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All microorganisms share basic nutritional needs- a source of the elements carbon and
nitrogen for example
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To such a solution, growth factors specific to the needs of a particular microorganism
ae added, for example, and energy source or vitamins
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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
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The addition of a gelling agent,
such as agar, to a liquid medium
makes it solidify
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In a glass flat-sides bottle often
called a medical flat, or a test tube
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Allows harvesting of any
useful metabolic products
from the microorganisms
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Provides a large surface
area for growth and gas
exchange with the air in
the dish
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Provides a greater depth
of agar than a petri dish,
reducing the risk of
dehydration and salt
crystallisation
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Continuous culture- Inoculation of microorganisms into a sterile container containing liquid
growth medium
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Only batch culture is possible using solidified culture media since it contains a limited mass of agar
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They can be used to grow a wide variety of microorganisms, and these are known
as general purpose/broad spectrum media
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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
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Environmental temperature is an important variable in determining the ecological niche of each
species
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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
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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
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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
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They are
ineffective against
bacterial spores,
though
...
No
microorganism can
survive this exposure
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Moist heat- Place
objects in an autoclave
at 121 degrees for at
least 15 minutes
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Using a filter of
pore size 0
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Expose items to UV or
ionising radiation
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Dispose of wet equipment
immediately, treat any spillages that
occur
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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
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The size of the pores involved makes
this unsuitable for all but the smallest
volumes of liquid
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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
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You then dilute
the inoculum by spreading it multiple times
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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
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Viable count- Count only the living cells in the culture
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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
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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
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An indirect count, using a colorimeter to measure the turbidity of a culture
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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
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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
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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
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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
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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
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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
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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
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The generation time (g)- The time between two consecutive divisions
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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
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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: fever develops with low blood pressure, progressing to a come and organ failure
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Most are derived from fungi and bacteria commonly found in the soil
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Narrow-spectrum antibiotics- Effective over a limited range of bacteria
...

The action of antibiotics:
•
•

Bacteriostatic antibiotics- Prevent the multiplication of, but do not kill, bacteria
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Bactericidal antibiotics- Kill bacteria
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Nucleic
acid
synthesis

•
•
•
•
•

Disrupt DNA replication or transcription
Prevent formation of precursors of nucleic acids
Bind to DNA molecule and break down its double helix
Bind to DNA and prevent its replication and transcription
Inhibit one or more enzymes that catalyse either DNA replication or
transcription

Protein
synthesis

•
•
•
•

Inhibit protein synthesis
Inhibit the binding of tRNA to bacterial ribosomes
Prevent movement of TRNA-peptide complex
Prevent formation of peptide bonds
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This is called primary resistance
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This is called secondary resistance, and is a major problem
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Production by the bacteria of enzymes that can modify or inactivate the antibiotic
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In many cases, resistance is the result of a single gene
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Some bacteria replicate and divide by binary fission every 30 minutes
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If this happened before the development of antibiotics, the cell would have had no
advantage over any other, but today in a mammal being treated with the antibiotic, the cell would
have an advantage
...
The entire population would then be resistant
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Conjugation
tubes form between them, and they pass either one of their plasmids or part of their own nucleoid
through the tube to a difference cell
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Horizontal gene transfer, accounts for the increasing number of bacteria
that show resistance to many antibiotics
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This includes the following
practices:
•
•
•

Sub-clinical doses of antibiotics are routinely used in agriculture as growth promoters
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In many countries they are sold without prescription, and so are often used inappropriately
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As they are in these low
concentrations, they do not destroy bacteria, but the presence of antibiotics does create a selective
pressure for resistance
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Measure

explanation

Problems that may be
encountered
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Animal feeds account for about
half of all antibiotic sales
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The agricultural industry forms a
strong lobby group and would
resist any restriction on practices
that increase the profitability of
farming or the quality of their
products
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If family doctors stop prescribing
antibiotics as a precautionary
measure and prescribe them only
when they are needed, the
selective pressure favouring
resistant bacteria will lessen
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Many patients are
unwilling to take this advice and
pressure doctors
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Many patients begin to feel
better as their own immune
system overcomes the infection,
so stop taking the antibiotic
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We rely on people acting in the
best interests of the community at
large
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Create new antibiotics
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The development takes years and
the success rate is very low, not
only making it a slow process but a
very expensive one
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Like most fungi,
the stem rust fungus produces dormant spores, which are carried in the air to new plants
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A
mass of this is called a mycelium
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The enzymes digest chemicals in the stem and the fungus
absorbs the products of this digestion
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Uses nutrients that would otherwise be stored in the plant’s seeds, reducing the harvest
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Fungicides can be used to kill the stem rust fungus, but these are usually expensive
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The most promising way of eradicating the fungus
is by gene manipulation, creating cereals resistant to stem rust
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It contains eight short strands of RNA
surrounded by a protein capsid
...
Glycoprotein spikes project
through this and cover the surface of the virus
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The virus then enters the
epithelial cells lining the bronchus and bronchioles by endocytosis
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Viral antigens attach to receptors on the epithelium cell membranes and trigger endocytosis
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The goblet cell secretes mucus which protects the epithelium
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Copies of RNA segments move to the cytoplasm, where the viral proteins are replicated, and
the new virus particles are assembled
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The virus exits the host cell, picking up the lipid coat in the process, and goes on to cause
new infections
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When in human tissues, these act as antigens
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Often humans have little or no resistance to these new antigens,
with no memory cells with complementary receptors present in their lymph nodes
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From time to time, mutations lead to an extremely virulent strain of the influenza virus
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A pandemic must involve a novel virus strain, unfamiliar to human immune systems, an ability to
replicate in humans and cause disease, and an ability to be efficiently transferred between humans
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It is estimated that about 400
million people are infected each year, of whom 1
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It is caused by four species of
plasmodium, a single-celled protocist
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Although the male feeds on plant juices, the female drinks the
blood of humans by inserting her piercing mouthparts like a mini-hypodermic needle, inserting them
through the skin into a blood vessel, injecting saliva
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The saliva causes
inflammation and itching, and if the female was infected, the saliva also contains the infective stage

called sporozoites
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Eventually the infected liver
cells burst, releasing the merozoites into the
blood
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Each undergoes several cell
cycles to produce between 8 and 32 new
merozoites
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The cycle of infection of red blood cells
and merozoites being released then follows
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Each red blood cell that bursts releases
toxins produced during the breakdown of its contents by merozoites
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Prevention of infection includes a
number of different methods
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Not possible to drain large lakes, which are also breeding
grounds for mosquitoes
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Successful drainage might
involve inter-governmental co-operation which is not always
possible
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governments or areas occupied by warring groups
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Cover beds with fine-mesh
nets, coated with insecticide
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Mosquitoes that bite during the day are, however, emerging in
many areas
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Since releasing more insects in order to reduce the number of
insects seems counter-intuitive, local groups of people are
reluctant
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Vaccination
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Countries that would need this are often too poor
to afford this service
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Pathogen: An agent of infection that invades another organism, causing harm to it
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Antigenic variability: Variation in the chemical nature of the same type of antigen resulting from
frequent gene mutations and, in eukaryotes, different splicing of the pre-MRNA molecules
transcribed from the same gene
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The outbreak is widespread within one community at a
particular time
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Endemic: A disease that occurs frequently or is constantly present at a low level of infections, in
particular areas or among people of a particular population
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Response to infection
An infectious disease is caused when another organism or virus invades the body and lives there
parasitically
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There are three
ways in which pathogens can enter our tissues – via our skin, our lungs, and intestines
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INTERFACE PROTECTIVE MECHANISMS
SURFACE
OF BODY

Our skin is normally an effective physical barrier
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Cells in the outer layer are dry and filled with a tough,
indigestible protein, keratin
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LUNGS

The linings of the trachea, bronchi, and bronchioles secrete copious volumes of
mucus, which trap microorganisms
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INTESTINE

Our saliva contains lysosomes: enzymes that hydrolyse components of bacterial
cells
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Our intestines are also colonised by hundreds of different species of commensal
bacteria
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Once the pathogens have entered the body, they are much harder to eliminate
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Non-specific Inflammatory Response:
This response is non-specific, meaning it is the same for all pathogens
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These chemicals have the following effect in that area:
•
•
•

The smooth muscle of the arterioles relaxes, increasing blood flow
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This causes localised swelling
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This means that the volume of blood in the damaged area is increased, and we suffer local oedema
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Macrophages and neutrophils are phagocytic leucocytes, meaning they engulf and destroy the
pathogens
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Bindi

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