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1
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All microorganisms share basic nutritional needs- a source of the elements carbon and
nitrogen for example
...
To such a solution, growth factors specific to the needs of a particular microorganism
ae added, for example, and energy source or vitamins
...

Type of culture medium
Liquid (Broth culture)

Solidified

Usual method of cultivation in a
lab
In a partially filled conical flask, or
a similar flask that enables
maintenance of a large surface
area in contact with the air
...

The addition of a gelling agent,
such as agar, to a liquid medium
makes it solidify
...


In a glass flat-sides bottle often
called a medical flat, or a test tube
...

Allows harvesting of any
useful metabolic products
from the microorganisms
...

Provides a large surface
area for growth and gas
exchange with the air in
the dish
...

Provides a greater depth
of agar than a petri dish,
reducing the risk of
dehydration and salt
crystallisation
...

Continuous culture- Inoculation of microorganisms into a sterile container containing liquid
growth medium
...


Only batch culture is possible using solidified culture media since it contains a limited mass of agar
...
They can be used to grow a wide variety of microorganisms, and these are known
as general purpose/broad spectrum media
...

Some media will allow the growth of only a few, or one, species of microorganism
...
If you inoculated a mixed culture onto a selective medium, only
the specific organism, for which the medium had been designed, will grow
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The main use of selective media is in diagnostic work, as you would know that any sampled bacteria
that grew on a certain selective medium is a certain bacterium, so a suitable drug could be
prescribed once the genus is identified
...

Environmental temperature is an important variable in determining the ecological niche of each
species
...
On basis of the
temperature at which they grow best, bacteria are commonly classified into three groups
•
•
•

Psychrophilic- Grow best at low temperatures in the range -10 to 20 degrees
...

Thermophilic- Grow best at high temperatures in the range 55 to 85 degrees
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You normally grow a pure culture, containing only
one type of microorganism
...

Contamination of yourselves or other workers
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When culturing microorganisms, you must work as though the microorganisms in your culture are
particularly harmful
...

Sterilisation methods:
The only way to be sure that bench surfaces and items of laboratory equipment are free of
microorganisms is to kill them or remove them, by sterilising any equipment used
...
They are
ineffective against
bacterial spores,
though
...
No
microorganism can
survive this exposure
...

Moist heat- Place
objects in an autoclave
at 121 degrees for at
least 15 minutes
...
Using a filter of
pore size 0
...

Expose items to UV or
ionising radiation
...

Dispose of wet equipment
immediately, treat any spillages that
occur
...

Needles and forceps can also be
flamed during the manipulation of
cultures
...

The neck of a glass bottle, flask or
tube containing a culture of
microorganisms is sterilised by
passing it through a Bunsen flame
without allowing it to become redhot
...

This is the preferred method for many
items of laboratory equipment and
for culture media that are not heatsensitive
...

The size of the pores involved makes
this unsuitable for all but the smallest
volumes of liquid
...

Ionising radiation such as gamma
rays, cannot be used in a college or
laboratory since industrial facilities
are needed
...

Streak plating:
If you leave an agar plate open to the air, a number of microorganisms will land
and start to grow, and you end up with a mixed culture
...
You can do this using
a technique called streak plating
...
You then dilute
the inoculum by spreading it multiple times
...

Measuring the growth of bacterial cultures:
To measure the growth of a bacterial population, you need to find out how many bacterial cells are
in your culture medium at repeated time intervals
...

Viable count- Count only the living cells in the culture
...
Even with the best light microscopes, bacterial are difficult to see and
you can’t distinguish between a dead bacterium and a living one just by looking at it
...
Then,
knowing your dilution factor, you can multiply the count by
this dilution factor to obtain an estimate of the number of
cells in the undiluted culture
...

Making total counts:
The total count estimates the number of cells in a culture, regardless of whether they are alive or
dead
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•
•
•

A direct count, using a counting chamber and light microscope
...

An indirect count, using a colorimeter to measure the turbidity of a culture
...
It has a grid etched into it and it is thicker than a normal glass
slide
...
The grid is formed of squares of known dimensions, making it very
accurate, and the volume can be calculated
...

Some of the bacterial cells end up overlapping the lines around the edge of the haemocytometer
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2 x 0
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DO NOT include in your count any cell that touches or overlaps the middle of the three lines
at the bottom and left-hand side of the 0
...
2mm square
...
Since the technique involves filtering a culture to
remove the bacterial cells, it is only useful with small volumes of liquid media
...
You
would then heat the filter membrane, with the bacteria on its surface, in the oven at 100 degrees
until its mass remained constant
...
Finally, knowing the volume of medium you
filtered, you can calculate the mass of cells per unit volume
...

You can measure the degree of this turbidity using a colorimeter
...
You can then
either measure the amount of light absorbed by the contents of the cuvette, or the amount of light
that passes through the contents
...

This technique can only be used with liquid cultures, and you cannot distinguish between living and
dead cells
...
Once
plated onto a solid culture medium, the growth of each viable cell will produce a visible colony
...
You would use a constant volume of each dilution and create a spread plate
for each
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Method of estimating cell
number
Direct microscopic count
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Turbidity measurement
...

A relatively slow method
...

Useful for obtaining data to produce a calibration curve
for turbidity measurements
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A slow method, so can be used only with small volumes of
culture, making results unreliable
...

Can only be used for total counts
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Does not kill the cells
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Can be used for viable counts
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Does not kill cells being examined
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If you plotted
the number of bacteria against time, it would result
in a curve showing exponential growth
...
Therefore, the growth curve
of bacteria in batch culture is not exponential, and
can be split into 4 stages
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The length of this phase depends on the culture medium used and the activity of the
bacteria before they were inoculated
...
This time taken differs from species to
species, but is usually very short
...
As a result, the
conditions become no longer optimal for bacterial growth and cells begin to die
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•

The death phase- As the conditions of the culture medium become less and less suitable for
growth, an increasing number of the cells die
...


Calculations involving the log phase of the growth curve:
There are three ways to analyse the log phase quantitatively:
•
•

•

Finding the number of cells- This involves the knowledge that bacteria divide by binary
fission
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If the number of cells at time t0 is N0 and the number of cells at the
later time tx is Nx, you can find the exponential growth rate constant using the formula
u=2
...

The generation time (g)- The time between two consecutive divisions
...
It can be calculated using
the same symbols as the previous equation
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301(tx-t0)/logNx-logN0

Definitions:
Agar: An extract from seaweed that is used as a gelling agent to solidify culture media
...

Petri dish: A shallow glass or plastic dish with a slightly larger dish that fits over it as a lid
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Inoculum: The small sample of a microbial culture that, using aseptic techniques, is transferred to a
new medium
...

Aseptic technique: A way of working with microorganisms that ensures that only one type of
microorganism grows in each culture, and that none escape
...

Autoclave: A machine that works similarly to a pressure cooker
...

Plating: The transfer of an inoculum to the surface of a sterile agar medium
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2
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It is a very common commensal in the guts of
mammals including humans, and is a major component of
faeces
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The damage can result from either:
•
•

The release of toxins- Substances produced by the
pathogen,
Invasion and destruction of the host’s tissues

Pathogenic effects produced by toxins:
A toxin is a poison produced by an organism
...
Pathogenic
bacteria produce toxins that we can classify into endotoxins and exotoxins
...
Each endotoxin is a lipopolysaccharide
that is embedded in the cell surface membrane
...

The O-specific component lies outside the membrane
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It also has antigenic properties- it can cause the production of antibodies against it
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A large number of bacteria must be ingested in order to cause symptoms in healthy
adults
...
On death and lysis, the endotoxins are released, causing inflammation and
reduces the ability of the cells lining the intestinal wall to stop the movement of water and ions into
the lumen of the intestine, resulting in diarrhoea
...
Once
ingested in contaminated food, it invades the body via the lymphatic system, usually by the lymph
nodes in the tonsils or small intestine
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Their release brings about the symptoms of typhoid fever, which
includes temperatures of up to 40 degrees, ulcerations of the gut, and sometimes death
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The products of this digestion are
used by the bacteria as nutrients
...

Superantigens- Polypeptides that stimulate large numbers of cells of the immune system,
resulting in a massive release of cytokines into the blood
...


Antibiotics:
An antibiotic is a chemical substance that, in low concentrations, kills or inhibits the growth of
microorganisms
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•
•

Broad-spectrum antibiotics- Effective against a wide range of pathogenic bacteria
...


As antibiotic resistance develops, the range of broad-spectrum antibiotics decreases
...
They
prevent an infection spreading, but the hosts immune system kills the bacteria
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Bacterial
mechanism
targeted

Effects of antibiotics

Cell wall
synthesis

•
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Disrupt the synthesis of bacterial cell walls
Inhibit the enzyme that catalyses the formation of cross-linkages between
the peptidoglycan molecules in the cell wall
...


Antibiotic resistance in bacteria:
Since narrow-spectrum antibiotics only affect a few groups of bacteria, the others are naturally
resistant
...
Recently, groups of bacteria that were once susceptible to
an antibiotic have become resistant to it
...

Bacteria can acquire secondary resistance by one of three mechanisms:
•
•
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A decrease in the uptake, or an increase in the expulsion of an antibiotic by the bacteria
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Development by the bacterial cell of a biochemical pathway that bypasses the reaction
affected
...
During DNA replication errors occur at an
estimated rate of 1 in 1,000,000 base pairs, and these are gene mutations that produce different
alleles
...
Therefore, it is clear to
see how a gene mutation in a single bacterial cell might produce a new allele of a gene that confers
resistance
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The antibiotic kills or stops the growth of the susceptible cells, but the cell with
the mutant allele would be unaffected, eventually giving rise to millions of daughter cells by binary
fission with the same allele
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Bacteria can also acquire new genes by passing genetic material between each other
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Passing on the gene through binary fission is vertical gene
transfer, this is horizontal
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Controlling the spread of antibiotic resistance:
Given their rapid rate of growth, the evolution of bacterial resistance was almost inevitable,
however it is occurring more rapidly than expected due to their misuse
...

Doctors prescribe antibiotics for patients with viral infections despite it having no effect
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Animals or humans often take sub-clinical concentrations of antibiotic
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If the full treatment of antibiotic is not taken after the patient believes they
are no longer needed, some bacteria are still able to survive and pass on their resistance
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Restrict addition of
antibiotics to animal
feeds
...
Used at
sub-clinical concentrations they
do not destroy bacteria but
favour those that develop
resistance
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Doctors to restrict
prescriptions of
antibiotics
...


This might mean that doctors
advise patients to go home to bed
for a few days until their own
immune systems overcome the
infection
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Patients must complete
their full course of
antibiotics
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This
creates the selection pressure
without destroying the bacteria
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In general, we do whatever
we feel like doing
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Make new antibiotics faster than
bacteria can become immune to
them
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Stem rust fungus:
This parasite infects the plants stem, producing a covering that is the colour of rust
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On
landing on a new plant, the spores germinate and produce threadlike structures called hyphae
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Like all fungi, it secretes digestive enzymes from its hyphae onto
the material on which it is growing
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Infection by stem rust fungus:
•
•
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Weakens the stem, often causing the plant to fall over, making mechanical harvest
impossible
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Breaks the outer epidermis o the stem, increasing the rate of water loss and making the
stem more susceptible to infection by other plant pathogens
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As associations
between plant roots and fungi are vital to the roots’ efficient absorption of inorganic ions from the
soil, fungicides can damage delicate ecosystems
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Influenza:
Influenza is caused by a virus
...
Outside the capsid is an envelope with an
outer lipid layer and inner protein layer
...
The influenza virus enters
the human host lungs via droplets inhaled through the nose or mouth
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•
•
•
•
•
•
•
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Virus particles taken into lungs in droplets
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Antigen is an enzyme hydrolysing mucus, allowing contact between the virus and the
membrane of the epithelial cell
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RNA segments enter the nucleus, transcription and replication of nucleic acid occurs here
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The infected host cell undergoes lysis
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Influenza viruses are classified into antigenic groups according to the H and N glycoproteins on their
capsids
...
The influenza virus shows great antigenic
variability, meaning mutations frequently occur that result in new forms of the antigens appearing
on the outer surface of the virus
...
This often results
in a major epidemic
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This leads to
a pandemic in which millions of humans die
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The malarial parasite:
Malaria is endemic in tropical and sub-tropical regions of the world
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5 million die
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Transmission of plasmodium from an infected person to
another person is by the mosquito
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The saliva contains anticoagulants that enable
the mosquito to draw blood through its mouthparts without the blood clotting
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The injected sporozoites enter cells in the liver of their new human host, and
rapidly divides to form thousands of daughter cells called merozoites
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The released merozoites enter red blood
cells, where they digest the haemoglobin as a
food source
...
The red blood cells burst,
releasing these new merozoites into the
blood, where each infects another red blood
cell
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Every 3-4 days, increasing numbers of red blood cells
burst, releasing merozoites that infect new red blood cells
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These toxins cause the
symptoms, including a very high fever, and a swollen spleen
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Method to reduce incidence
of malaria

Issues

Drain wetland areas where
mosquitoes breed
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Some people earn their living or derive
their main food source from wetlands
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Spray areas where malaria is
The areas are vast, much larger than areas treated in Europe,
endemic with insecticide to kill and would often involve co-operation between different
mosquitoes
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Insecticide
would damage other wildlife, including beneficial insects
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This is cheap and effective since most mosquito’s bite at night
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Released sterilised male
mosquitoes that would mate
with the females but not
result in viable eggs being laid
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Large scale education programmes are needed
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A vaccine would have to be suitable for use with babies and
young children, since malaria is most severe during the first
years of life
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Definitions:

Commensal: A partner in an association between two organisms of different species where one
benefits and the other neither benefits nor is harmed
...

Antibiotic: A substance that, in low concentrations, kills or inhibits the growth of microorganisms
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Epidemic: An outbreak of disease in which the number of new infections is much greater than would
be expected from recent experience
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Pandemic: A human disease that has spread worldwide
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3
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First Lines of Defence against Infectious Diseases:
In order to cause an infection, the pathogen must first be able to enter the body
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Each of these
has evolved mechanisms that protect us from infection
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It has many layers of cells, making
it difficult to penetrate
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The waxy sebum on the skins surface contains fatty acids that are toxic to many
pathogens
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There are hair-like extensions of surface
membranes of cells lining these organs, cilia, moving the mucus to the pharynx,
where we swallow it
...

Other enzymes operating in the intestine, including lipids and proteases, together
with the very low pH of the stomach, contribute to the destruction of pathogens
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Competition with the bacteria already present further reduces the ability of
pathogenic bacteria to become established in the intestine
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The body must
distinguish them from its own cells and destroy them without damaging its own tissues
...
Inflammation is the rapid,
localised response of our tissues to damage, and is triggered when damage cells release chemicals
which act as an alarm, including histamine
...

Cells in the walls of capillaries draw away from each other (diapedesis), making them ‘leaky’
and producing more tissue fluid
...

Sensory neurones become more sensitive
...

The increased blood flow also results in more leucocytes (white blood cells) being brought to the
area
...
To do so, they must be able to distinguish foreign cells, and they do this with the aid of
complement proteins, which have several effects:
•
•
•

Attracting more phagocytes to the site of infection
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Binding to surface membranes of foreign cells, aiding the attachment of the surface
membrane of the phagocyte to a foreign cell
...
These bind to healthy cells nearby, and trigger
synthesis of antiviral proteins, slowing replication
...
There are
two distinct types, based on the ways they function:
1
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2
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Both types of lymphocyte are produced by multipotent stem cells in bone marrow
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T cells leave the bone marrow and
complete their maturation in the thymus gland
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In both cases, during maturation, any lymphocytes that would attack the body’s
own cells are selectively destroyed
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Recognition of ‘non-self’ Antigens by Lymphocytes:
Lodged within the ‘fluid-mosaic’ structure of the cell surface membranes, are glycoprotein
molecules
...
Cells in each tissue of the
body have markers that are different from those in other tissues, and from other organisms,
including other people
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They are encoded by genes on chromosome 6, so each persons MHC proteins are
genetically determined, and inherited
...

B and T cells both have molecules on their surface that are antigen
receptors, which are complementary to antigens – molecules that
trigger an immune response – that occur on the surface
membranes of other cells
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We produce several
million different antigen-receptor molecules, due to fast mutations
and crossing over during the encoding process
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They are a type of protein, called an immunoglobulin,
which can be abbreviated to Ig
...
It contains four polypeptides linked by disulphide bridges to form a ‘Y’
shape
...
Only two
small parts of the antibody molecule bind to a complementary antigen
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Similarly, to those of B-cells, the receptors are almost infinitely
variable, but there is only one type on the surface of each T-cell
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The Humoral Immune Response:
This response involves the release of antibodies by B-cells
...
The process occurs in a number of steps,
1
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This pathogen
randomly collides with a corresponding B-cell with a complementary antigen receptor
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The other cells of the pathogen
are attacked by macrophages
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The B-cell engulfs and digests the pathogen, then displays fragments of the antigen on its
surface
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Meanwhile, the macrophage from step one digests the antigen-carrying pathogen
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4
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This activates the T cell and it becomes an ‘activated T-helper cell
...
An activated T-helper cell binds to a B cell displaying the same antigen, activating the B cell
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Stimulated by cytokine secretion from the Thelper, the activated B-cell divides rapidly by
mitosis, forming a clone of cells called plasma cells,
which are packed with RER
...
It is produced in such numbers that
the antigen is overcome
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7
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However, some of these activated cells are retained as memory cells, which are long-lived,
and in the event of re-infection by the same antigen, these memory cells make an early and
effective response possible
...
T-cells will bind to an antigen-presenting cell that
has antigens from a pathogen on its surface
...
The release of cytokines stimulates the activated T-helper cell to divide
repeatedly to form a clone
...
T-killer cells: Destroy body cells infected by viruses
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T-helper cells: Release cytokines that stimulate production of B-cells
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Memory cells: Remain in the body and bring abut the secondary response
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ARTIFICIAL
IMMUNITY

NATURAL
IMMUNITY

LONGEVITY OF IMMUNITY

ACTIVE
IMMUNITY
(ANTIBODIES
MADE BY
SUBJECT)

Antibodies made
following
administration of
a vaccine
...


Production of
antibodies following
infection by, and
recovery from, a
disease
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Long-lasting since memory cells
are maintained throughout life
...


Mother’s antibodies
cross placenta to
foetus or ingested by
baby in mothers’
milk
...


Vaccination is the deliberate administration of antigenic material to stimulate the recipient to
develop active immunity against a pathogen
...

Purified components of the pathogen that have been found to have antigenic properties but
not cause disease
...

Genetically engineered DNA, designed to stimulate particular target cells of the immune
system
...
They then retain the
appropriate memory cells for life
...

It is not necessary for everyone in a population to be immunised against a disease for it to be
controlled, as the herd immunity theory proposes that the risk of someone who is susceptible to a
contagious disease becoming infected gets less, the greater the proportion of people in a community
who are immune
...

Definitions:
Diapedesis: A localised response to damage in which cells lining capillaries move part creating gaps,
through which plasma can leave the capillaries
...
It also allows phagocytes to leave the capillaries
...

Cytokines: A group of small proteins released by one type of cell that affect the behaviour of other
cells
...

Major histocompatibility complex proteins: Glycoproteins found on the cell surface membrane of a
cell that are unique to that individual
...
Small molecules, like amino acids, sugars,
and triglycerides, do not trigger an immune response
...
The surface membrane of cells is coated with antigens
...
The ability
of this glycoprotein to cause an immune response is reduced by the body destroying any randomly
produced lymphocytes with a complementary antigen-receptor
...

Herd immunity: Provided a large enough proportion of a population are immune to a particular
disease, the likelihood of the pathogen being transmitted to a member of the population who is not
immune is negligible



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