoculation can be used to transfer microorganisms between media, e.g.
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From agar gel into a liquid culture flask YOUR NOTES
From a liquid culture flask onto agar gel 

The new medium should be sealed or covered to avoid contamination from
microorganisms in the air; if growing aerobic microorganisms any seal or cover should not
be airtight
Flasks can be sealed with a sterile cotton wool stopper
Petri dishes can be covered with a lid
Label the medium clearly and incubate at around 20 °C to prevent the growth of
pathogenic microorganisms
Microorganisms that are pathogenic to humans will grow best at around 37 °C
In a hospital or research laboratory a higher temperature might be used to obtain faster
results
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YOUR NOTES
A metal inoculating loop can be used to transfer microorganisms from a liquid broth medium
onto an agar gel medium. A Bunsen burned enables sterilisation of the loop between uses.
Growing a single type of microorganism
In order to grow a single type of microorganism, or a pure culture, the specific
microorganism must be isolated
This can be done by using knowledge about the needs of the microorganism to be cultured
or those of microorganisms that may contaminate the culture
Examples include
Growing the culture under either aerobic or anaerobic conditions to reduce the
variety of microorganisms in the culture
Using a selective medium that is tailored to the specific requirements of the desired
microorganism
Indicator media can provide a colour change to distinguish desired colonies from the
rest
Being able to isolate pathogenic microorganisms is useful in the diagnosis and treatment
of diseases
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6.2 Measuring the Growth of Microorganisms YOUR NOTES

Measuring the Growth of Microorganisms
Bacteria and most other microorganisms are too small to count with the naked eye
There are a variety of methods that can be used to count microorganisms and it is
important to be able to make an appropriate choice when conducting investigations
These methods include
Cell counts
Dilution plating
Measuring area and mass
Optical methods
Cell counts
A microscope and haemocytometer can be used to count single-celled microorganisms
A haemocytometer is a microscope slide with a rectangular chamber that is marked
with grid lines
The chamber can hold a standard volume of 0.1 mm3
Haemocytometers were originally used to count blood cells
Haemo = blood
Counting cells using a haemocytometer involves the following
A nutrient broth is diluted with an equal volume of trypan blue
This is a dye that will stain dead cells blue
It enables the investigator to only count the living cells
The chamber of the haemocytometer is filled with the stained nutrient broth
The number of living cells in the four corner squares of the grid are counted
Each corner square consists of 16 smaller squares
Consistency needs to be used when deciding whether to count cells that are on
the lines that border the corner squares
E.g. Counting cells on the top and right-hand borders and ignoring cells on
the bottom and left-hand borders
The mean number of cells from the four corner squares can be calculated
The haemocytometer is calibrated to allow the calculation of the number of cells in a
known volume of broth
Because the haemocytometer chamber can hold exactly 0.1 mm3 of liquid, it is
possible to estimate of the cell count in 1 ml of nutrient broth using the following
calculation
No. of cells per ml nutrient broth = mean cell count x dilution factor x 104
Multiplying by the dilution factor enables calculation of the bacterial cell count in the
original broth rather than the diluted broth
The multiplication by 104 enables calculation of the bacterial cell count in 1 ml rather
than in 0.1 mm3
1 ml = 1 cm3
1 cm3 = 0.1 mm3 x 10 000
10 000 = 1 x 104
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E.g. in a scenario where a nutrient broth is diluted by a factor of 100 and the four corner grid YOUR NOTES
squares contain 20, 14, 19, and 16 cells 

This gives a mean cell count of 17.25
No. cells per ml nutrient broth = 17.25 x 100 x 104 = 17 250 000
The living cells in the corner squares of a haemocytometer grid can be counted to determine
the number of microorganisms in a standard volume of nutrient broth
Dilution plating
This method can be used to determine the total viable cell count in a nutrient broth
The nutrient broth is transferred to agar where the bacteria use nutrients in the agar gel
to reproduce
A single cell that lands on agar reproduces by cloning itself, resulting in a mass of
identical cells known as a colony
Each microbial colony that grows on agar gel originated with one viable
microorganism, so can be counted as one viable cell
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Individual microbial colonies can be difficult to identify on an agar plate as they tend to form YOUR NOTES
one large mass 

This problem can be overcome by diluting the original cultures before transferring the
samples to agar; this reduces the number of cells in the original sample so that individual
colonies are visible on an agar plate
This is why the technique is known as dilution plating
To calculate the total viable cell count, the number of colonies are multiplied by the
dilution factor
A mean can be determined if more than one plate is used
Dilution plating can provide a way to determine the total viable cell count of a microbial
culture
Area and mass of fungi
Fungi do not always live as single-celled organisms, but can form a mass of elongated cells
known as a fungal mycelium (plural mycelia); this means that the methods described
above can be unsuitable for measuring the growth of some fungi
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Measuring the diameter of individual areas of the mycelium can be used to determine the YOUR NOTES
growth of fungi 
Petri dishes of agar are inoculated with fungal spores and incubated at a suitable
temperature
The resulting areas of fungal mycelia are then measured
This can be used to compare growth rates in different conditions, e.g. at different
temperatures
The larger the mean diameter, the greater the growth of the fungi
Testing the dry mass of fungi is another effective way to measure fungal growth
A liquid nutrient broth is inoculated with fungal spores
Samples of the nutrient broth are removed at set time intervals
The fungal mycelia are removed by filtering or centrifugation
The material is dried in an oven overnight and its mass measured
The higher the mass, the more fungal growth has occurred
Optical methods
Turbidimetry is a specialised form of colorimetry that can be used as an alternative
method to measure the number of cells in a sample
Turbidity is a measure of how cloudy a solution is
More turbid = more cloudy
Less turbid = less cloudy
Colorimetry uses a machine called a colorimeter to shine a beam of light at a sample
and measure the amount of light that is either transmitted through or absorbed by the
sample
The higher the number of cells the more turbid the solution becomes
More turbid solutions will absorb more light and allow less light through; this can be
measured by a colorimeter
This provides an indirect measure of the number of microorganisms present
A calibration curve can be constructed by measuring the turbidity of a series of control
cultures while also counting the cells in each culture using a haemocytometer; the results
are plotted in a graph of turbidity against cell count
This curve can then be used to estimate the cell count of unknown samples by measuring
their turbidity and then reading their cell count from the graph
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YOUR NOTES
A colorimeter can be used to determine the turbidity of a solution containing

microorganisms. The solution containing bacteria is placed into a container called a cuvette
and the amount of light that can pass through, or is absorbed by, the solution can be
measured.
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6.3 The Bacterial Growth Curve YOUR NOTES

The Bacterial Growth Curve
Bacteria divide using the process of binary fission during which one cell will divide into two
identical cells
The process is as follows
The single, circular DNA molecule undergoes DNA replication
Any plasmids present undergo DNA replication
The parent cell divides into two cells, with the cytoplasm roughly halved between the
two daughter cells
The two daughter cells each contain a single copy of the circular DNA molecule and a
variable number of plasmids
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YOUR NOTES
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YOUR NOTES

The process of binary fission produces two identical daughter cells
Bacterial growth curve
The growth of a bacterial population follows a specific pattern over time; this is known as a
growth curve
There are 4 phases in the population growth curve of a microorganism population
Lag phase
The population size increases slowly as the microorganism population adjusts to
its new environment and gradually starts to reproduce
Exponential phase
With high availability of nutrients and plenty of space, the population moves into
exponential growth; this means that the population doubles with each division
This phase is also known as the log phase
Stationary phase
The population reaches its maximum as it is limited by its environment, e.g. a lack
of resources and toxic waste products.
During this phase the number of microorganisms dying equals the number being
produced by binary fission and the growth curve levels off
Death phase
Due to lack of nutrients and a build up of toxic waste build up, death rate exceeds
rate of reproduction and the population starts to decline
This phase is also known as the decline phase
There are four phases in the standard growth curve of a microorganism

Using logarithms in growth curves
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During the exponential growth phase bacterial colonies can grow at rapid rates with very YOUR NOTES
large numbers of bacteria produced within hours 

Dealing with experimental data relating to large numbers of bacteria can be difficult when
using traditional linear scales
There can be a wide range of numbers reaching from single figures into millions
This makes it hard to work out a suitable scale for the axes of graphs
Logarithmic scales can be very useful when investigating bacteria or other
microorganisms
The numbers in a logarithmic scale represents logarithms, or powers, of a base number
If using a log10 scale, in which the base number is 10, the numbers on the y-axis
represent a power of 10, e.g. 1=101 (10), 2=102 (100), 3=103 (1000) etc.
Logarithmic scales allow for a wide range of values to be displayed on a single graph
For example, if yeast cells were grown in culture over several hours the number of cells
would increase very rapidly from the original number of cells present
The results from such an experiment are shown in the graph below using a log scale
The number of yeast cells present at each time interval was converted to a logarithm
before being plotted on the graph
This can be done using a log function on a calculator
The log scale is easily identifiable as there are not equal intervals between the
numbers on the y-axis
The wide range of cell numbers fit easily onto the same scale
When a log10 scale is used, the scale increases by a factor of 10 each time; this allows large
increases in numbers to be shown on a single graph
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Exam Tip YOUR NOTES
 You won’t be expected to convert values into logarithms or create a log scale graph

in the exam. Instead you might be asked to interpret results that use logarithmic
scales or explain the benefit of using one! Remember that graphs with a logarithmic
scale have uneven intervals between values on one or more axes.
Exponential growth rate constants

To calculate the number of bacteria in a population the following formula can be used
N t = N 0 x 2kt
Where
Nt = the number of organisms at time t
N0 = the number of organisms at time 0
k = the exponential growth rate constant
t = the time for which the colony has been growing
To use this equation the exponential growth rate constant k must be calculated
This refers to the number of times the population doubles in a given time period
The following formula can be used to calculate the exponential growth rate constant
log10 N t − log10 N 0
k =
log10 2 x t
 Worked Example
A bacterial colony started with 2 individuals and after 3 hours of growing there were
926 bacteria in the colony.
1. Calculate the exponential growth rate constant of this colony
2. Calculate the number of bacteria in the colony after 5 hours
Step 1: Calculate the exponential growth rate constant

log10 N t − log10 N 0
k =
log10 2 x t
log10 ( 926) − log10 (2)

k =
log10 2 x 3
2. 67
k =
0. 30 x 3
k = 3
Step 2: Calculate the number of bacteria after 5 hours
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N t = N 0 x 2kt YOUR NOTES

N t = 2 x 2 (3) (5)
N t = 2 x 215
N t = 65 536
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6.4 Core Practical 13: Rate of Growth of Microorganisms YOUR NOTES

Rate of Growth of Microorganisms
There are several ways to investigate the growth of microorganisms
In this example the change in turbidity of a yeast solution will be used as a measure of the
growth of microorganisms in a liquid culture over time
Turbidity = cloudiness of a solution
Apparatus
Cloths and antimicrobial solution
Conical flask
Glucose solution
Dried baker's yeast
Bunsen burner
Cotton wool
Magnetic stirrer
Aluminium foil
Colorimeter
Dropper pipette
Cuvettes
Optional
Microscope
Microscope slides with cover slips and graph paper photocopied on to acetate OR a
haemocytometer
Method
1. Use antimicrobial solution to sterilise the work area
2. Place 250 cm3 glucose solution into a conical flask
This solution will be the liquid culture medium
3. Inoculate the glucose solution with 1.25 g dried yeast using aseptic techniques
Work next to a Bunsen flame to ensure that microorganisms in the air are drawn away
from the nutrient broth
4. Seal the flask using a cotton wool stopper immediately after inoculation is complete to
prevent contamination
5. Swirl to mix the yeast with the glucose solution and place on a magnetic stirrer
6. While stirring continuously, loosely cover the stopper with aluminium foil and incubate the
flask at 20 °C
7. Fill a cuvette with plain glucose solution and use this as a blank to calibrate the colorimeter
This ensures that the colorimeter is reset to zero before each measurement is taken
8. Transfer about 3 cm3 of the yeast suspension into a cuvette using a dropping pipette
9. Use the colorimeter to measure the absorbance and record this in a suitable table against
time
10. Repeat steps 7-9 at intervals over a 12 hour period
E.g. this could involve taking samples every 30 minutes for the first two hours and then
every 2-3 hours after this
11. Plot a graph of absorbance against time
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The temperature or concentration of the glucose solution can be changed and the YOUR NOTES
experiment repeated to investigate the effect these changes would have on the growth of 
the yeast
Optional extension to practical
Turbidity measurements are useful as the turbidity of the medium is mainly a measure of
the number of living cells in suspension; however, the measurement can be affected by
the presence of dead cells and other suspended particles, so other methods of cell
counting can be used to back up the turbidity findings, e.g.
Use a haemocytometer to estimate the number of yeast cells in the glucose solution
Calculate the area of the microscope's field of view using graph paper copied on to
acetate, and use the number of cells in the field of view to estimate the number of
cells in the glucose solution
If using the area of the microscope's field of view the following steps can be used to
estimate the number of cells in the growth medium
Count the number of squares of acetate graph paper visible when using the x4
objective lens and use this to calculate the area of the field of view under a x40
objective lens; this will be 0.01 of the area of the x4 objective lens
Stain the yeast suspension with methylene blue and place one drop of the suspension
on a microscope slide
View under the x40 objective lens and count the number of yeast cells in the field of
view
Calculate the volume of this drop by measuring the volume of 10 drops and dividing by
10
To calculate the volume under the field of view when using the x40 objective lens, use
the following formula
volume = (area of field of view of x40 objective lens ÷ area of cover slip) x volume of one
drop
To calculate the number of yeast cells in 1 mm3 of medium the following formula can be
used
number of cells per mm3 = average cell count in field of view ÷ volume of field of view
Note that if many cells overlap when viewed under x40 objective lens a serial dilution of
the yeast suspension will be required
Safety
Eye protection should be worn
Care should be taken around Bunsen burners
Aseptic techniques should be used when transferring microorganisms
Cultures should be incubated at a safe temperature for school laboratory work; 20 °C and
not 37 °C
Work spaces and hands should be thoroughly disinfected at the end of the experiment
Cultures should be safely destroyed at the end of the experiment
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6.5 Comparison of Bacterial & Viral Structure YOUR NOTES

Comparison of Bacterial & Viral Structure
Bacteria
Bacteria are single-celled prokaryotes
Prokaryotic cells are much smaller than eukaryotic cells
They also differ from eukaryotic cells in having
A cytoplasm that lacks membrane-bound organelles
Ribosomes that are smaller (70 S) than those found in eukaryotic cells (80 S)
No nucleus, instead having a single circular bacterial chromosome that is free in the
cytoplasm and is not associated with proteins
A cell wall that contains the glycoprotein murein
Murein is sometimes known as peptidoglycan
In addition, many prokaryotic cells also have the following structures
Loops of DNA known as plasmids
Capsules
This is sometimes called the slime capsule
It helps to protect bacteria from drying out and from attack by cells of the
immune system of the host organism
Flagella (singular flagellum)
Long, tail-like structures that rotate, enabling the prokaryote to move
Some prokaryotes have more than one
Pili (singular pilus)
Thread-like structures on the surface of some bacteria that enable the bacteria
to attach to other cells or surfaces
Involved in gene transfer during sexual reproduction
A cell membrane that contains folds known as mesosomes; these infolded regions
can be the site of respiration
Some bacteria are disease-causing, or pathogenic, but not all bacteria cause harm to
other organisms
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YOUR NOTES
Prokaryotic cells have a peptidoglycan cell wall, no membrane-bound organelles, a circular

chromosome, and 70S ribosomes
Viruses
Viruses are non-cellular infectious particles
They are relatively simple in structure, and much smaller than prokaryotic cells
Structurally they have
A nucleic acid core
Their genomes are either DNA or RNA, and can be single or double-stranded
A protein coat called a ‘capsid’ made of repeating units known as capsomeres
They do not possess a plasma membrane, cytoplasm, or ribosomes
Some viruses have an outer layer called an envelope formed from the membrane-
phospholipids of the cell they were made in
The fact that lipid envelopes are formed from the membrane of a viral host cell means
that very few plant viruses have lipid envelopes
Some contain proteins inside the capsid which perform a variety of functions
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E.g. HIV contains the enzyme reverse transcriptase which converts its RNA into DNA YOUR NOTES
once it has infected a cell 

Viruses also contain attachment proteins, also known as virus attachment particles, that
stick out from the capsid or envelope
These enable the virus to attach itself to a host cell
Viruses can only reproduce by infecting living cells and using the protein-building
machinery of their host cells to produce new viral particles
Viruses are classified on the basis of the genetic material they contain and how they
replicate
They can be classified into the following categories
DNA viruses
RNA viruses
Retroviruses
HIV contains RNA as its genetic material. It is surrounded by a protein capsid, as well as
having an outer lipid envelope and attachment proteins
DNA viruses
They contain DNA as genetic material
Viral DNA acts as a direct template for producing new viral DNA and mRNA for the
synthesis of viral proteins
Examples: smallpox, adenoviruses, and bacteriophages
Bacteriophages are viruses that infect bacteria, such as the λ (lambda) phage
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YOUR NOTES
Bacteriophage viruses, such as the λ phage, are examples of DNA viruses

RNA viruses
They contain RNA as genetic material
Most have a single strand of RNA
They do not produce DNA at all
Mutations are more likely to occur in RNA viruses than DNA viruses
Examples: tobacco mosaic virus (TMV), ebola virus
Retroviruses
Special type of RNA virus that does produce DNA
They contain a single strand of RNA surrounded by a protein capsid and lipid envelope
Viral RNA controls the production of an enzyme called reverse transcriptase
This enzyme catalyses production of viral DNA from the single strand of RNA
The new viral DNA is incorporated into the host DNA using integrase enzymes where it acts
as a template to produce viral proteins and RNA
Example: HIV (Human Immunodeficiency Virus)
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Lytic & Latency YOUR NOTES
Viruses can only reproduce within a host cell as they lack the cellular machinery to do so on 
their own
They can enter a host cell in a variety of different ways
Bacteriophages inject their genetic material into bacteria
Some animal viruses enter the cell via endocytosis by fusing their viral envelope with
the host cell surface membrane
Plant viruses will often use a vector such as an insect to breach the cell wall
Once inside the host cell one of the following pathways can occur
Lysogenic
Lytic
Lysogenic pathway
Some viruses will not immediately cause disease once they infect a host cell
Viral DNA known as a provirus is inserted into the host DNA, but a viral gene coding for a
repressor protein prevents the viral DNA from being transcribed and translated
Every time the host DNA copies itself, the inserted viral DNA will also be copied
This is called latency and the time during which it occurs is known as a period of lysogeny
Viruses in a lysogenic state may become activated and enter the lytic pathway
Activation may occur as a result of, e.g. host cell damage or low nutrient levels inside a
cell
Lytic pathway
The viral genetic material is transcribed and translated to produce new viral components
These components are assembled into mature viruses that accumulates inside the host
cell
Eventually the host cell bursts which releases large numbers of viruses, each of which can
infect a new host cell
Cell bursting is known as cell lysis
This typically results in disease
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YOUR NOTES
The life cycle of the λ bacteriophage includes a lysogenic and a lytic pathway
Page 23 of 63
Immunity YOUR NOTES

6.6 Tuberculosis & HIV
Tuberculosis
A disease is an illness or disorder of the body or mind that leads to poor health
Each disease is associated with a set of signs and symptoms
Infectious diseases are caused by pathogens and are transmissible, meaning that they
can be spread between individuals within a population
Pathogens may include certain species of bacteria, some fungi and all viruses
Note that not all viruses are pathogenic to humans!
An example of a pathogen is the bacteria Mycobacterium tuberculosis which causes the
disease tuberculosis, also known as TB
Transmission of TB
When infected people with the active form of TB cough or sneeze, the Mycobacterium
tuberculosis bacteria enter the air in tiny droplets of liquid released from the lungs
TB is transmitted when uninfected people inhale these droplets
TB spreads more quickly among people living in overcrowded conditions
Once inside the lungs, TB bacteria are engulfed by phagocytes
The bacteria may be able to survive and reproduce while inside phagocytes
Individuals with a healthy immune system will not develop TB at this stage
This is known as the primary infection
Over time the infected phagocytes will become encased in structures called tubercles in
the lungs where the bacteria will remain dormant
It is possible for the bacteria to become activated and overpower the immune system at a
later stage, such as during an HIV infection when the immune system is compromised; the
person will then develop TB
This is known as the active phase of TB
The length of time between infection and developing the disease can vary from a few
weeks to a few years
The first symptoms of TB will include developing a fever, fatigue, coughing and lung
inflammation
If left untreated the bacteria will cause extensive damage to the lungs which can result in
death due to respiratory failure
TB may also spread to other parts of the body where it can lead to organ failure if not
treated promptly
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HIV YOUR NOTES
Transmission of HIV 
HIV is the human immunodeficiency virus
Be careful not to refer to it as the HIV virus, as that would mean that you would be using
the word 'virus' twice!
HIV contains RNA and is a retrovirus
HIV can be transmitted in body fluids in the following ways
Sexual intercourse
Blood donation
Sharing of needles used by intravenous drug users
From mother to child across the placenta
Mixing of blood between mother and child during birth
From mother to child through breast milk
Replication of HIV
When the virus enters the bloodstream it infects helper T cells, a type of white blood
cell that is normally responsible for activating antibody-producing B cells
It enters the helper T cells by attaching to a receptor molecule on the host cell
membrane
The capsid enters the helper T cell and releases the RNA it contains
The viral RNA is used as a template by reverse transcriptase enzymes to produce
a complementary strand of DNA
Once this single-stranded DNA molecule is turned into a double-stranded
molecule it can be successfully inserted into the host DNA
From here it uses the host cell's enzymes to produce more viral components which are
assembled to form new viruses
These bud from the host cell and enter the blood, where they can infect other helper T
cells and repeat the process
At this stage, the individual is HIV positive and may experience flu-like symptoms
This is known as the acute HIV syndrome stage
After the initial infection period, during which HIV replication is rapid, the replication rate
drops and the individual enters the asymptomatic or chronic stage
During this period the person will not show any symptoms, often for years
Gradually the virus reduces the number of helper T cells in the immune system
B cells are no longer activated
No antibodies are produced
The patient begins to suffer from HIV-related symptoms and are now in the
symptomatic disease stage of the infection
The lack of T helper cells decreases the body’s ability to fight off infections, eventually
leading to the final stage of an HIV infection, which is known as advanced AIDS (Acquired
immune deficiency syndrome)
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YOUR NOTES
HIV attaches to helper T cells (also called CD4 T-lymphocytes) and uses their cell machinery
to replicate. This leads to decreased lymphocyte numbers which then affects the body's
ability to respond to infection. Note that HIV should not be referred to as the 'HIV virus' as it
is here.
Symptoms of AIDS
Immediately after infection with HIV a patient often suffers mild flu-like symptoms
These symptoms pass and for a period of time infected people might not know they
are infected
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After several months or years, the viral DNA replicated by the HIV particles becomes active YOUR NOTES
Virus particles gradually destroy and reduce the number of helper T cells present in a host 
This is detrimental as helper T cells play an important role in the specific immune response
They stimulate B cells, the production of antibodies and increased rates of
phagocytosis
As a patient can no longer produce antibodies against pathogens, they are
immunocompromised and unable to fight off infections
They begin to suffer from diseases that would usually cause very minor issues in healthy
individuals
These diseases are described as opportunistic
Tuberculosis (TB) is a common example
An HIV infection will progress to AIDS when
An individual starts suffering from constant opportunistic infections
The helper T cell count drops below a critical level
The length of time that it takes for an HIV infection to progress to AIDS can vary between
individuals but the disease will follow a standard sequence of symptoms
Initially an AIDS sufferer will only have mild infections of the mucous membranes due
to the low helper T numbers
Over time, however, infections will become more severe e.g. diarrhoea, TB
During the final stages of AIDS a person will suffer from a range of more serious
opportunistic infections
It is these opportunistic diseases that cause an individual with advanced AIDS to die
Several factors affect how quickly HIV will progress into AIDS and how long a person with
AIDS will survive
The number of existing infections
The strain of HIV the person is infected with
Their age
Access to healthcare
 Exam Tip
Try not to confuse the terms HIV and AIDS. Many people use them interchangeably
when they actually mean different things.
HIV is a virus
AIDS is the disease caused by HIV
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6.7 Pathogens: Routes of Entry YOUR NOTES

Pathogens: Routes of Entry
In order for a pathogen to cause disease it must enter the body of the host
Body openings, e.g. the mouth, eyes, and urinary tract, provide easy access for
pathogens to enter
Pathogens may enter directly into the bloodstream through breaks in the skin
Pathogens may be transmitted in a variety of ways
Vectors
These are living organisms that carry pathogens and transmit them between hosts
Insects, such as flies and mosquitoes, are common vectors for diseases like malaria and
yellow fever
Inhalation
Droplets from the respiratory tract will be suspended in the air when an infected person
coughs, sneezes or talks
These droplets contain pathogens that can be inhaled by healthy people
The airways provide an entry point into the respiratory system of a new host and another
infection occurs, e.g. flu, measles, tuberculosis
Ingestion
Pathogens can enter through the digestive system when we ingest contaminated food or
drink
This is especially probable if food is undercooked, as heat destroys most of the pathogens
These pathogens can make their way through the lining of the gut and cause disease (e.g.
cholera, Salmonella poisoning)
Indirect contact
Inanimate objects can contain large numbers of pathogens that may be transferred
between hosts
An infected individual may touch or cough on an object which is later touched by a
healthy individual who transfers the pathogens to their mouth or nose by touching their
face
Examples include bedding, towels, and surfaces
Direct contact
Pathogens that spread this way will require some part of the host, e.g. skin, body fluids, to
come into direct contact with a healthy individual
Pathogens that spread by this route can then pass through the mucous membranes and
enter the bloodstream, e.g.
When shaking hands with another person who then puts their hand to their nose or
mouth
During sexual transmission
Examples include HIV, ebola, syphilis
Inoculation
Page 28 of 63
This typically occurs when a pathogen enters the body through broken skin, providing it YOUR NOTES
with a direct route into the bloodstream 

Transmission could be through sexual contact, sharing needles during drug use, or bites or
scratches from infected animals
Examples include hepatitis B, HIV, tetanus, and rabies
Page 29 of 63
Barriers to Pathogenic Entry YOUR NOTES
Skin 
The skin provides a physical barrier against infection
If the skin is damaged it leaves the exposed tissue beneath vulnerable to pathogens
The blood clotting mechanism of the body plays an important role in preventing pathogen
entry in the case of damage to the skin
Blood clotting takes time, however, so a few pathogens may still enter before a clot forms
Microorganisms of the gut and skin
Collectively these harmless microorganisms are known as the gut or skin flora
They compete with pathogens for resources, thereby limiting their numbers and therefore
their ability to infect the body
Stomach acid
The hydrochloric acid that makes up a large part of the gastric juices in the stomach
creates an acidic environment that is unfavourable to many pathogens present on food
and drink
Sometimes a few of these pathogens may survive and make their way to the intestines
where they infect the gut wall cells and cause disease
Lysozyme
Secretions of the mucosal surfaces, e.g. tears, saliva, and mucus, contains an enzyme
called lysozyme
This enzyme will damage bacterial cell walls, causing them to burst, or lyse
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YOUR NOTES
The body has various barriers that prevent the entry of pathogens
Page 31 of 63
6.8 Non-Specific Immune Responses YOUR NOTES

Non-specific Immune Responses
There are two types of immune response in the body once a pathogen enters
Non-specific
This response is the same, regardless of the pathogen that invades the body
Specific
This is a response specific to a particular pathogen
The immune system is able to recognise specific pathogens due to the presence
of antigens on their cell surface
Antigens are molecules such as proteins or glycoproteins located on the
surface of cells; their role is to act as an ID tag, identifying a cell as being
'self' or 'non-self'
Pathogens have non-self antigens, so the immune system recognises
them as not belonging to the body
When a pathogen invades tissue the non-specific immune response begins immediately;
this includes
Inflammation
Interferons
Phagocytosis
Inflammation
The surrounding area of a wound can sometimes become swollen, warm and painful to
touch; this is inflammation
Body cells called mast cells respond to tissue damage by secreting the
molecule histamine
Histamine is a chemical signalling molecule that enables cell signalling, or
communication between cells
Histamine stimulates the following responses
Vasodilation increases blood flow through capillaries
Capillary walls become 'leaky', or more permeable, allowing fluid to enter the tissues
and creating swelling
Some plasma proteins leave the blood when the capillaries become more
permeable
Phagocytes leave the blood and enter the tissue to engulf foreign particles
Cells release cytokines, another cell signalling molecule that triggers an immune
response in the infected area
Interferons
Cells infected by viruses produce anti-viral proteins called interferons
Interferons prevent viruses from spreading to uninfected cells
They inhibit the production of viral proteins, preventing the virus from replicating
They activate white blood cells involved with the specific immune response to
destroy infected cells
They increase the non-specific immune response e.g. by promoting inflammation
Phagocytosis
Page 32 of 63
Phagocytes are a type of white blood cell responsible for removing dead cells and YOUR NOTES
invasive microorganisms; they do this by engulfing and digesting them 

The process of engulfing and digesting is known as phagocytosis
Phagocytes travel throughout the body and can leave the blood by squeezing through
capillary walls
During an infection they are released in large numbers
Mode of action
Chemicals released by pathogens, as well as chemicals released by the body cells
under attack, e.g. histamine, attract phagocytes to the site where the pathogens are
located
They move towards pathogens and recognise the antigens on the surface of the
pathogen as being non-self
The cell surface membrane of a phagocyte extends out and around the
pathogen, engulfing it and trapping the pathogen within a phagocytic vacuole
This part of the process is known as endocytosis
Enzymes are released into the phagocytic vacuole when lysosomes fuse with it
These digestive enzymes, which includes lysozyme, digest the pathogen
After digesting the pathogen, the phagocyte will present the antigens of the
pathogen on its cell surface membrane
The phagocyte becomes what is known as an antigen presenting cell
The presentation of antigens initiates the specific immune response
Phagocytes engulf pathogens in the process of phagocytosis, enclosing them in a

phagocytic vacuole. Lysosomes fuse with the vacuole, releasing enzymes which digest the
pathogen
Page 33 of 63
6.9 Specific Immune Responses YOUR NOTES

Specific Immune Responses
Antigens
Every cell in the human body has markers on its cell surface membrane that identify it
Microorganisms such as bacteria and viruses also have their own unique markers
These markers are called antigens and they allow cell-to-cell recognition
Antigens are found on cell surface membranes, bacterial cell walls, or the surfaces of
viruses
Some glycolipids and glycoproteins on the outside of cell surface membranes act as
antigens
Antigens can be either self antigens or non-self antigens
Antigens produced by the organism's own body cells are known as self antigens
Self antigens do not stimulate an immune response
Antigens not produced by the organism’s own body cells are known as non-self
antigens
Non-self antigens stimulate an immune response
E.g. the antigens found on pathogenic bacteria and viruses, or on the surface of a
transplanted organ
After pathogens are engulfed by phagocytosis, phagocytes transfer the antigens of the
digested pathogen to their cell surface membrane, becoming antigen presenting cells
Antigen presenting cells such as macrophages activate the specific immune
response
This occurs when the white blood cells of the specific immune response, known
as lymphocytes, bind to the presented antigens with specific receptors on their
cell surface membranes
Note that macrophages are a type of phagocytic white blood cell
Antibody structure
Antibodies are secreted by specialised white blood cell known as plasma cells
Antibodies are Y-shaped molecules sometimes known as immunoglobulins
Antibodies consist of four polypeptide chains; two ‘heavy’ chains attached by disulfide
bonds to two ‘light’ chains
'Heavy' chains are long while 'light' chains are short
Each polypeptide chain has a constant region and variable region
The constant regions do not vary within a class of antibody
There are 5 classes of mammalian antibodies, each with different roles
The amino acid sequences in the variable region are different for each antibody
The variable region is where the antibody binds to an antigen to form an antigen-
antibody complex
At the end of the variable region is a site called the antigen binding site
The antigen binding sites vary greatly, giving the antibody its specificity for
binding to antigens
The ‘hinge’ region, where the disulfide bonds join the heavy chains, gives flexibility to the
antibody molecule, allowing the antigen binding site to be placed at different angles when
binding to antigens
Page 34 of 63
This region is not present in all classes of antibodies YOUR NOTES
Antibodies can be either membrane-bound or secreted directly into the blood 

Membrane-bound antibodies are attached to the surface of lymphocytes
The membrane-bound antibodies have an extra section of polypeptide chain within
their heavy chains which forms the attachment to lymphocytes
Antibodies are Y-shaped molecules consisting of four polypeptide chains. Note that the
term epitope here refers to the part of the antigen that is recognised by the immune system;
the variable regions of the antibody are complementary to the epitope of the antigen,
allowing them to bind
Antibody function
Antibodies bind to specific antigens that trigger the specific immune response
Antibodies function to disable pathogens in several ways
Pathogens enter host cells by binding to them using receptors on their surface;
antibodies can bind to these receptors, preventing pathogens from infecting host
cells
Antibodies can act as anti-toxins by binding to toxins produced by pathogens, e.g.
the bacteria that cause diphtheria and tetanus; this neutralises the toxins
Antibodies cause pathogens to clump together, a process known as agglutination;
this reduces the chance that the pathogens will spread through the body and makes it
possible for phagocytes to engulf a number of pathogens at one time
Page 35 of 63
YOUR NOTES
Antibodies cause agglutination, which makes it difficult for the pathogens to infect host
cells. This also makes it easier for the phagocytes to engulf the trapped pathogens
Page 36 of 63
6.10 Lymphocytes: Types & Roles YOUR NOTES

Lymphocytes: Types & Roles
There are two types of lymphocyte that play a particular role in the specific immune
response
T cells
B cells
Note that lymphocytes are a type of white blood cell
T cells
T cells, sometimes known as T lymphocytes, are produced in the bone marrow and finish
maturing in the thymus, which is where the T in their name comes from
Mature T cells have specific cell surface receptors called T cell receptors
These receptors have a similar structure to antibodies and are each specific to a
particular type of antigen
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YOUR NOTES
Mature T cells have many different types of receptor on the cell surface membrane; these
receptors will bind to different antigens on antigen presenting cells
T cells are activated when they encounter and bind to their specific antigen on the
surface of an antigen presenting cell
This antigen-presenting cell might be a macrophage, an infected body cell, or
the pathogen itself
These activated T cells divide by mitosis to increase in number
Dividing by mitosis produces genetically identical cells, or clones, so all of the
daughter cells will have the same type of T cell receptor on their surface
There are three main types of T cell
T helper cells
Release chemical signalling molecules that help to activate B cells
Release chemical signalling molecules that help to activate T killer cells
Page 38 of 63
Release chemicals called opsonins that label pathogens and infected cells for YOUR NOTES
phagocytosis 
T killer cells
Bind to and destroy infected cells displaying the relevant specific antigen
T memory cells
Remain in the blood and enable a faster specific immune response if the same
pathogen is encountered again in the future
B cells
B cells, also known as B lymphocytes, are a second type of white blood cell in the specific
immune response
B cells remain in the bone marrow as they mature, hence the B in their name
B cells have many specific receptors on their cell surface membrane
The receptors are in fact antibodies, and are known as antibody receptors
Each B cell has a different type of antibody receptor, meaning that each B cell
can bind to a different type of antigen
Page 39 of 63
YOUR NOTES
Mature B cells each have different types of antibody receptors on their cell surface
membrane
If the corresponding antigen enters the body, B cells with the correct cell surface
antibodies will be able to recognise it and bind to it
When the B cell binds to an antigen it forms an antigen-antibody complex
The binding of the B cell to its specific antigen, along with the cell signalling molecules
produced by T helper cells, activates the B cell
Once activated the B cells divide repeatedly by mitosis, producing many clones of the
original activated B cell
There are two main types of B cell
Effector cells, which differentiate into plasma cells
Plasma cells produce specific antibodies to combat non-self antigens
Page 40 of 63
Memory cells YOUR NOTES
Remain in the blood to allow a faster immune response to the same pathogen in 
the future
During a primary immune response B cells divide by mitosis to form plasma cells and
memory cells. Note that a primary response occurs the first time an individual comes into
contact with a particular pathogen
Page 41 of 63
6.11 Developing Immunity YOUR NOTES

Developing Immunity
Developing immunity
The immune system is activated when a new antigen is encountered
This launches a primary immune response consisting of a non-specific immune response
followed by a specific immune response
The primary response occurs the first time an antigen is encountered by the immune
system
Since it is the first time the immune system has encountered the antigen, the numbers of T
and B cells with the correct membrane receptors present in the blood will be low
It will take time for the correct T and B cells to be activated and to divide and differentiate
into different cell types
It can take several days before plasma cells develop and are able to start producing
antibodies against an antigen
This is the reason why an infected person will experience symptoms of the disease the
first time they contract it
Both T and B cells produce memory cells during the primary response, which will remain in
the blood after an infection is over
The presence of memory cells means that a person is said to be immune to the
pathogen
Should the immune system encounter the same antigen again in the future it will launch a
secondary immune response which will be much faster and stronger than the primary
response
Memory cells are present in larger quantities than the mature lymphocytes at the start
of the primary response, so the correct memory cells are able to detect an antigen,
activate, divide by mitosis, and differentiate much more quickly
Antibodies are produced more quickly and in larger quantities in a secondary
response
This will often eliminate the pathogen before the infected person can show symptoms
Page 42 of 63
YOUR NOTES
The secondary response is much larger and faster than the primary response due to the
presence of memory cells in the blood
Active immunity
Active immunity is acquired when an antigen enters the body triggering a specific immune
response
Active immunity can be
Natural; acquired through exposure to pathogens
Artificial; acquired through vaccination
In both cases the body produces memory cells, giving the person long-term immunity
Passive immunity
Passive immunity is acquired without an immune response; antibodies are gained from
another source, not produced by the infected person
Passive immunity can be
Natural
Foetuses receive antibodies across the placenta from their mothers
Babies receive antibodies in breast milk
Artificial
People can be given an injection / transfusion of antibodies e.g. the tetanus
antitoxin
The antibodies will have been collected from people or animals whose immune
system had been triggered by a vaccination to produce antibodies
As the person’s immune system has not been activated, there are no memory cells that
can enable antibody production in a secondary response; if a person is reinfected they
would need another infusion of antibodies
Comparing Active & Passive Immunity Table
Page 43 of 63
YOUR NOTES
Vaccines
A vaccine contains antigens that are intentionally put into the body to induce artificial
active immunity
Vaccines can contain dead or weakened pathogens, less harmful strains of a
pathogen, antigens alone, or a piece of genetic material that codes for the antigens
Vaccines are administered either by injection or by mouth
Vaccinations produce long-term immunity as they cause memory cells to be created.
The immune system recognises the antigen when re-encountered and produces
antibodies in a faster, stronger secondary response
This is the main reason why vaccinated individuals typically do not show symptoms of
the diseases they were vaccinated against
Antigenic variation can mean that vaccinations need to be constantly modified to keep up
with the changes to a pathogen's antigens
Antigenic changes are the result of mutation
Page 44 of 63
6.12 Pathogens vs Hosts: An Evolutionary Race YOUR NOTES

Pathogens vs Hosts: An Evolutionary Race
Vertebrates have evolved over millions of years to have immune systems that are capable
of dealing with a wide range of different pathogens
Pathogens, however, have also evolved and have developed different ways of
evading their host's immune system
This battle between host and pathogen is known as an evolutionary race; each organism
develops new ways in which to have an advantage over the other
This evolutionary race is sometimes referred to as an evolutionary arms race
Evasion mechanisms developed by pathogens serve as support for this theory
HIV evasion mechanisms
The virus kills helper T cells after it infects them which reduces the number of cells that
could detect the presence of the virus and activate the production of antibodies
HIV shows antigenic variability due to the high mutation rate in the genes coding for
antigen proteins
This forms new strains of the virus which each require a new primary immune response
Memory cells for one strain will not recognise the antigens of another strain
The virus prevents infected cells from presenting their antigens on the cell surface
membrane, making it very difficult for the relevant white blood cells to recognise and
destroy the infected cells
Mycobacterium tuberculosis evasion mechanisms
Once engulfed by phagocytes in the lungs the bacteria produce substances that will
prevent a lysosome from fusing with the phagocytic vacuole
This prevents the bacteria from being broken down by digestive enzymes, leaving
them to multiply within the phagocyte
As with HIV the bacteria can disrupt antigen presentation in infected phagocytes, making
it difficult for the immune system to recognise and destroy these cells
Page 45 of 63
Antibiotics YOUR NOTES

6.13 Antibiotics
Antibiotics
When humans experience a bacterial infection they are often prescribed antibiotics
Antibiotics are chemical substances that damage bacterial cells with little or no harm to
human tissue
Penicillin is a well-known example; it was discovered in 1928 by Sir Alexander Fleming
Antibiotics are either
Bactericidal; they kill bacterial cells
Bacteriostatic; they inhibit bacterial growth processes
Note that bacteriostatic antibiotics given at a high enough dose will result in the
death of bacterial cells
Antibiotics work by interfering with the growth or metabolism of the target
bacterium e.g.
Inhibiting bacterial enzymes needed to form bonds in the cell walls; this prevents
bacterial growth and can cause death
Cell walls are weakened and burst under the pressure of water entering the cell by
osmosis
Binding to ribosomes and preventing protein synthesis; this inhibits enzyme
production, stopping metabolic processes in the bacterial cell
Damaging cell membranes, leading to loss of useful metabolites or uncontrolled
entry of water
Preventing bacterial DNA from coiling into rings, meaning that it no longer fits into the
bacterial cell
Since mammalian cells are eukaryotic, they will not be damaged by antibiotics
They do not have cell walls
They have different enzymes
They have different ribosomes
Viruses do not have cellular structures such as enzymes, ribosomes, and cell walls so
they are not affected by antibiotics
Page 46 of 63
YOUR NOTES
Penicillin prevents the formation of a strong cell wall in prokaryotes, ultimately leading to
the death of the cell by lysis
Page 47 of 63
6.14 Core Practical 14: The Effects of Different Antibiotics YOUR NOTES

The Effects of Different Antibiotics
The effectiveness of different antibiotics in preventing the growth of specific types of
bacteria can be studied using aseptic techniques
Aseptic techniques involve the use of sterile equipment and require a researcher to work in
a sterile environment
Sterile refers to the absence of microorganisms
Equipment can be sterilised by washing at high temperatures or with antibacterial
chemicals
Work benches can be wiped with antibacterial chemicals
An updraft can be created to ensure sterile air around a work space e.g. by placing
a Bunsen burner next to the work bench
Apparatus
Bacterial culture
Disinfectant
Bunsen burner
Agar plates
Pipettes
Plastic spreader or metal inoculating loop
Antibiotics and paper discs, or antibiotic infused paper discs
Distilled water
Forceps
Method
1. Set up your sterile work area by wiping surfaces with disinfectant and setting up a Bunsen
burner
The Bunsen burner will create an updraft and prevent microorganisms in the air from
landing in the work area
2. Transfer some of the bacterial culture on to an agar plate using a sterile pipette and spread
it out using a sterile spreader or inoculating loop
Plastic equipment can be set aside for washing after each use
Metal equipment, e.g. inoculating loops, can be sterilised in a Bunsen flame after each
use
3. Soak similar sized paper discs in different types of antibiotics, e.g. Methicillin, Tetracycline
and Streptomycin
An alternative to using different types of antibiotics could be to use different
concentrations of the same antibiotic
Note that you can use paper discs that have been pre-treated with antibiotics as an
alternative to soaking
4. Add a negative control to the investigation by soaking a disc in distilled water
This provides a point of comparison, demonstrating that any results gained are due to
the changes in antibiotic type and not any other factor
5. Space the discs apart on the agar plate using sterile forceps
Place the forceps in a container filled with disinfectant after use, or sterilise in a
Bunsen flame
Page 48 of 63
6. Lightly tape a lid onto the petri dish, invert and incubate at 25 °C for 24 to 48 hours YOUR NOTES
Lightly taping rather than sealing ensures that oxygen is available to the bacteria 
Inverting the dish, i.e. storing it upside down, prevents condensation from dripping
onto the agar, potentially contaminating the dish
Incubating at around room temperature prevents the growth of harmful pathogens;
research laboratories may use warmer temperatures to achieve faster results
Bacteria will grow to form a 'lawn' on top of the agar
Any clear patches in the lawn will indicate where bacteria could not grow
This is called the clear zone
Interpretation of results
The larger the clear zone, the more effective the antibiotic was at inhibiting bacterial
growth
If no clear zone is present, it means that the bacteria is resistant to that type of antibiotic
The sizes of the clear zones can be compared between the different types of antibiotics,
as well as the different concentrations of each
The presence and size of clear zones around the paper discs indicates the effectiveness of
an antibiotic against the type of bacteria used
Page 49 of 63
6.15 Current Usage of Antibiotics YOUR NOTES

Current Usage of Antibiotics
Hospital acquired infections
Infections that are contracted by a patient while in hospital are known as hospital acquired
infections (HAIs)
The transmission of HAIs is increased by poor hygiene practices which might include
Staff and visitors not washing hands regularly
Uncontained coughing and sneezing
Failing to disinfect equipment and surfaces after use
Patients in hospital are likely to already be ill and to have weakened immune systems,
putting them at high risk of contracting infectious disease
Hospitals have measures in place that aim to reduce the spread of HAIs
Staff and visitors must wash hands regularly while visiting patients
If a person contracts a HAI they should be moved to an isolation ward to prevent
spread of the infection
Surfaces and equipment must be disinfected after every use
HAIs and antibiotic resistance
Some HAIs are caused by antibiotic resistant bacteria
A well known example of an antibiotic resistant HAI is methicillin-
resistant Staphylococcus aureus, also known as MRSA; in this case the bacterial strain
of S. aureus has developed resistance to the antibiotic methicillin
Antibiotic resistant infections are difficult to treat as they do not respond to regular
antibiotics
These HAIs can cause serious health complications or death
The risk of antibiotic resistant bacterial strains arising is high in hospital environments
Antibiotics are widely used in hospitals to treat disease which provides a selection
pressure for resistant strains of bacteria to develop
Selection pressures are factors in the environment that drive natural selection
In ecology the presence of a predator would be an example of a selection
pressure
Certain hospital practices have been developed to reduce the risk of antibiotic resistant
HAIs
No antibiotic prescriptions for minor infections or viral diseases
No use of antibiotics as a preventative measure against infections
Prescription of a narrow-spectrum antibiotic to treat the infection
Narrow-spectrum antibiotics are active against a narrow range of bacterial
infections, as opposed to broad-spectrum antibiotics which are effective
against many types of bacteria
The advantage of using narrow-spectrum antibiotics is that any resistance genes
that arise will not cause problems if they are transferred to other types of bacteria,
as those other types of bacteria will be treated with a different antibiotic
Bacteria are able to exchange genes with each other by a process of
horizontal gene transfer
Page 50 of 63
Rotate the use of different antibiotics to decrease the chance of bacteria developing YOUR NOTES
resistance against one antibiotic 
Antibiotic resistance in bacteria arises by natural selection; it is therefore more likely to

occur in environments where antibiotics are frequently used, such as hospitals
Page 51 of 63
Decomposition & Decay YOUR NOTES

6.16 The Role of Microorganisms
Microorganisms & Decay

Dead plants and animals are broken down by microorganisms such as bacteria and fungi
These organisms are known as decomposers
They play an important role in recycling the chemical components needed by living
organisms
These decomposers secrete enzymes that break large organic molecules, such as
cellulose, down into smaller ones
These small molecules, such as glucose, can be broken down further
during respiration
During decomposition they also release waste products which provides nutrients to
plants
The microorganisms involved in decomposition produce CO2 and methane which
are released into the atmosphere
Carbon dioxide can then be absorbed by green plants which will fix the carbon back into
carbohydrates during photosynthesis
Microorganisms use biological molecules within dead tissue to fuel their respiration,
releasing carbon back into the atmosphere in the process
Page 52 of 63
Forensics YOUR NOTES

6.17 Polymerase Chain Reaction
Polymerase Chain Reaction

Every person, with the exception of identical twins, has a unique DNA sequence which can
be used to create a DNA profile
This is very useful in forensic science as it provides a way to identify individuals
DNA profiling can also be used to determine the genetic relationships between
different organisms e.g.
Paternity and maternity testing
Ancestry kits
Determining evolutionary relationships between different species
DNA profiles can be created using the following steps
Isolating a sample of DNA e.g. from saliva, skin, hair, or blood
Producing more copies of the DNA fragments in the sample using the polymerase
chain reaction (PCR)
Carrying out gel electrophoresis on the DNA produced by PCR
Analysing the resulting pattern of DNA fragments
The polymerase chain reaction
PCR is a common molecular biology technique used in most applications of gene
technology e.g.
DNA profiling
Genetic engineering
It can be described as an in vitro method of DNA replication
PCR produces many copies of a piece of DNA; this can be referred to as DNA amplification
It is used to produce large quantities of specific fragments of DNA or RNA from very
small quantities; even just one molecule of DNA or RNA is enough to run PCR
By using PCR scientists can produce billions of identical copies of the DNA or RNA
sample within a few hours
In each PCR cycle the DNA is doubled, so in a standard run of 20 cycles a million DNA
molecules are produced.
The process is carried out in a PCR machine, or thermal cycler, which automatically
provides the optimal temperature for each stage and controls the length of time spent at
each stage
Each PCR reaction requires
DNA or RNA to be amplified
Primers
These are short sequences of single-stranded DNA that have base sequences
complementary to the 3’ end of the DNA or RNA being copied; they define the
region that is to be amplified, identifying where the DNA polymerase enzyme
needs to bind
DNA polymerase
The enzyme used to build the new DNA or RNA strand.
Page 53 of 63
The most commonly used polymerase is Taq polymerase, which comes from YOUR NOTES
thermophilic bacterium Thermus aquaticus 

Taq polymerase does not denature at the high temperature required during
the first stage of the PCR reaction
The optimum temperature of Taq polymerase is high enough to prevent
annealing of the DNA strands that have not been copied yet
Free nucleotides
Enable the construction of new DNA or RNA strands
Buffer solution
Ensures the optimum pH for the reactions to occur in
There are three main stages of the PCR reaction
Denaturation
The double-stranded DNA is heated to 95 °C which breaks the hydrogen bonds
that hold the two DNA strands together
Annealing
The temperature is decreased to 50-60 °C so that primers can anneal to the ends
of the single strands of DNA
Elongation / Extension
The temperature is increased to 72 °C, as this is the optimum temperature
for Taq polymerase to build the complementary strands of DNA to produce the
new identical double-stranded DNA molecules
These three stages make up a single PCR cycle, and many cycles can be completed
Each PCR cycle doubles the amount of DNA
Page 54 of 63
YOUR NOTES
PCR can be used to amplify a fragment of DNA. Note that you don't need to know about
forward and reverse primers
After PCR is completed the DNA is treated with restriction endonuclease enzymes and
a fluorescent tag can be added; both in preparation for gel electrophoresis
Restriction endonucleases break the DNA up into fragments of different length
Fluorescent tags enable the DNA fragments to be seen under UV light
Page 55 of 63
6.18 Gel Electrophoresis in Forensics YOUR NOTES

Gel Electrophoresis in Forensics
Gel electrophoresis is a technique used widely in the analysis of DNA, RNA and proteins
DNA fragments are created, e.g. using enzymes known as restriction endonucleases
that cut DNA at specific restriction sites
The resulting fragments are inserted into a well at the end of a piece of agar gel, before
a current is passed through the gel
Molecules move through the agar due to the difference in charge across the gel
Positively charged molecules will move towards the cathode (negative pole)
while negatively charged molecules will move towards the anode (positive pole)
DNA is negatively charged due to the phosphate groups and so when placed in an
electric field the molecules move towards the anode
The molecules are separated according to their size / mass
Different sized molecules move through the gel at different rates
The tiny pores in the gel allow smaller molecules to move quickly, whereas larger
molecules move more slowly
The process of gel electrophoresis involves the following stages
An agarose gel plate is created and wells are cut into the gel at one end
The gel is submerged in a tank containing electrolyte solution; this is a salt solution
that conducts electricity
The DNA samples are transferred into the wells using a micropipette, ensuring that a
sample of DNA standard is loaded into the first well
The purpose of the standard is to produce a set of known results with which
to compare any new results
The negative electrode is connected to the end of the plate with the wells and the
positive anode is connected at the far end
The DNA fragments move towards the anode due to the attraction between the
negatively charged phosphates of DNA and the anode
The smaller mass / shorter pieces of DNA fragments move faster and therefore
further from the wells than the larger fragments
Probes are then added, after which an X-ray image is taken or UV-light is shone onto
the paper producing a pattern of bands which can be compared to the control, or
standard, fragments of DNA
Probes are single-stranded DNA sequences that are complementary to the
regions of interest; they can be
A radioactive label which causes the probes to emit radiation that makes the
X-ray film go dark, creating a pattern of dark bands
A fluorescent dye which fluoresces when exposed to UV light, creating a
pattern of coloured bands
Page 56 of 63
YOUR NOTES
Gel electrophoresis can be used in DNA profiling

Analysing the results of gel electrophoresis
Gel electrophoresis produces a pattern of bands on the gel that represent DNA fragments
of different length
The fragments were produced after PCR by cutting the DNA samples into pieces using
restriction endonuclease enzymes
Restriction endonucleases cut DNA at specific locations in the DNA base sequence,
so will always cut in between sections of repeated bases known as variable number
tandem repeats (VNTRs)
VNTRs are known as micro- or mini-satellites depending on the number of repeats
that occur; micro-satellites have fewer repeats than mini-satellites
Page 57 of 63
Different people have different numbers of repeats in their VNTR regions, so YOUR NOTES
the fragments will differ in length depending on whether there are few or many 
repeats
Different individuals will have different lengths of DNA fragments, so a different pattern
of banding will form on each profile
Every banding pattern will be unique to an individual, so comparisons of DNA from crime
scenes with that of suspects is a reliable way of finding out who was present at a crime
scene
 Exam Tip
Don't get too bogged down in the details regarding restriction enzymes and VNTRs,
but you should be able to explain how gel electrophoresis works to separate DNA
fragments of different length.
Page 58 of 63
6.19 DNA Profiling YOUR NOTES

DNA Profiling
A DNA profile can be produced using the process of gel electrophoresis; the result is a
series of bands of DNA of different length which can be seen due to the addition of
radioactive or fluorescent labels
DNA profiles can be used to determine the genetic relationships between people, e.g.
in paternity tests
During a paternity test the DNA profile of a child is compared with a variety of
candidates that could be the potential father
If many bands of the child's DNA profile match with the bands in a paternity
candidate's profile, this could indicate that they are the most likely biological father
During fertilisation half of the DNA comes from each parent, so a child will
share half of their DNA with a parent
When comparing DNA profiles the more bands that match between the profiles,
the greater the genetic similarity between those individuals and the closer the
relationship
DNA profiles can be compared to determine relationships in paternity testing. Here the child
shares bands 1, 3 and 5 with the mother and bands 2, 4 and 6 with candidate B, so he is the
most likely father
DNA profiling is a useful tool in forensic science where it can be used to link possible
suspects to a crime scene
Regions of DNA, known as short tandem repeats, are examined
These short tandem repeats are a type of non-coding, repeated sequence of
bases known as a variable number tandem repeat, or VNTR; short tandem repeats
consist of short repeating sections, and are also known as micro-satellites
Page 59 of 63
The greater the number of these regions examined, the more reliable the evidence YOUR NOTES
provided 
If only a few regions are analysed then there is a greater chance that closely
related individuals will have an identical profile; an analysis of 11 or more sites is
considered to be reliable evidence in a law court
DNA profiles can be created using DNA samples found at crime scenes and then compared
with the profiles of suspects to show who was present at a crime scene. VNTRs are shown
beneath each individual in blue and green; different individuals have different numbers of
VNTRS. In this example the DNA profile of suspect 3 is the closest match with the DNA found
at the crime scene, though none of the suspects is a perfect match.
DNA profiling can also be useful in selective or captive breeding programmes of animals
or cultivation of plants
DNA profiles of the particular organisms can be compared to determine which
are genetically the most different from each other
These organisms will then be crossbred, ensuring that the individuals that breed
together are not closely related
Breeding between closely related individuals is known as inbreeding, and can
cause genetic problems at an individual and population level
In individuals there can be an accumulation of harmful recessive alleles that
might otherwise have been masked by healthy dominant alleles
Inbreeding leads to a smaller gene pool within a population, which can
reduce a population's ability to adapt to change
Page 60 of 63
6.20 Types of Data Provided by Forensic Analysis YOUR NOTES

Useful Data Provided by Forensic Analysis
Forensics is the use of science in the investigation of criminal activities
It involves the collection and analysis of evidence from a potential crime scene e.g.
the scene at which a dead body is found
Determining the time of death (TOD) of a body is a very important component of forensic
science
The TOD can be used during a police investigation to provide information about
the circumstances surrounding the death of a person
In order to accurately estimate TOD, there are several factors that must be established
Extent of decomposition
Stage of succession
Forensic entomology
Body temperature of the deceased
The degree of muscle contraction
Extent of decomposition
The process of decomposition begins soon after death
Decomposition is carried out by organisms known as decomposers e.g. bacteria and
fungi
Enzymes secreted from the cells of these organisms break down biological
molecules in dead tissue
The time since death can often be established visually by looking at the appearance of a
body that is decomposing
Decomposers break down cells and tissues over the course of a few days
At this stage in decomposition the appearance of the skin can be a helpful
indication of time since death; skin will often appear greenish in colour
The next stage of decomposition involves the breakdown of tissues and organs by
micro-organisms over the course of a few days or weeks
This process produces gases, such as methane, which will lead to bloating
The skin will blister and fall off the rest of the body
A few weeks after death the remains of the soft tissues will turn to liquid which
becomes visible as it leaves the body
This process will continue over the course of months or years until only
a skeleton remain
After a few decades or centuries, the skeleton will disintegrate until nothing remains
The rate of decomposition will be affected by factors such as temperature and availability
of oxygen
Decomposition would be slower in anaerobic conditions and at lower temperatures
but would be faster at high temperatures
Stage of succession
Succession refers to the change in the types of organisms found in a habitat over time
This is often an ecology term that is applied to a habitat such as a pond or woodland,
but in this case the habitat is the dead body
Page 61 of 63
The difference between succession in ecology and in forensics is that in an YOUR NOTES
ecosystem the early pioneer species are out-competed and disappear as the 
system matures, while in a dead body all of the newly arriving species remain as
decomposition progresses
The stage of succession of a body can provide information about the estimated TOD
Above ground the body would undergo the following stages of succession
Bacteria will be found in and on the dead body immediately after TOD
As tissue decomposition sets in it creates ideal conditions for flies to lay eggs and
their larvae to hatch
As more soft tissue is consumed by the fly larvae it creates favourable conditions
for beetles to establish
When tissue dries out over time flies will leave the body as they prefer a moisture-rich
environment
Beetles, however, can decompose dry tissue so they will remain on the body
Once all tissues have been decomposed most organisms will leave the body
These succession stages will differ depending on where the body is located as
the accessibility to insects and availability of oxygen will be affected e.g.
Buried in soil
Buried in a coffin
Under water
Forensic entomology
A dead body provides an ideal habitat for many species of insects; the study of these
insect colonies is known as forensic entomology
Different insect species will colonise a body at different times after death, providing
information about the TOD
Flies will be found on the body within a few hours after death, while beetles will only
colonise the body later
Another clue that insects can provide is the stage of life cycle they are at
E.g. blowfly eggs will hatch after about 24 hours so if larvae are present on the body it
indicates that the person died more than 24 hours ago
Other insects have longer life cycles, so if only blowfly larvae are found it indicates
that only 24 hours has passed since TOD
Factors that might affect the progression of insect life cycles include
Drugs that may be present in the body
Humidity of the surroundings
Oxygen availability
Temperature
Body temperature
Respiration and other metabolic processes produce heat in living organisms
This heat is necessary for maintaining our body temperature at around 37 °C during life
Once a person dies metabolic reactions will eventually come to an end
Since no more heat is produced the body temperature drops until it reaches the
temperature of the surrounding environment
This process of cooling is known as algor mortis
Page 62 of 63
Body temperature decreases by 1.5-2.0 °C per hour, therefore providing forensic YOUR NOTES
scientists with a way to determine the TOD based on the temperature of the body 
Certain conditions will affect the rate at which body heat is lost e.g.
Air temperature
Surface area : volume ratio
Presence of clothing
Percentage body fat
Degree of muscle contraction
Muscles in the body begin to contract about 4-6 hours after TOD, leading to a general
stiffening of the body known as rigor mortis
Rigor mortis comes about as a result of changes to the proteins in muscle cells after death
Since no more oxygen reaches the muscle cells after death they will start to respire
anaerobically, producing lactic acid
The accumulation of lactic acid decreases the pH in the muscle cells, denaturing the
enzymes that produce ATP
Without ATP the myosin heads cannot be released from the actin filaments, locking
the muscles in a contracted state
Muscles contract due to the action of two protein filaments; myosin and actin
The binding of myosin heads to actin proteins followed by the bending of the
myosin heads causes muscle contraction
ATP is required to allow the myosin heads to detach from the binding sites on actin
This leads to the stiffness that is the main characteristic of rigor mortis
Rigor mortis will begin in the smaller muscles of the head and end in the larger muscles of
the lower body, meaning that forensic experts can determine TOD by the progress of rigor
mortis through the body
Rigor mortis would have taken place in every muscle between 12 and 18 hours after
death, but will wear off again after about 24 to 36 hours from TOD
The process is affected by the level of muscle development and the temperature of the
surroundings
Higher temperatures will speed up the rate of rigor mortis
 Exam Tip
Remember that the time of death is only an estimate and cannot be pinpointed
exactly. There are so many factors that will affect a dead body that it is nearly
impossible to provide a time of death that will be precisely accurate; keep this in
mind when answering questions on this topic.
Page 63 of 63

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International A Level Biology Edexcel 

6. Microbiology, Immunity & Forensics

Microbiology YOUR NOTES

From agar gel into a liquid culture flask YOUR NOTES

From a liquid culture flask onto agar gel 

6.2 Measuring the Growth of Microorganisms YOUR NOTES

squares contain 20, 14, 19, and 16 cells 

one large mass 

A colorimeter can be used to determine the turbidity of a solution containing

6.3 The Bacterial Growth Curve YOUR NOTES

There are four phases in the standard growth curve of a microorganism

large numbers of bacteria produced within hours 

Exam Tip YOUR NOTES

Exponential growth rate constants

Step 1: Calculate the exponential growth rate constant

log10 ( 926) − log10 (2)

N t = N 0 x 2kt YOUR NOTES

6.4 Core Practical 13: Rate of Growth of Microorganisms YOUR NOTES

6.5 Comparison of Bacterial & Viral Structure YOUR NOTES

Prokaryotic cells have a peptidoglycan cell wall, no membrane-bound organelles, a circular

once it has infected a cell 

Bacteriophage viruses, such as the λ phage, are examples of DNA viruses

Lytic & Latency YOUR NOTES

Immunity YOUR NOTES

HIV YOUR NOTES

6.7 Pathogens: Routes of Entry YOUR NOTES

with a direct route into the bloodstream 

Barriers to Pathogenic Entry YOUR NOTES

6.8 Non-Specific Immune Responses YOUR NOTES

invasive microorganisms; they do this by engulfing and digesting them 

Phagocytes engulf pathogens in the process of phagocytosis, enclosing them in a

6.9 Specific Immune Responses YOUR NOTES

This region is not present in all classes of antibodies YOUR NOTES

Antibodies can be either membrane-bound or secreted directly into the blood 

6.10 Lymphocytes: Types & Roles YOUR NOTES

Memory cells YOUR NOTES

6.11 Developing Immunity YOUR NOTES

6.12 Pathogens vs Hosts: An Evolutionary Race YOUR NOTES

Antibiotics YOUR NOTES

6.15 Current Usage of Antibiotics YOUR NOTES

resistance against one antibiotic 

Antibiotic resistance in bacteria arises by natural selection; it is therefore more likely to

Decomposition & Decay YOUR NOTES

Microorganisms & Decay

Forensics YOUR NOTES

Polymerase Chain Reaction

thermophilic bacterium Thermus aquaticus 

6.18 Gel Electrophoresis in Forensics YOUR NOTES

Gel electrophoresis can be used in DNA profiling

6.19 DNA Profiling YOUR NOTES

6.20 Types of Data Provided by Forensic Analysis YOUR NOTES

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